CLINICAL BACTERIOLOGY|LECTURE

PRELIMS LESSON 1: INTRODUCTION AND HISTORICAL BACKGROUND

TOPIC OUTLINE Evidence Pro and Con
 Introduction and Historical Background 1745: John Needham put boiled nutrient broth into covered flask.
1. Spontaneous Generation 4. Koch’s Postulates Condition Results
2. Fermentation and Pasteurization 5. Vaccination Nutrient broth heated, then placed in sealed flask. Microbial growth
3. The Germ Theory of Disease 6. Birth of Modern Chemotherapy
ROLES OF A CLINICAL MICROBIOLOGIST
• Culture of organisms from specimens (different body fluids, blood culture etc.).
- Snapshot of specimen
• Classification and identification of organisms after they have been isolated.
- possible cause of disease (Etiologic agents).
• Prediction and interpretation of antimicrobial susceptibility patterns. (Culture and
sensitivity testing – antibiotic sensitivity testing. - Determining which antibiotic is
more appropriate.)
- Improve treatment.
BACTERIAL RELATIONSHIP TO ISOLATION
• Growth requirements of bacteria
- Allows microbiologist to select the correct medium for primary culture The experiment was flawed because Needham did not sterilized the Flask, so the
Increases likelihood of pathogen isolation. flask is contaminated abling the growth of organism.
• Steps in bacterial identification ▪ Boiled Mutton broth became cloudy after pouring in
(Phenotypic characteristic) John Needham flask and sealed tightly.
- Determine staining characteristics based on cell wall structure, organism ▪ An advocate of spontaneous generation (prons)
size, and organism shape. Evidence Pro and Con
- Observe metabolic biochemical reactions. 1765: Lazzaro Spallanzani boiled nutrient solution in flask
Condition Results
HISTORICAL PERSECTIVE
Nutrient broth placed in flask, heated, then sealed. No microbial growth
SCIENTIST CONTRIBUTION
Lucretius
and ▪ Suggested that a disease was caused by “invisible
Girolamo living creatures”
Fracastoro
▪ A Dutch lens maker and biologist.
▪ First true Microbiologist.
Antonie Van ▪ First person to observe and describe
Leeuwenhoek microorganisms accurately (Father of Protozoology
and Microbiology).
▪ Discovered “wee beasties”/ “animalcules”.
The debate over SPONTANEOUS GENERATION
▪ The hypothesis that living organisms arise from nonliving matter is
called spontaneous generation. According to spontaneous generation, a ▪ Improved the previous experiments of Needham by
Lazzaro
"vital force" forms life. heating broth placed in a sealed jar.
Spallanzani
- those who believed are called Pros and are (fake news peddler). ▪ No growth as long as flask remains sealed.
▪ The alternative hypothesis, that the living organisms arise from Rudolf Virchow ▪ Challenged spontaneous generation with the concept
preexisting life, is called biogenesis. of “biogenesis”.
- those who don’t believe in the spontaneous Generation. Evidence Pro and Con
Evidence Pro and Con 1861: Louis Pasteur demonstrated that microorganisms are present in the air.
1668: Francisco Redi Filled six jars with decaying meat. Condition Results
Condition Results Nutrient broth placed in flask, heated, not sealed Microbial growth
Three jars covered with fine net. No maggots Nutrient broth placed in flask, heated, then sealed No microbial growth
Three open jars Maggots appeared

▪ Debunked Spontaneous Generation by using

Francesco Redi maggots and decaying meat in different jars.
▪ An advocate for Biogenesis
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CLINICAL BACTERIOLOGY|LECTURE
PRELIMS LESSON 1: INTRODUCTION AND HISTORICAL BACKGROUND
▪ Microorganisms are present in the air and can KOCH’S POSTULATES
contaminate sterile solutions, but air does not create
microbes.
▪ Microbial life can be destroyed by heat (Basis of
Antiseptic technique)
▪ Developed vaccines against anthrax (1881) and
rabies (1885)
Louis Pasteur
▪ Improved wine industry (Theory of Fermentation)
▪ Pasteurization: Heating the drink just enough to kill
most of the bacteria
▪ Vaccine: For cultures of avirulent microorganism use
for preventive inoculation
▪ Father of Microbiology and Bacteriology (Theory
of biogenesis)
The Golden Age of Microbiology – Pasteur
▪ Beginning with Pasteur’s work, discoveries included
1857-1914 the relationship between microbes and diseases,
immunity, and antimicrobial drugs.
FERMENTATION and PASTEURIZATION
Fermentation - anaerobic respiration (no oxygen) 1. The microorganism must be present in every case of the disease but absent
from healthy organisms.
▪ Saccharomyces cerevisiae (Yeast) – will convert the grape juice (CHO) into an 2. The suspected microorganism must be isolated and grown in a pure culture.
alcohol. 3. The same disease must result when the isolated microorganism is inoculated
▪ If the grape juice is contaminated with bacteria (yeast +bacteria) the grape juice into a healthy host.
will be converted into vinegar instead of alcohol. – Pasteurization 4. The same organism must be isolated again from the diseased host.
▪ Pasteur showed that microbes are responsible for fermentation. Fermentation EXCEPTIONS TO KOCH'S POSTULATES
is the conversion of sugar to alcohol to make beer and wine.
▪ Microbial growth is also responsible for spoilage of food Bacteria that use a. Microorganisms that are unable to be cultured on artificial media (example:
alcohol and produce acetic acid spoil wine by turning it to vinegar (acetic acid). Treponema pallidum) - Syphillis
b. 2 or more organism work in synergy to cause a disease. – multiple infection
▪ Pasteur demonstrated that these spoilage bacteria could be killed by heat that
(ex. COVID-19 (primary infection) and TB (2nd))
was not hot enough to evaporate the alcohol in wine.
c. Symptoms and diseases can be causes by any one of several microbes. (ex.
▪ Pasteurization is the application of a high heat for a short time. Diarrhea)
▪ Classical Method of Pasteurization is Low temperature Holding (LTH) Fannie Eilshemius ▪ Suggested use of agar as a solidifying agent. Before?
▪ 63 Centigrade for 30 mins for grape juice Hesse _____________
Theodore ▪ Yeast cells were responsible in converting sugars to Julius Richard ▪ Developed the Petri dish.
Schwann alcohol. Petri ▪ Before? ___________________
THE GERM THEORY OF DISEASE – Robert Koch Martinus
For ever disease there is an ethologic/ causative agent Beijerinck and ▪ Developed the enrichment-culture technique and the
1835 ▪ Agostino Bassi showed that a silkworm disease was Sergei use of selective media
caused by a fungus. Winogradsky
1865 ▪ Pasteur believed that another silkworm disease was VACCINATION
caused by a protozoan.
▪ Edward Jenner inoculated a person with cowpox
▪ Ignaz Semmelweis advocated hand washing to virus.
1840s prevent transmission of puerperal fever from one OB
▪ The person was then protected from smallpox.
patient to another. 1796
▪ Vaccination is derived from vacca for cow.
▪ Joseph Lister used a chemical disinfectant to
prevent surgical wound infections after looking at ▪ The protection is called immunity.
1860s
Pasteur's work showing microbes are in the air, can ▪ First virus utilized by Vaccine is the Vaccinia Virus.
spoil food, and cause animal diseases. ▪ Father of Immunology
▪ Robert Koch proved that a bacterium causes ▪ First to discovered Vaccination.
1876 anthrax and provided the experimental steps, Koch's ▪ Experimented on how people can be protected
postulates to prove that a specific microbe causes a Edward Jenner against smallpox.
specific disease. Anthrax – zoonotic disease
▪ Scrapings from ________________, inoculated in a
Ignaz Semmelweis ▪ Doctor who first endorsed handwashing. healthy volunteer.
▪ Developed the antiseptic system of surgery: Phenol. ▪ Basis? _______________________
Joseph Lister ▪ Discovered Salvarsan (Arsphenamine) to treat
▪ Father of Aseptic surgery Paul Ehrlich
Syphilis
▪ First proof that bacteria indeed cause diseases
▪ Discovered Bacillus anthracis and Mycobacterium THE BIRTH OF MODERN CHEMOTHERAPY

Robert Koch tuberculosis. ▪ Treatment with chemicals is chemotherapy.

▪ Developed culture media for observing growth of ▪ Chemotherapeutic agents used to treat infectious disease can be synthetic
bacteria: Boiled potatoes, gelatin, and used meat drugs or antibiotics.
extracts and protein digests
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CLINICAL BACTERIOLOGY|LECTURE
PRELIMS LESSON 1: INTRODUCTION AND HISTORICAL BACKGROUND
▪ Antibiotics are chemicals produced by bacteria and fungi that inhibit or kill Selected Nobel Prizes in Physiology or Medicine
other microbes. Quinine from tree bark was long used to treat malaria.  1901 von Behring Diphtheria antitoxin
▪ Paul Ehrlich developed a synthetic arsenic drug,  1802 Ross Malaria transmission
salvarsan, to treat syphilis.  1905 Koch TB bacterium
1910 ▪ Agent 606 because he failed 606 times because of  1908 Metchnikoff Phagocytes
the Selective toxicity – any drug that you will be  1945 Fleming, Chain, Florey Penicillin
drinking will only be harmful to the microorganism/  1952 Waksman Streptomycin
bacteria and not to the human host.  1069 Delbrück, Hershey, Luria Viral replication
1930s ▪ Sulfonamides were synthesized  1987 Tonegawa Antibody genetics
 1997 Prusiner Prions
▪ Alexander Fleming discovered the first antibiotic.
* The first Nobel Prize in Physiology or Medicine.
1928 ▪ He observed that Penicillium fungus made an
antibiotic, penicillin, that killed S. aureus.
▪ Discovered Penicillin
▪ Fortunate accident – the bacteria that he is culturing
go infected by a fungus, and the fungus are
Penicillium notatum his observation shows that there
is a clear zone between the fungus and the bacteria.
Alexander Fleming
Zone of inhibition (clear zone) – a zone where the
growth of bacteria is inhibited. He extracted the
component from the fungi and called it Penicillin.
▪ Penicillin the first natural antibiotic
▪ Reason why he will the noble price
Howard Florey and
Ernst B. Chain ▪ Made the purification process for Penicillin

1940s ▪ Penicillin was tested clinically and mass

MODERN DEVELOPMENT IN MICROBIOLOGY

• Bacteriology is the study of bacteria.
• Mycology is the study of fungi.
• Parasitology is the study of protozoa and parasitic worms.
• Recent advances in genomics, the study of an organism's genes, have provided
new tools for classifying microorganisms.
• Immunology is the study of immunity.
• Vaccines and interferons are being investigated to prevent and cure viral
diseases.
• The use of immunology to identify some bacteria according to serotypes
(variants within a species) was proposed by Rebecca Lancefield in 1933.
- SEROTYPES – Lancefield group streptococcus (GROUP A B C D)
• Virology is the study of viruses.
• Recombinant DNA is DNA made from two different sources. In the 1960sPaul
Berg inserted animal DNA into bacterial DNA and the bacteria produced an
animal protein.
• Recombinant DNA technology or genetic engineering involves microbial
genetics and molecular biology.
• Using microbes
George Beadle and
Edward Tatum ▪ showed that genes encode a cell's enzymes (1942)

Oswald Avery,
Colin MacLeod,
▪ showed that DNA was the hereditary material (1944).
and Maclyn
McCarty

Francois Jacob
and Jacques ▪ discovered the role of mRNA in protein synthesis
Monod (1961)

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CLINICAL BACTERIOLOGY|LECTURE(Sir Gelo)
PRELIMS LESSON 2: INFECTION CONTROL AND LABORATORY SAFETY
TOPIC OUTLINE ▪ Close-fronted BSC with an airtight system, completely
 Biosafety separating worker from the sample.
BIOSAFETY ▪ Also known as the glove-box cabinet.
• FUNDAMENTAL OBJECTIVE: Containment of potentially hazardous biological CLASS III
▪ Provides the highest level of safety to the laboratory worker.
agents and toxins ▪ Interior of the cabinet is under a negative pressure.
• Logo of Biosafety: ▪ Air coming in or out of the cabinet is sterilized using HEPA
filter.
▪ Developed by Charles L. Baldwin and Robert S. Runkle in
1966 ▪ Preferred for BSL 4 agents.
▪ May be black, fluorescent orange, or orange-red
▪ Background: Optional, sufficient contrast needed CLASSIFICATION OF BIOLOGICAL AGENTS
THE BIOSAFETY CABINET ▪ Agents that have no known potential for infecting healthy
▪ Device (Primary containment) that encloses a working area to protect people and immunocompetent adult humans
BSL 1
workers from aerosol exposure and/or infectious disease agents. ▪ Used for Laboratory activities of students (minimal threat)
▪ AKA ventilated cabinet or ventilated enclosure. ▪ EXAMPLES: Bacillus subtilis, Escherichia coli
▪ Engineering control that is utilized in areas handling biological samples. ▪ Acquired by: Ingestion/Exposure to percutaneous or
mucous membranes.
▪ Considered a standard device in the academic and clinical laboratory to ▪ Include all common agents of infectious diseases, pose
contain hazardous biochemical agents and its products. moderate potential hazard.
▪ Air is sterilized either by heat, UV light, or passage through a High- BSL 2 ▪ Considered indigenous and cause severe diseases.
efficiency particulate air (HEPA) filter removing particles larger than 3 um. ▪ Personnel handling these agents must receive
TYPES OF BIOSAFETY CABINETS immunization.
▪ Open-fronted, uses an exhaust fan to allow movement of air ▪ EXAMPLES: Bacillus anthracis, Yersinia pestis (BSL 2 or
going through the open front. 3), Salmonella, Shigella, HIV (BSL 2 or 3), HBV
▪ Allows room (unsterilized) air into the cabinet, to circulate ▪ Potential agents for aerosol transmission.
around the workspace, and expose the material within: ▪ Either indigenous or exotic, transmitted through respiratory
CLASS I Samples exposed to contamination. route.
▪ Only the air to be exhausted is sterilized using HEPA filters. BSL 3 ▪ Air movement in Laboratory: controlled to contain infectious
▪ Protection: Laboratory worker environment only. materials.
▪ Mostly used for: BSL 1 agents, can be used for BSL 2 ▪ EXAMPLES: Mtb, Francisella tularensis, Brucella, Coxiella
agents. burnetti, St. Louis Encephalitis virus, systemic fungi
▪ Provides protection for the worker, environment, and ▪ Life-threatening infections and diseases, dangerous or
sample. exotic.
▪ Air sterilized using HEPA filter. ▪ Aerosol transmission with pressure: very high or unknown
▪ Mostly for BSL 1 and 2 agents, provisions available for risk of transmission.
BSL 3 organisms. ▪ Maximum containment and decontamination strictly
▪ 2 TYPES: BSL 4
observed (Class III BSC).
o Class II A: Air exhausted to the room/thimble ▪ No exact treatment or vaccine available.
connection. ▪ Laboratory must have strict access control.
▪ Class II, Type A1
▪ EXAMPLES: arbovirus, arenavirus, filovirus (Ebola and
• Only utilized for biological samples.
Marburg).
• Airflow: 70% recirculated, 30%
exhausted.
CLASSIFICATION OF INFECTIVE MICROORGANISMS BY RISK GROUP
▪ Class II, Type A2
• Most commonly used BSC. Risk Group 1
▪ Without/Low Individual and Community Risk.
• For biological and clinical (infectious) ▪ Unlikely to cause human or animal disease.
sample. ▪ Moderate Individual Risk, Low Community Risk.
CLASS II • For specimens treated with minimal Risk Group 2 ▪ Can cause disease, unlikely to be a serious hazard.
concentration of volatile chemicals. ▪ Laboratory exposures may cause serious infection, but
• Airflow: 70% recirculated, 30% with effective treatment and preventive measures.
exhausted. ▪ High Individual Risk, Low Community Risk.
o Class II B: Air exhausted outside the facility through a ▪ Causes serious human or animal disease, does not
hard duct. Risk Group 3
spread from one infected individual to another, with
▪ Class II, Type B1 effective treatment and preventive measures.
• For biological samples and minimal ▪ High Individual and Community Risk
concentration of volatile or toxic
Risk Group 4 ▪ Causes serious disease, can be readily transmitted;
chemicals
effective treatments/preventive measures not usually
• Airflow: 30% recirculated, 70% available
exhausted
▪ Class II, Type B2 CLASSIFICATION OF INFECTIOUS SUBSTANCES
• For processing chemicals, AS TO TRANSPORT OF SAMPLES
radioisotopes, carcinogens aside from CATEGORY A ▪ Capable of causing permanent disability, lifethreatening or
biological samples INFECTIOUS fatal disease
• Airflow: 100% exhausted SUBSTANCES

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CLINICAL BACTERIOLOGY|LECTURE(Sir Gelo)
PRELIMS LESSON 2: INFECTION CONTROL AND LABORATORY SAFETY
▪ Pose the greatest biosafety and biosecurity risks; subject is less than the ▪ Factors affecting Hydrogen Peroxide,
to the most stringent risk control procedures; Requires “sterility assurance effectiveness: Antibacterial soaps).
trainings and certifications for handling level” or one in one − Nature & Number of ▪ Sterilize: Reduce the
▪ Capable of causing infections but do not meet Category A million (10-6) Contaminating number of
CATEGORY B ▪ Mostly used in: microorganisms to
inclusions. Microorganisms
INFECTIOUS safe hygienic levels,
SUBSTANCES ▪ Strict regulations but lower risk and severity compared to − Culture Media, − Amount of Organic
Category A. Pipette Tips Matter present less effective than
− Tubes, Plates disinfection.
EXEMPT ▪ Derived from human/animal patients with minimal − Type &
HUMAN/ likelihood that infectious biological agents are present. − Non-flammable concentration of
ANIMAL ▪ Still require triple packaging. reagents germicide used,
SPECIMENS − Heat-sensitive duration &
stock solutions temperature of
CATEGORIES OF POTENTIAL AGENTS OF BIOTERRORISM ▪ Can be achieved thru: germicide contact
▪ Pose the greatest public health threat; Easily transmitted − Heat, Ethylene − Type and condition
and highly infectious. Oxide Gas of instruments,
▪ High mortality rates, may cause public panic and social − Hydrogen devices, and
CATEGORY A disruption. materials
Peroxide Gas
▪ EXAMPLES: Bacillus anthracis, Clostridium botulinum, (Spaulding
− Ozone, Radiation classification)
Francisella tularensis, Yersinia pestis, smallpox, Ebola,
Marburg, Lassa, Machupo viruses
▪ Moderate morbidity and low mortality; Not as easily
transmitted.
▪ Require surveillance and diagnostic assistance from CDC
CATEGORY B ▪ EXAMPLES: S. aureus enterotoxin B, Clostridium
perfringens, E. coli O157:H7, Salmonella, Shigella, Vibrio
cholerae, Burkholderia mallei and pseudomallei, Brucella,
Chlamydia psittaci, Coxiella burnetti, Rickettsia prowazekii,
Cryptosporidium parvum
▪ Emerging pathogens, easily produce and disseminate,
CATEGORY C potential for high morbidity and mortality.
▪ EXAMPLES: Hanta virus, Nipah virus
HAZARDOUS WASTES
• Substances, which in singly or in combination, have a significant hazard to human
health or the environment. STERILIZATION METHODS
• Require special handling, processing, or disposal. APPLICATION ▪ Heat is the most used method for the removal of
LABORATORY WASTE RECOMMENDED TREATMENT OF HEAT microorganisms
UNCONTAMINATED, NON- Can be reused/recycled/disposed as general ▪ It is the method of choice for sterilization of antibiotic
INFECTIOUS waste FILTRATION solutions, toxic chemicals, radioisotopes, vaccines and
Collected in puncture-proof containers with METHOD carbohydrates (heat-sensitive).
CONTAMINATED SHARPS ▪ It maybe used with both liquid and air
covers, treat as infectious
Decontaminate first (Chemical/Physical) then ▪ It is considered bacteriostatic, because it reduces the rate
CONTAMINATED, FOR of metabolism (0°-7°C).
wash; Can be treated after as uncontaminated
RECYCLE/ RECYCLING ▪ Deep Freezing: -50 to -95°C for foods and drug
material LOW/ COLD
CONTAMINATED, FOR Decontaminate onsite or stored safely before preservation.
TEMPERATURES
DISPOSAL transportation to another site for decontamination ▪ it is important in food microbiology.
CONTAMINATED, FOR Incinerated onsite or stored safely before ▪ Example: the agent of Syphilis is killed when exposed to
INCINERATION transportation to another site for incineration 2-8°C for 72 hours
LIQUID WASTE, FOR Should be decontaminated before disposal in a DESSICATION AND LYPHILIZATION
DISPOSAL IN SEWER sanitary sewer system ▪ Destroys bacteria by disruption of metabolism; it involves
DESSICATION
DECONTAMINATION removing water from microbes (bacteriostatic).
• Used to describe a process of: ▪ The most effective method for long term preservation of
microbial cultures.
▪ Inactivating or reducing contaminants to an acceptable level
▪ Reasonably free from risk of transmitting disease and can be handled LYPHILIZATION − Neisseria gonorrhoeae - viable for one hour
safely − Mycobacterium tuberculosis - viable for several months
• Incineration is NOT an option: Philippine Clean Air Act and Ecological Solid − Bacillus and Clostridium - viable for ten years (decade)
Waste Management ▪ The use of high concentrations of salts and sugars in food
STERILIZATION DISINFECTION ANTISEPSIS create a hypertonic environment.
OSMOTIC
▪ Plasmolysis: As water leaves the cell, plasma membrane
▪ A Process that ▪ Less lethal process ▪ Antiseptic: A PRESSURE
shrinks away from the cell wall (cell may not die, but
eliminates all viable than Sterilization chemical agent used usually stops growing)
microorganisms, ▪ Eliminates nearly all on the skin to safely
▪ Principle: when it passes through the cells, it creates free
including the most recognized Pathogenic reduce the number
hydrogen and hydroxyl radicals and some peroxidases
resistant forms microorganisms but not of microorganisms RADIATION
which in turn cause different intracellular damage.
▪ Microorganism necessarily all and reduce risks of
▪ Biological indicator: Bacillus pumilus
surviving on an item microbial forms on infection (Iodine,
subjected to treatment inanimate objects.
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CLINICAL BACTERIOLOGY|LECTURE(Sir Gelo)
PRELIMS LESSON 2: INFECTION CONTROL AND LABORATORY SAFETY
TYPES OF PASTEURIZATION
HIGH TEMP SHORT
LOW TEMP HOLDING ULTRA HIGHT
TIME (HTST)/ FLASH
(LTH)/ BATCH METHOD TEMPERATURE (UHT)
PASTEURIZATION
▪ Reduce spoilage of ▪ Quick heating then ▪ Milk can be stored at
food without affecting immediate cooling RT for 2 months
taste. ▪ 72˚C for 15 seconds without affecting
▪ Destroys milk-borne flavor; Applicable for
pathogens coffee creamers.
▪ 63˚C for 30 minutes ▪ 140˚C for 3 seconds
(Cooled in vacuum
chambers)

DRY HEAT PROCEDURE

MOIST HEAT PROCEDURE
▪ Destroy by coagulation of enzymes and structural proteins, degenerate
nucleic acids.
BOILING
▪ Destroys vegetative bacteria (nonsporulating)
▪ Time & Temp of exposure: 100˚C, 10-15 mins
▪ Chamber filled with hot steam under pressure (Steam
under pressure)
▪ Fastest and simplest method of sterilization (Except
Prions) and spores are killed within 15 minutes.
▪ Principle: steam under pressure
AUTOCLAVE ▪ Biological Indicator: Geobacillus stearothermophilus
▪ Use: sterilize biohazardous trash and heat-stable
objects
a. 121˚C, 15 PSI, 15 minutes: Media, Liquids,
Utensils, Glass Pipettes, Instruments for Assay
b. 132˚C, 15 PSI, 30-60 minutes: Medical waste
▪ Naked pieces of protein, like a virus, but without the
Prions nucleic acid; the most resistant to the actions of heat,
chemicals, and radiation
▪ Destroys vegetative cells & spores after 3 consecutive
TYNDALLIZATION days of sterilization.
(Fractional/
Intermittent ▪ Temp, Time of exposure: 100˚C for 30 mins for 3
consecutive days
Sterilization)
▪ Instrument: Arnold’s Sterilizer (Free flowing steam)
▪ It is used to sterilize protein-rich medium such as
Lowenstein Jensen Medium.
INSPISSATION ▪ Principle: it involves thickening through evaporation.
▪ Temp, Time of exposure: 70-80˚C for 2 hours for 3
consecutive days.
▪ Used to sterilize milk, dairy products, alcoholic
beverages.
PASTEURIZATION/
partial sterilization ▪ Eliminates foodborne pathogens and organisms
responsible for food spoilage.
▪ This method cannot eliminate bacterial endospores.
▪ Kills microorganisms by denaturation of proteins; Sterilization without water.
o Use: glassware, oil products and powders
o Not applicable for heat-sensitive materials (rubber and plastic).
o Biological Indicator: Bacillus atrophaeus NRS 1221A (ATCC 9372).
FLAMING
▪ Direct heating
▪ Sterilization of inoculating loops and needles
OVEN
▪ For glassware, oil, petrolatum, powders
▪ 160-170˚C for 1.5-2 hours
▪ Most common method of treating infectious waste and
infected laboratory animal.
INCINERATION
▪ Burning materials into ashes (300-400˚C)
▪ Hazardous materials: 870-980˚C
CREMATION ▪ Control spread of communicable diseases
INCINERATION NOT OPTION: Philippine Clean Air Act and Ecological Solid
Waste Management
• Terminologies:
▪ THERMAL DEATH POINT: Lowest temperature at which a suspension of
bacteria is killed in 10 minutes.
▪ THERMAL DEATH TIME: Shortest period needed to kill bacteria at a
prescribed temperature and under specific conditions.
▪ DECIMAL REDUCTION TIME (DRT/D/D VALUE): Time in minutes to
reduce the bacterial population or spores by 90% at a specified
temperature.
o Used or observed in the food industry
FILTRATION METHODS
• Method of choice for antibiotic solutions, toxic chemicals, radioisotopes,
vaccines, carbohydrates (May be used for liquid or air)
▪ 2 TYPES OF FILTERS
o Depth Filters
▪ Consist of fibrous or granular material
▪ Examples: Berkefield filter (Diatomaceous Earth),
Chamberlain filter (unglazed porcelain), and asbestos
o Membrane Filters (Circular Filters)
▪ Porous membranes (almost 0.1 um thick)
▪ Composed of cellulose acetate or polycarbonate.
▪ Use: Pharmaceuticals, ophthalmic solutions, culture
media, antibiotics and oil products
TYPES OF FILTERS
▪ It uses cellulose acetate/ cellulose nitrate membrane
LIQUID
with a vacuum
AIR
▪ Uses high-efficiency particulate air (HEPA) filters.
▪ HEPA FILTERS - remove organisms larger than 0.3um
FILTRATION OF ▪ Uses 0.2 to 0.45um pores of membrane filters
BACTERIA,
YEAST, MOLDS
▪ Uses 0.22um membrane filters for parenteral solutions.
CRITICAL
▪ 0.22um membrane filters are used to remove vegetative
STERILIZING
cells but not viruses
− HEPA FILTER – 99.97% of 0.3 microns
− ULPA FILTER – 99.999% of MPPS
− SULPA FILTER – 99.9999% of MPPS
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CLINICAL BACTERIOLOGY|LECTURE(Sir Gelo)
PRELIMS LESSON 2: INFECTION CONTROL AND LABORATORY SAFETY
RADIATION
• Creates free hydrogen and hydroxyl radicals and some peroxidases, cause
intracellular damage
• Biological Indicator: Bacillus pumilus
▪ DNA mutation: Generates peroxides
▪ Destroys vegetative cells and endospores of both
prokaryotes and eukaryotes
IONIZING
RADIATION (COLD ▪ Uses: Plastic syringe, sutures, catheters, gloves,
hormone solutions, antibiotics
STERILIZATION)
▪ Used to “pasteurize” meat products
▪ Examples: Gamma Rays (1500 to 2500 radiation),
X-Rays
▪ DNA mutation: Produce thymine dimers
NON-IONIZING
▪ Uses: Exposed surfaces, air, operating rooms,
nurseries, cafeterias
RADIATION
▪ Examples: UV Rays (10-400nm), (260 nm is the
most lethal)
▪ Effective against DNA, little to no activity on RNA
CHEMICAL METHODS
▪ Mechanisms by which chemicals destroy microorganisms:
o Reaction with components of the cytoplasmic membrane
o Denaturation of cellular proteins
o Reaction with thiol groups of enzymes
o Damage to DNA and RNA
▪ Factors influencing activity of disinfectants:
o Type of organism present and load
o Temperature and pH level of process
o Concentration of disinfectant
o Number of organics and biofilms
o Contact time.
o Type of water used for disinfection.
CHEMICAL METHODS
▪ Common chemical agents used in microbial control.
ACID AND
ALKALINE ▪ Hydrolyzes and coagulate proteins
SOLUTIONS
▪ First widely used antiseptic and disinfectant
▪ Destroy plasma membrane and denatures cell
proteins.
▪ Effective even in presence of organics
▪ 5% Phenol, 10-30 mins contact time: Tuberculocidal
▪ Effect: Disruption of plasma membrane, denaturation
PHENOL of enzymes
▪ Structure: 5-Carbon aromatic ring
▪ Examples: Cresol, Xylenol, Hexachlorophene,
Amphyl, Orhophenylphenol
▪ Active against: Most vegetative bacteria
(Mycobacterium), viruses (HepaB), inactive against
spores
▪ Cause denaturation of proteins and dissolution of lipid
membranes
▪ Used as antiseptic + disinfectant (Bactericidal +
Fungicidal)
▪ 60-90% concentration (100% Alcohol: Can dehydrate
cells but limited effectivity because it cannot coagulate
ALCOHOL
proteins)
(Non-sporicidal)
▪ Should be allowed to evaporate from surface to
achieve complete antisepsis.
▪ Examples: Isopropanol and Ethanol
▪ Disadvantage: Poor activity against non-enveloped
viruses, ineffective in the presence of organic
materials
▪ Destroy microorganisms through oxidation
▪ Examples: Chlorine, Iodine, Fluorine, Bromine,
Astatine
▪ NaOCl: Freshly prepared everyday, 10-30 mins
contact time to make it tuberculocidal (1:10 or 1:100
dilution)
o Action: Hypochlorous acid oxidizes the
sulfhydryl group on amino acids
▪ Halozone (Parasulfone Dichloroaminobenzoic Acid):
contains Chloride: Used to disinfect drinking wate
HALOGENS
▪ PREPARATION OF IODINE: TINCTURE &
IODOPHOR
o Tincture of Iodine (Antiseptic): 2% Iodine +
70% Alcohol
o Iodophor (Antiseptic/Disinfectant): Iodine +
Neutral Polymer carrier
▪ 70% Alcohol followed by Iodophor: Most common form
of antisepsis before drawing blood for culture or
surgery
▪ 30-60 seconds contact time prior to blood collection
▪ A combination of iodine and neutral polymer.
▪ Antibacterial effect: oxidative effects of molecular
iodine and hypoiodic acid.
IODOPHOR ▪ Improperly diluted iodophors may not kill
microorganisms - lack of free iodine in solution.
▪ Free iodine - Degrades microbial cell walls,
cytoplasm, denatures enzymes and coagulates
chromosomal material.
▪ Antibacterial effect: Oxidative effects of hypochlorous
acid (formed when chloride ions are dissolved in
water)
▪ 1:10 dilution of 5.25% solution– recommended by
CDCs for cleaning blood spills
CHLORINE
▪ Contact time: 3 mins. (longer in the presence of
organic material); 10-30 mins. = Mycobacteria
▪ Made fresh daily, loses its effectiveness in the
presence of a large amount of protein material.
COMMON CHEMICAL AGENTS USED IN MICROCIAL CONTROL
▪ Destroy microorganisms by inactivating and
precipitating cell proteins
SALTS OF
▪ Example: Copper, Arsenic, Mercury, Silver, Zinc
(Bacteriosatatic)
HEAVY
METALS o AgNO3: Eye drop against Neisseria
gonorrhoeae (Opthalmia neonatorum)
o HgCl2: Antiseptic
o CuSO4: Algicide
▪ Non-tuberculocidal, Non-sporicidal.
▪ Widely used as surface-active agent
▪ Effect: Disruption of the cell membrane resulting in the
leakage of cell contents
QUATERNARY
AMMONIUM ▪ Used on Laboratory bench-tops and floors, cleaning
COMPOUNDS dairy utensils, food outlet facilities
▪ Examples: Zephiran (Benzalkonium chloride +
Ceepryn), Cetylpyridinium chloride.
▪ Pseudomonas aeruginosa cultures in ammonium
acetate medium: resistant to quats
▪ Derivative of phenol with reduced toxicity.
▪ Stable surface disinfectant, effective in presence of
PHENOLIC organics.
COMPOUNDS ▪ Effects: Disrupts microbial cell walls and membranes,
(non-sporicidal) precipitates proteins.
▪ Common phenolics: Orthophenylphenol, and Ortho-
benzyl-para-chlorophenol
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CLINICAL BACTERIOLOGY|LECTURE(Sir Gelo)
PRELIMS LESSON 2: INFECTION CONTROL AND LABORATORY SAFETY
▪ Examples: Chlorhexidine gluconate, Chloroxylenol, ▪ Test organisms: S. aureus and S. serotype Typhi (20 or 37˚C)
Triclosan ▪ Solving for the PC:
▪ Chlorhexidine Gluconate (Biguandines):
o Inactivates certain lipid-enveloped viruses,
Disrupts plasma membranes ▪ Result: PC >1, disinfectant more effective than Phenol
o Binds to the skin and remains active for at ▪ Interpretation: The higher the PC value, the more effective the disinfectant
least 6 hours MICROBIAL RESISTANCE
o Effective antiseptic, used in hospitals prior to LEVEL OF RESISTANCE
surgery • Most resistant: Bacterial Endospores (Clostridium tetani)
▪ Chloroxylenol: • Moderate resistance: Fungal spores, Parasite cysts, Mycobacterium tuberculosis,
o Effective for surface cleansing contaminated Naked viruses, Hepatitis B, Polio
with body fluids (Blood, Sputum); Antiseptic • Least resistance: Most vegetative bacteria, Parasite trophozoites, Enveloped
▪ Triclosan: viruses (Influenza A), Fungi
MICRBIAL CONTROL
o More effective than CHG, utilized for clothing
and furniture decontamination, additive to Bacterial populations die at a constant logarithmic rate.
dental care liquid (Broad spectrum, most
effective against Gram positive)
▪ Inactivation of proteins and nucleic acids
ALDEHYDE ▪ Recommended for sterilizing medical instruments
(Cold/ Chemical o Used as Formalin (37% aqueous) or HCHO gas
Sterilant) o Not for routine disinfection/sterilization
o 3-8% HCHO, 30 mins: Tuberculocidal
o Sterilize HEPA filters in BSCs
TWO TYPES OF ALDEHYDES
▪ Generally used as Formalin – 37% aqueous solution
or HCHO gas.
FORMALDEHYDE
▪ It is not recommended for routine disinfection and
sterilization (carcinogenic).
▪ 3-8% HCHO, 30 mins: Tuberculocidal
▪ Use: Sterilize HEPA filters in BSCs(formaldehyde
vapor)
▪ Pseudomonacidal, Tuberculocidal, Fungicidal,
Virucidal
▪ Disinfectant or a sterilant: Contact time longer when EFFECTIVENESS OF ANTIMICROBIAL TREATMENT
used as a sterilant.
• Depends on: Number of microbes.
GLUTARALDEHYDE ▪ 2% Glutaraldehyde in 10 minutes and Sporicidal in 3-
• Environment (Organic Matter, Temperature, Biofilms)
10 hours (10 hours is needed to kill all spores).
• Time of Exposure
▪ Effective against HIV and HBV: 10 minutes exposure.
• Microbial characteristics
▪ Use: Medical equipment (heat-labile), plastic and
rubber materials
▪ Ethylene Oxide (EtO)
o Most commonly used gas for sterilization
o Explosive: Mixed with Nitrogen/CO2 before use
o Effect: Alkylation of nucleic acids in spores and
vegetative cells
o Concentration: 450-700 mg/L of chamber space
at 55-60˚C for 2 hours
o Use: Plastic petri dishes, sutures, catheters,
heart-lung machines
o Biological Indicator: Bacillus
GAS
atrophaeus(Formerly Bacillus subtilis subsp.
STERILANTS
globigii)
▪ Periacetic Acid
o Active against all vegetative bacteria and fungal
spores
o Strong oxidizing agent acting on cell walls,
proteins, enzymes.
o For medical equipment
o Limited role due to toxicity, mutagenicity,
reduced activity in large organic materials
EVALUATION OF A DISENFECTANT
especially blood
• Use dilution test
DISINFECTANT SCREENING TEST: Phenol Coefficient (PC) ▪ Metal rings dipped in test bacteria are dried
▪ Highest dilution that kills bacteria after a 10-minute exposure ▪ Dried cultures are placed in disinfectant for 10 minutes at 20°C
▪ Rings are trans red to culture media to determine whether bacteria survived
▪ Potency of a disinfectant compared with Phenol
treatment
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CLINICAL BACTERIOLOGY|LECTURE(Sir Gelo)
PRELIMS LESSON 2: INFECTION CONTROL AND LABORATORY SAFETY
• Disk Diffusion Method ▪ Limitations:
o Corrosive to some metals
o Destructive to nylon, rubber
o Limited surface sterilization (Does not penetrate lumens well)
o Inability to be used on highly absorptive material
TUBERCULOCIDAL AND SPORICIDAL ANTIMICROBIALS

SURFACE ACTIVE AGENTS

• Soap: Mechanical removal through scrubbing
• ACID ANIONIC DETERGENTS
▪ Enzyme activation or disruption, sanitizers in dairy and food processing
industries
Soap Degerming
Acid-anionic detergents Sanitizing
• DETERGENTS/SURFACTANTS
▪ Action: Disrupt cell membranes, inhibiting active transport and energy
metabolism of the microbial cell
▪ Classified as:
o Anionic Compounds: Negative charge (Soaps)
o Cationic Compounds: Positive Charge (Quats)
▪ All detergents have a similar basic structure: Hydrocarbon chain + Charged
polar head
o Hydrophobic tail: Fat soluble and inserts into the membrane
• SOAP
▪ Effectivity: Poor disinfectants
o Useful when: Mechanical action of scrubbing, combined with the
physical properties of soaps as wetting agents
o Certain Gram-negative organisms (E.g., Pseudomonas aeruginosa)
can grow on soap.
▪ Increase effectivity thru:
o Combination with other agents (Alcohols, lodine, or Chlorhexidine)
 Biguanides (Aka Chlorhexidine)
− Disruption of plasma membrane
− Skin Disinfection
− Bactericidal to Gram + & -
− Non-toxic
CHEMICAL FOOD PRESERVATIVES
• Organic Acids
▪ Metabolic Inhibition
▪ Mostly affect molds
▪ Examples: Sorbic Acid, Benzoic Acid
o Calcium Propionate: Bread
▪ Used in: Cosmetics, Shampoos
• Nitrite: Prevent endospore germination
• Antibiotics:
▪ Nisin and Natamycin (Spoilage of cheese)
Gases
Hydrogen Peroxide Gas Plasma
▪ Drawn into an autoclave under vacuum to create a vapor phase
▪ Once vapor has covered and penetrated the items to be sterilized, radio
waves are applied: Breaks the vapor into reactive free radicals that form
the "Gas Plasma"
o Free Radicals effective against: Cell membranes, Enzymes, nucleic
acids
Vapor Phase Hydrogen Peroxide
▪ Forms free radicals which are toxic to many cells
▪ High concentrations of Hydrogen Peroxide required for sterilization are
extre caustic.
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CLINICAL BACTERIOLOGY|LECTURE
PRELIMS LESSON 2: MICROBIAL TAXONOMY
TOPIC OUTLINE Genotype
Phenotype
 Microbial Taxonomy (Genetic makeup)
DIFFERENT MICROOGRANISMS ▪ Genotypic ▪ Observable physical and functional features of
Membership: characteristics an organism expressed by genotype.
 Bacteria  Fungi ▪ Comparison of the ▪ Macroscopic and microscopic morphology
base composition ▪ Staining characteristics
 Parasites  Viruses
▪ Ratio to measure ▪ Nutritional requirements
BACTERIA
degree of ▪ Antigenic markers susceptibility or resistance to
• Bacterium (singular); Bacteria (plural) relatedness of two antibiotics or chemicals
• Prokaryotes (no organelles, unicellular) organisms. ▪ Physiologic and biochemical characteristics
3 STRUCTURES, INTERRELATED CATEGORIES
• “Before nucleus”
1. Classification
• Primitive nucleus – no nuclear membrane/no nucleus 2. Nomenclature
• Has nucleoid 3. Identification
BASIS OF GRAM STAIN: Cell Wall (Peptidoglycan) PHYLOGENETIC TREE OF LIFE (Woes Classification)
GRAM STAIN • Karl Woes
▪ Basic stain, differential stain 3 DOMAINS:
TWO (2) DIVISIONS: 1. Bacteria
1. GRAM + 2. Archaea
o peptidoglycan layer is thicker (Teichoic acid) - in between the bacteria and eucaryota
2. GRAM – 3. Eucaryota
o peptidoglycan layer is thinner (Lipopolysaccharides, LPS) VIRAL CLASSIFICATION/TAXONOMY (Non-living things) is based on:
PARASITES ▪ Genome Replication
• Eukaryotic (organelles present) - true nucleus ▪ Virion structure
• single/multicellular International Committee on Taxonomy of Viruses (ICTV)
▪ single (protozoa) ▪ More than 2000
▪ multicellular (worms) FORMAL LEVELS OF BACTERIALS CLASSIFICATION
Domain
• Motile or Non-motile
Kingdom
FUNGI
Division (Phylum)
• Heterotrophic eukaryotes (obtain nutrients through absorption) Class
• Saprophytes (live on decaying organic matter) Order
• Yeast (unicellular, asexual reproduction); Ex. Candida albicans Family (-aceae)
Tribe
• Most fungi (multicellular, asexual and sexual reproduction); Ex. Molds
Genus (capitalized)
• Dimorphic (yeast at human body; filamentous at room temperature) - 2 Species (lowercase) sp. singular or spp. plural (specific epithet)
morphologic forms; Ex. Histoplasma capsulatum Subspecies (when appropriate)
VIRUSES Dear King Philip Cried Out For Goodness Sake
• Smallest infectious particles THE TAXONOMY HIERARCH
• Contain DNA or RNA, rarely both (may be single or double stranded). • Kingdom: Animal
• Needs to use electron microscope to see viruses. • Phylum: Chordata
• Filterable agents (smaller than bacteria that they can pass through the • Class: Mammalia
bacteriologic filter). • Order: Primates
• Acellular (non-biological entity) non-living things but still infectious. • Family: Hominidae
• Obligate intracellular parasites (require host cells for replication). • Genus: Homo (underline)
VIRAL EMERGENCE
• Species: H. sapiens (italicized; abbreviated)
• becoming better known by: NOMENCLATURE
▪ DNA or RNA makeup
• Provides naming assignments for each organism in companion textbook.
▪ Host disease signs and symptoms STANDARD RULES
▪ Chemical makeup - family name is capitalized and ends in "-aceae"
▪ Geographic distribution - genus name is capitalized followed by species (epithet), which begins with
▪ Resistance to lipid solvents and detergents (non-enveloped viruses) a lowercase letter
▪ Resistance to pH and temperature changes • Some genera have the same first letter, so the first syllable is used.
▪ Antigenicity (serologic methods) - viral proteins is the basic for the viral - Staph. for Staphylococcus
antigenicity; to detect viral proteins from the sample; ex. antigen test - Strept. for Streptococcus
CLASSIFICATION/TAXONOMY - Esch. coli (bacteria)
• Orderly/systematic groupings of organisms into taxa (categories) - Ent. coli (parasite)
CLASSIFICATION BY PHENOTYPIC AND GENOTYPIC CHARACTERISTICS
• Carl Linnaeus (Father of Taxonomy) He systematically classified all of the
SPECIES
organisms.
• Subspecies
• Based on similarities and differences in:
• Serovarieties (serovar): serologic differences

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CLINICAL BACTERIOLOGY|LECTURE
PRELIMS LESSON 2: MICROBIAL TAXONOMY
• Biovarieties (biovar): biochemical test result differences ARCHAEA
• Phase typing: based on susceptibility to specific bacterial phages • Most closely related to eukaryotic cells
(Bacteriophages - bacteria eating virus) • Contains microorganisms that grow under extreme environmental conditions.
STRAIN • Cell walls lack peptidoglycan.
• Species with different susceptibility/resistance (basis) patterns • Can stain gram-positive or gram-negative.
• Ex. susceptibility or resistance to penicillin • Cell envelope and enzymes present allow organisms to survive hash conditions.
GENETIC RELATEDNESS
• rRNA HOW TO WRITE SCIENTIFIC NAMES
• Led to some classification. GENUS: CAPITAL LETTER
16s rRNA species: small letter
• Primers for PCR (Prokaryotic organisms) • Name should be written italics when 'typed'.
18s rRNA • When written by hand, the genus and species names should be underlined
• For eukaryotic organisms separately.
CLASSIFICATION BY CELLULAR TYPE • Name can be written in a short form.
3 DOMAINS: ▪ When the name comes for the second time
1. Bacteria ▪ When referring another species of the same genus
▪ Classic prokaryotic cell encountered in clinical microbiology. • Species Plantarum is the book initiated by Carl Linnaeus in 1753
2. Eukarya ▪ The book lists every species of plant known at that time.
3. Archaea TAXONOMY
▪ Extremely thermophilic (live at a very high temperature environment) Taxonomy: Area of biologic science comprising of three distinct principles:
▪ Extremely halophilic (live at a temperature with high salt concentration)
• Classification: Groups or taxa based on similar morphologic, physiologic, and
▪ Methanogens (lives on a high methane gas environment; wetlands)
genetic traits
▪ Taq polymerase (most important enzyme in PCR from Thermus aquaticus)
- Resist high temperature
- Extremophiles • Nomenclature: Based on established rules and guidelines by the International
- Not encountered in clinical microbiology Code of Nomenclature of Bacteria (ICNB) or the Bacteriological Code.
2 MAJOR KINGDOM: BINOMIAL NAME:
1. Prokaryotes (No nuclear membrane) o Genus: First letter capitalized, remaining letters in lower case, all
▪ Bacteria italicized/underlined
▪ Archaea o Species: NO capitalized letters, all italicized/underlined
2. Eukaryotes ▪ When bacteria are referred to as a group, names are neither capitalized nor
▪ Fungi, protozoa underlined (staphylococci, streptococci, gonococci)
▪ Algae, animals, plants • Identification of organisms: Key features are delineated.
TWO CATEGORIES:
Pathogenic bacteria (Disease-causing) o Genotypic: Genetic makeup (Nucleic Acids)
▪ Prokaryotes that infect eukaryotic hosts o Phenotypic: Observable characteristics
Species Epithet: Proper word for the name of the species

IDENTIFICATION CRITERIA AND CHARACTERISTICS FOR MICROBIAL CLASSIFICATION

PHENOTYPIC CRITERIA EXAMPLES
MACROSCOPIC MORPHOLOGY
MICROSCOPIC MORPHOLOGY
STAINING CHARACTERISTICS
ENVIRONMENTAL REQUIREMENTS
NUTRITIONAL REQUIREMENTS
RESISTANCE PROFILES
ANTIGENIC PROPERTIES
SUBCELLULAR PROPERTIES
GENOTYPIC CRITERIA EXAMPLES
DEOXYRIBONUCLEIC ACID (DNA)
base composition ratio
NUCLEIC ACID
(DNA and ribonucleic acid [RNA])
base sequence analysis, including
hybridization assays
PRINCIPLES
Characteristics of microbial growth patterns on artificial media as observed when inspected with the unaided eye. Examples of such characteristics
include the size, texture, and pigmentation of bacterial colonies.
Size, shape, intracellular inclusions, cellular appendages, and arrangement of cells when observed with the aid of microscopic magnification.
Ability of an organism to reproducibly stain a particular color with the application of specific dyes and reagents
Staining is used in conjunction with microscopic morphology for bacterial identification For example, the Gram stain for bacteria is a critical criterion
for differential identification.
Ability of an organism to grow at various temperatures, in the presence of oxygen and other gases, at various pH levels, or in the presence of other
ions and salts, such as NaCl.
Ability of an organism to utilize various carbon and nitrogen sources as nutritional substrates when grown under specific environmental conditions.
Exhibition of a characteristic inherent resistance to specie antibiotics, heavy metals, or toxins by certain microorganisms.
Establishment of profiles of microorganisms by various serologic and immunologic methods for determining the relatedness among various microbial
groups
Establishment of the molecular constituents of the cell that are typical of a particular taxon, or organism group, by various analytic methods Some
examples include cell wall components, components of the cell membrane, and enzymatic content of the microbial cell
PRINCIPLES
DNA comprises four bases (guanine, cytosine, adenine, and thymine). The extent to which the DNA from two organisms is made up of cytosine and
guanine (i.e., G+C content) relative to their total base content can be used as an indicator of relatedness or lack thereof. For example, an organism
with a G+C content of 50% is not closely related to an organism with a G+C content of 25%.
The order of bases along a strand of DNA or RNA is known as the base sequence The extent to which sequences are similar (homologous)
between two microorganisms can be determined directly or indirectly by various molecular methods The degree of similarity in the sequences may
be a measure of the degree of organism relatedness specifically, the ribosomal RNA (rRNA) sequences that remain stable in comparison to the
genome as a whole

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CLINICAL BACTERIOLOGY|LECTURE(Sir Gelo)
PRELIMS LESSON 3: BACTERIAL PHYSIOLOGY
TOPIC OUTLINE ACID-FAST CELL WALL

Bacterial Physiology  Bacterial Genetics ▪ Has a gram-positive cell wall structure
BACTERIAL PHYSIOLOGY ▪ Contains Glycolipids and Fatty acids (Hydroxymethoxy acid or
PROKARYOTES Mycolic acid) bound to exterior of cell wall (Hydrophobic)
• Organisms without a true nucleus ABSENCE OF CELL WALL
• Do not contain: Mitochondria, ER, Golgi Apparatus (All happen in the cytoplasm) ▪ Contain sterols in their cell membrane
• “Pro”: Before ; “karyon”: Nucleus, Nut, or Kerne ▪ Mycoplasma and Ureaplasma
BACTERIAL CELL STRUCTURE ENZYMES THAT ATTACK THE CELL WALL
CELL ENVELOPE ▪ Lysozyme
▪ Outermost structure of the bacterial cell o Found in tears, saliva, nasal secretions
▪ Composed of an outer membrane (G- only), cell wall, periplasm (G- only), o Cleaves peptidoglycan of cell wall
plasma membrane ▪ Autolysins
CELL WALL o Catalyze turnover/degradation of peptidoglycan
▪ AKA Peptidoglycan/Murein layer o Probably participate in cell wall growth
▪ Rigid structure maintaining shape of the cell PLASMA (INNER MEMBRANE)
▪ Composed of: Disaccharide-pentapeptide subunits, teichoic acid ▪ Deepest layer of the cell envelope
(Lipoteichoic acid) ▪ Phospholipid bilayer with embedded proteins that surrounds the cytoplasm
▪ Synthesis and structure are a primary target of antimicrobial agents. ▪ Functions:
Functions: ▪ Acts as mitochondria, golgi complexes, and lysosomes of prokaryotic cells
o Prevent bacterial cells from rupturing. o Regulates transport of solutes across the membrane
o Point of anchorage for flagella o Site for generation of chemical energy
o Determine staining characteristics. RIBOSOMES
GRAM-POSITIVE CELL WALL ▪ Consist of RNA and Proteins
▪ Very thick protective peptidoglycan ▪ Site of protein biosynthesis; Cytoplasm’s granular structure
▪ Glycan chains of N-acetyl-DGlucosamine and N-Acetyl-D-Muramic ▪ 70S in size, separated into 2 subunits
Acid
o Prokaryotes: 50S and 30S subunits
▪ Has antigenic polysaccharides o Eukaryotes: 60S and 40S
▪ Contains teichoic acid (- charge) GENOME
o Composed of Alcohol (Glycerol or Ribitol) and Phosphate ▪ Single, circular chromosome
(Resposible for the negative charge)
▪ Appears as a diffuse nucleoid or chromatin body, attached to a mesosome
o Two kinds: Lipoteichoic Acid (Bound to plasma membrane) and (sac-like structure)
Wall Teichoic Acid (Bound to Peptidoglycan) PLASMIDS
GRAM-NEGATIVE CELL WALL
▪ Extrachromosomal, double-stranded elements of DNA associated with
▪ Outer membrane and inner (thin) peptidoglycan membrane virulence
▪ Has porins (Contribute to permeability of cell) ▪ Mostly circular, double-stranded except Borrelia burgdorferi and Borrelia
▪ Has Periplasmic space: Peptidoglycan synthesis hermsii
▪ Does not contain Teichoic acid ▪ Exist independently of chromosome (Replicate independently)
▪ Outer membrane ▪ Serve as a site for the genes that code for antibiotic resistance and toxin
o Composed of proteins, phospholipids, and LPS production
o Contribute to (-) charge of bacterial surface to stabilize the ▪ Not essential for bacterial growth
membrane structure ▪ Can transform bacteria to become pathogenic, mostly gram-negative
o Allow hydrophilic compounds to enter cell through the porins bacteria
o Vital in evading host defenses (Strong negative charge) ▪ Types of Plasmids:
o Considered as an endotoxin o Large Plasmid: Responsible for the production of B-Lactamases that
▪ Inner membrane: Reason for high susceptibility to mechanical provide resistance to B-Lactam antibiotics like Penicillin and Oxacillin
breakage o Small Plasmid: Resistant to Tetracyclines and Chloramphenicol
▪ Lipopolysaccharide INCLUSION BODIES
o 3 layers: Lipid A (Major constituent; Endotoxin); Core ▪ Serve as energy source (food reserve)
Polysaccharide; Antigenic O (Specific Polysaccharide) ▪ Composed mainly of Polysaccharides, lessens osmotic pressure
o Vital in evading host defenses o Examples: Glycogen, Poly-BHydroxybutyrate granules, Carboxysome
o Contribute to negative charge. (cyanobacteria, nitrifying bacteria, thiobacilli), gas vacuoles
o Considered an endotoxin. (cyanobacteria, halobacterium, Thiothrix)
▪ Polyphosphate granules:
o Corynebacterium diphtheriae: BabesErnst bodies
o Mycobacterium tuberculosis: Much granules
o Yersinia pestis: Bipolar bodies
ENDOSPORES/ASEXUAL SPORES (RESISTANT STRUCTURES)
▪ Small, dormant; Develop inside bacterial cell as a means of survival
▪ Composed of: Dipicolinic acid and Calcium ions (Calcium Dipicolinate)
▪ Location of spores:
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CLINICAL BACTERIOLOGY|LECTURE(Sir Gelo)
PRELIMS LESSON 3: BACTERIAL PHYSIOLOGY
o Terminal spore: Clostridium tetani o Common/Somatic Pili (Fimbriae): Virulence factor/Organ of
o Sub-terminal spore: Clostridium botulinum attachment
o Central spore: Bacillus anthracis o Sex Pili: Essential for genetic transfer (Conjugation)
GLYCOCALYX
▪ Outward complex of polysaccharide from bacterial surface and other cells
▪ Helps on the attachment of bacteria to surfaces
▪ Composed of capsule and slime layer
▪ Capsule
o Neatly organized material attached to cell wall (Thick glycocalyx)
o Virulence factor: Resists phagocytosis and desiccation
▪ Slime Layer
o Unorganized material loosely attached to cell wall
o Made up of thin glycocalyx
o Aids in adherence to host tissues
FLAGELLUM
▪ Exterior protein filament rotating, causing bacteria to be motile EUKARYOTES
▪ Important for survival and ability to cause disease • Microorganisms with “true nucleus”
▪ Monotrichous: Single polar flagellum • Contain many membrane-bound organelles
▪ Lophotrichous: Tuft of flagella on one end ARCHAEOBACTERIA (ARCHAEA)
▪ Amphitrichous: Single flagella at both ends • From “archaios”: Ancient
▪ Cephalotrichous: Tuft of flagella on both ends • Closely related to eukaryotes than prokaryotes
▪ Peritrichous: All around the organism • Cell walls do not contain peptidoglycan, hence why separated from bacteria
▪ Atrichous: Without flagella
• Do not contain nucleus and membrane-bound organelles but have gas vesicles
PILI (FIMBRIAE)
▪ Hairlike, proteinaceous structures that extend from the cell membrane into • May be stained as G+ or G- in various shapes
external environment (2 um in length) • Examples: Methanospirillum, Halobacterium, Sulfolobus
▪ Aid in attachment to surfaces
▪ 2 Types:

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CLINICAL BACTERIOLOGY|LECTURE
PRELIMS LESSON 3: MICROBIAL CELL STRUCTURE
TOPIC OUTLINE - Covered by polymers factors.
 Microbial Cell Structure - Some pathogenic bacteria produce a discrete organized protective
1. Cytoplasmic Structure 5. Surface Polymers (against phagocytosis) covering known as a CAPSULE.
2. Cell Envelope Structures 6. Cell Appendages - Slime layer (not neatly organized); Capsule (neatly organized)
3. Gram Negative Cell Wall 7. Eukaryotic Cell Structure - Main component: Peptidoglycan (murein) layer
4. Acid-Fast Cell Wall 8. Granules and Endospores o Glycan (polysaccharide)
MICROBIAL CELL STRUCTURE o Chains of alternating NAG and NAM
CYTOPLASMIC STRUCTURE o NAG: N-acetyl-D-glucosamine
• Bacteria lack a membrane-bound nucleus. o NAM: N-acetyl-D-muramic acid
GRAM NEGATIVE CELL WALL
• Genome: singular circular chromosome
2 LAYERS:
• Diffuse nucleoid or chromatin body (attached to mesosome - sac-like structure in 1. Inner Peptidoglycan (thinner) layer
cell membrane) ▪ Has a periplasmic space
• Spores when present: highly refractile ▪ Not present in Gram Positive
• Bacteria (Endospores: function of endospore for bacteria - survival in extreme 2. Outer membrane
conditions like extreme dryness or extreme temperature ▪ Unique to the Gram-Negative cell wall
• Highly refracting bodies (spores ‘location) ▪ Not present in Gram-Positive
• Fungi: Spores (function: reproduction) ▪ Proteins
• Bacterial ribosomes of RNA and protein ▪ Phospholipids
▪ Site of protein synthesis; found free in cytoplasm and attached to ▪ Lipopolysaccharide (LPS)
cytoplasmic membrane. - Major virulence factor for some gram = site of synthesis of endotoxin
▪ Bacterial ribosomes are made of 70s ribosome-complex (50s and 30s) ▪ Porins
subunits. - Channel that molecules that pass in and out.
o Eukaryotic (80s) ▪ LPS region
o Svedberg (S) units are sedimentation rates during high-speed - Antigenic O-specific polysaccharide
centrifugation (ultracentrifugation) - Main virulence factor
o Values are not additive, due to binding together resulting in surface - Lipid A (ENDOTOXIN)
area loss. - Core polysaccharide
o There are some antibiotics interfere protein synthesis; antibiotics ▪ Outer membrane functions
would act on the ribosomes of the bacteria (Bacterial ribosome) Ex. - Acts as a barrier to hydrophobic compounds and harmful substances.
Erythromycin - Acts as a sieve, allows-water soluble molecules to enter the porins.
PROKARYOTIC RIBOSOME - Enhances attachment to host cells.
• 70S • 80s ACID-FAST CELL WALL
▪ 50s ▪
60s • Main substance: Mycolic acid (ability to resist decolorization by acid alcohol)
▪ 30s ▪
40s • Has the ability to resists decolorization by acid alcohol (Ex. Mycobacterium -
CELL ENVELOPE STRUCTURES major; Nocardia - slightly acid fast)
• Consists of the membrane and structures • Mycolic acids contribute to the acid fastness of the bacterium.
• Bacteria = made up of plasma membrane (semi-permeability) and cell wall • Waxy layer of glycolipids and fatty acids
(shape; peptidoglycan) ▪ Major component is mycolic acid (strongly hydrophobic)
• Some bacterial species (produce capsules and slime layers) • For organisms that are difficult to Gram stain: lightly gram-positive
▪ Virulence Factor (makes the bacteria more harmful) 3 COMPONENTS OF ACID-FAST STAIN:
▪ Slime layer and capsule prevent phagocytosis. 1. Carbol Fuchsin (Primary stain)
1. PLASMA MEMBRANE (CELL MEMBRANE) 2. Acid Alcohol (Decolorizer)
▪ Phospholipid bilayer (hydrophobic and hydrophilic layer) embedded 3. Methylene blue (Counter stain)
proteins ABSENCE OF A CELL WALL
▪ MAKEUP: • HAS NO PEPTIDOGLYCAN
o Phospholipids and proteins; no sterols • Can't be classified through gram stain because it has no cell wall pertains to
o Exception: Mycoplasmatacaea sterols present bacteria in the genera of Mycoplasma and Urea plasma (PPLO -
▪ Acts as an osmotic barrier. Pleuropneumonia like organisms)
▪ Conserve as a channel protein so that cells can pass through. • Plasma membrane contains sterols.
▪ Function: for osmotic barrier. • Various shapes are seen due to lack of rigid cell wall, a concept known as
2. CELL WALL pleomorphic (ability to assume a variety of shape)
4 BASIC CATEGORIES: SURFACE POLYMERS
I. GRAM + Cell Wall
- Has teichoic acid (Gram stain) • Production of a capsule
II. GRAM – Cell Wall ▪ Organized polysaccharide or polypeptide structure (adherence to surface;
- Has LPS (Gram stain) prevent phagocytosis)
III. Acid-fast Cell Wall • Negative stain
- Has mycolic acid: cell wall is waxy (Acid fast stain) ▪ Staining the background but leaving the organism colorless
IV. Absence of Cell Wall ▪ Presents as clear halo-like structure
- No mycolic acid (Acid fast stain)
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CLINICAL BACTERIOLOGY|LECTURE
PRELIMS LESSON 3: MICROBIAL CELL STRUCTURE
• Serologic typing 2 PROCESSES INVOLVE IN BACTERIAL
▪ Antigenic structure ENDOSPORES DEPENDING ON CONDITION
SPORULATION GERMINATION
▪ Sometimes remove capsule to detect somatic antigens (found in cell wall), Extreme Condition
usually by boiling. Normal Condition
(From vegetative cell to form
CELL APPENDAGES (From endospore to vegetative cell)
endospore)
• Found outside the cell.
3 STRUCTURES
▪ motility
▪ Atrichous (Absence of flagella)
▪ external rotating filaments
▪ Monotrichous (single flagellum)
▪ Lophotrichous (multiple flagella in tufts, in bundle)
1. FLAGELLA ▪ Amphitrichous (flagella on both ends) - Axial
filament (Ex. Treponema pallidum)
▪ Peritrichous - flagella all over
▪ Amphilophotrichous - tuft of flagella at both ends
▪ Protein: FLAGELLIN
▪ Also called as the H antigen
▪ Non-motile long filamentous tubes
2. PILI ▪ For conjugation (transferring plasmid to one
bacterium to another)
3. FIMBRIAE
▪ Hair-like structure used for adhesion
▪ Ex. Neisseria gonorrhoeae

EUKARYOTIC CELL STRUCTURE

(Fungi and Parasite)
NUCLEUS
▪ DNA (discrete chromosomes)
▪ Histone (basic protein around chromosomes)
▪ Nucleolus (round, refractile body inside nucleus)
▪ Nucleus is site of rRNA synthesis
▪ Nucleus is bound by bilayered lipoprotein nuclear membrane
▪ Eukaryotic ribosomes - where synthesis occurs (80s)
PLASMA MEMBRANE
▪ Phospholipid bilayer with embedded proteins
CELL WALL
▪ To provide rigidity and strength to exterior of cell
MOTILITY ORGANELLES
▪ Cilia
▪ Flagella
GRANULES AND ENDOSPORES
PROKARYOTES
▪ Granules in the cytoplasm (Metachromatic granules/Volutin - food reservoir
of organisms): Virulence Factor
o Glycogen
o Poly-B-hydroxybutyrate
o Polyphosphates
▪ Endospores
o Bacterial endospores are different to destroy.
o Utilization on autoclave - steam under pressure
o Bacillus (Aerobic spore former) and Clostridium (Anaerobic spore
former) produce endospores.
o Highly resistant to chemical agents, temperature change, starvation,
dehydration, ultraviolet (UV) light, gamma radiation, and desiccation
(drying)
o Not involved in reproduction

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CLINICAL BACTERIOLOGY|LECTURE
PRELIMS LESSON 4: MICROBIAL MORPHOLOGY
TOPIC OUTLINE BACTERIAL
 Microbial Morphology ARRANGEMENT AND EXAMPLES
SHAPE
1. Microscopic Size and Shape
1. Singly or in pairs – Pneumococcus, Gonococcus, Meningococcus
2. Cytoplasmic Structure 2. Tetrads – Geffyka tetragena
3. Cell Envelope Structures COCCI 3. Sarcinate – Sarcinna lute
4. Gram Negative Cell Wall 4. Clusters – Staphylococcus
5. Acid-Fast Cell Wall 5. Chains – Streptococcus
BACTERIAL PHYSIOLOGY 1. Singly or in pairs – Klebsiella pneumoniae
2. Chains – Bacillus subtilis, Bacillus anthracis
PROKARYOTES
BACILLI 3. Palisade – Mycobacterium leprae
• Organisms without a true nucleus 4. Groups – Mycobacterium tuberculosis
• Do not contain: Mitochondria, ER, Golgi Apparatus (All happen in the cytoplasm) 5. L,V Chinese Character Arrangement – Corynebacterium diphtheriae
1. Singly or in pairs
• “Pro”: Before ; “karyon”: Nucleus, Nut, or Kerne SPIRILLUM 2. Groups
MICROSCOPIC SIZE AND SHAPE 3. No typical arrangement
TYPICAL SIZE:
▪ 0.4 to 2 μm COMMON STAINS USED FOR MICROSCOPIC VISUALIZATION
SHAPE: • Gram (different stain)
▪ Cocci (spherical) • Acid-fast (differential stain)
▪ Bacilli (rod-shaped) • Acridine orange (fluorescent stain) – special stain
▪ Spirochetes (spiral) • Calcofluor white (fungi) – special stain
SIZE AND ARRANGEMENT
COCCUS/ COCCI
• Methylene blue (capsule)

• Round/Spherical/Coffee-bean shaped/Lancet-shaped (E.g., staphylococci,

• Lactophenol cotton blue
streptococci, gonococci) • India ink - special stain (negative stain)
▪ IN CLUSTERS: Staphylococci 1. GRAM STAIN
▪ IN CHAINS: Streptococci ▪ Differential stain
▪ IN PAIRS: Diplococci ▪ Heat fix/fixation: kill the bacteria (methanol can be used)
o LANCET SHAPE: Streprococcus - Streptococcus ▪ Crystal violet (1min): Primary stain
pneumoniae ▪ Iodine/Gram's iodine (1min): fixes
o COFFEE-BEAN SHAPED: Neisseriae – Neisseria gonorrhoeae ▪ Iodine (Mordant – fix the CV to the smear; enhance the affinity of the
▪ IN TETRADS: Micrococcus tetragena reagent)
▪ CUBICAL PACKETS OF 8, 16, 32: Sarcinaea/Sarcina – sarcina ▪ Alcohol-acetone (quick on and rinse) – decolorizer
ventriculi(8) ▪ Safranin (30sec) – counterstain
▪ Lancet diplococci: Streptococcus pneumoniae **Note: Rinse with water between steps
▪ IN SQUARE OR RECTANGULAR SHAPED – Haloarcula quadrata GRAM + GRAM -
▪ STAR SHAPED – Stella vaculata CV: Purple CV: Purple
BACILLUS/ BACILLI GI: Purple GI: Purple
AA: Not Decolorized AA: Decolorized
• Rod-shaped, club-shaped, comma-shaped, filamentous (E.g., Safranin: Purple Safranin: Red/Pink
Enterobacteriaceae, Vibrio, Mycobacterium) Final color: Blue/Purple Final color: Red/Pink
▪ IN CHAINS: Streptobacilli RULES IN GRAM STAINING
▪ COCCOBACILLI 1. All cocci are Gram + except for Neisseria, Veilonella, Branhamella.
▪ Bacilli of various sizes 2. All bacilli are Gram - except for Bacillus, Clostridium, Mycobacterium,
▪ FUSIFORM BACILLI (end forms) – Spindle shape Corynebacterium, Nocardia, Erysipelothrix, Listeria, Lactobacillus, Kurthia,
Rothia, Propionibacterium
▪ PALISADING – side by side - corynaebacteria 3. Fungi are not usually stained by Gram stain but if they are stained, they are said
▪ SPORE-FORMER to be Gram +
▪ FLAGELLATE RODS 4. Spirochetes are not usually stained by Gram staining but if they are stained,
SPIRILLUM (SPIRILLA/ SPIROCHETES) they are said to be Gram -
• Spiral/Coiled (E.g., Treponema, Leptospira, Borrelia) 2. ACID FAST STAINING
▪ Vibrios (Comma shape) Vibrio cholerae ▪ Ability to resist decolorization by acid alcohol due to the presence of thick
▪ Spirochaetes mycolic acid or hydroxymethoxy acids in the cell wall
▪ Corynebacterium ▪ All bacteria are non-acid fast except for Mycobacterium
▪ Filamentous bacteria with fruiting bodies - Streptomycetes ▪ Nocardia is slightly acid fast
BIZARRE SHAPE 2 METHODS OF ACID-FAST STAINING:
▪ Haloarcula quadrata (Square-shape) A. ZIEHL-NEELSEEN (Steam Method)
▪ Stella vacuolata (star-shape) o Steaming (allows Carbol fuchsin to enter the cell)
o Primary: Carbol Fuchsin (Red/Pink)
o Decolorizer: Acid-alcohol (95% Ethyl Alcohol and Hydrochloric acid)
o Counter stain: Methylene Blue
o Acid Fast: Red (Positive)
o Non-acid fast: Blue (Negative)
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CLINICAL BACTERIOLOGY|LECTURE
PRELIMS LESSON 4: MICROBIAL MORPHOLOGY
B. KINYOUN’S
o Increase the concentration of Carbol fuchsin, prolong the contact of
CF, add wetting agent such as Tergitol
o Primary: Carbol Fuchsin (Red/Pink)
o Decolorizer: Acid-alcohol (95% Ethyl Alcohol and Hydrochloric acid)
o Counter stain: Malachite Green
o Acid Fast: Red (Positive)
o Non-acid fast: Green (Negative)
3. ACRIDINE ORANGE
▪ Stains nucleic acid bright orange under UV light.
▪ Bacteria: living or dead
▪ Useful to locate bacteria in blood cultures and other specimens where
discerning bacteria might otherwise be difficult to see.
4. CALCOFLUOR WHITE
▪ Compound binds to chitin in fungal cell walls
▪ Bright apple-green and blue-white fluorescence (required UV light).

OTHER SPECIAL STAINS

1. METHYLENE BLUE
▪ Metachromatic granules in Corynebacterium diphtheriae.
2. LACTOPHENOL COTTON BLUE
▪ For molds; stains fungal cells.
▪ Used to stain medically important fungi grown in slide culture; fungal cell
walls blue.
3. INDIA INK
▪ Negative stain to visualize capsules.
▪ Create a halo-like visual.
4. ENDOSPORE STAIN
• Malachite green stains endospores (terminal, central, sub-terminal).

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CLINICAL BACTERIOLOGY|LECTURE(Sir Gelo)
PRELIMS LESSON 4: BACTERIAL METABOLISM AND GROWTH
TOPIC OUTLINE ▪ Organism that can survive in the presence of oxygen
 Bacterial Metabolism and Growth but will not be able to perform metabolic processes
AEROTOLERANT
METABOLISM unless placed in an anaerobic environment.
ANAEROBE
• Metabolism: Sum of all chemical processes that take place in a living organism ▪ Examples: Cutibacterium, Lactobacillus, Clostridium
and results in its growth, energy generation, waste disposal, and other functions perfringens
in relation to cell nutrient distribution. ▪ Organism that requires 2-10% oxygen for growth.
TWO MAJOR TYPES MICROAEROPHILE ▪ Examples: Campylobacter, Treponema pallidum
ANABOLISM: CATABOLISM: subsp. pallidum, Helicobacter pylori
CONSTRUCTIVE PHASE DESTRUCTIVE PHASE
Large complex molecules are broken
Synthesis of complex molecules and
down into simpler, smaller molecules
energy production
accompanied by energy utilization.

ENERGY PRODUCTION
• Breakdown of chemical substrates through degradative process of catabolism
that is coupled with oxidation-reduction reactions
• GLUCOSE
▪ Essential nutrient for energy production in organisms
▪ To produce energy, 2 general processes:
RESPIRATION
▪ Efficient ATP-generating process in which molecules are oxidized,
resulting in an inorganic molecule as the final electron acceptor
▪ Glucose is completely broken down àHigh energy production
o Glucose + O2 à CO2 + Water
▪ Carried out by obligate aerobes and facultative anaerobes
o Aerobic Respiration: Oxygen is final electron acceptor ACCORDING TO CARBON DIOXIDE REQUIREMENT
o Anaerobic Respiration: Either Nitrate, Sulfate, or
Fumarate is the final electron acceptor
• CAPNOPHILES: Require 5-10% CO2 (Haemophilus influenzae, Neisseria
gonorrhoeae, Streptococcus pneumoniae)
FERMENTATION
▪ Does not require Oxygen (Anaerobic), Kreb’s cycle, or an electron • 0.03% CO2: Needed by Aerobic bacteria.
transport chain
ACCORDING TO NUTRITIONAL REQUIREMENT
▪ Release energy from sugars or other organic molecules (Amino
As to CARBON SOURCE
Acids, Purines)
▪ Autotrophs: Use CO2 as the sole source of Carbon by reducing it
▪ Forms a mixture of end products (Lactate, Butyrate, Ethanol, Acetoin)
▪ Photoautotrophs (Energy source: Light): Photosynthetic bacteria,
▪ Analysis of products useful in identifying anaerobic bacteria
cyanobacteria
▪ Carried by Obligate and Facultative Anaerobes
▪ Heterotrophs: Use reduced, preformed, organic molecules from other
bacteria
PHYSIOLOGIC REQUIREMENTS OF BACTERIA
As to ENERGY SOURCE
ACCORDING TO OXYGEN REQUIREMENTS
▪ Photoheterotrophs (Energy source: Light): Purple non-sulfur bacteria,
▪ Is an organism that require oxygen and grow well in Green non-sulfur bacteria
room air.
AEROBE
▪ Air contains 15-21% oxygen and 1% CO2. ▪ Chemotrophs: Organisms that use energy produced by oxidation of
organic or inorganic compounds
▪ Bordetella, Brucella, Mycobacterium, Pseudomonas
As to ELECTRON SOURCE
▪ Organism that strictly does not require the presence
OBLIGATE
of Oxygen; die in the presence of Oxygen. ▪ Lithotrophs: Reduce inorganic molecules
ANAEROBE ▪ Organotrophs: Require organic substances (CHO, CHON, Lipids) for
▪ Clostridium, Bacteriodes
▪ The most clinically significant organism. growth and multiplication; all bacteria that inhabit the human body fall into
this group
▪ Organism that grows either in the presence or
absence of oxygen – aerobes which can grow NOTES TO REMEMBER:
anaerobically. ▪ Autotrophs are also lithotrophs, and they obtain energy either
▪ Organism that do not require oxygen but grow better photosynthetically or oxidatively.
in the presence of oxygen. ▪ Heterotrophs are also organotrophs and they obtain energy by oxidation or
FACULTATIVE
▪ Routinely cultured in an aerobic atmosphere – fermenting organic substances such as glucose.
ANAEROBE
because aerobic culture is easier and less expensive ▪ All bacteria that inhabit the human body fall into the heterotrophic or
than anaerobic culture. organotrophic group.
▪ Example: Enterobacteriaceae ▪ Fastidious bacteria require additional substances for growth and survival.
▪ Obligate aerobes and facultative anaerobes contain ACCORDING TO TEMPERATURE REQUIREMENT
protective enzymes against the toxic effect of
oxygen:
• 35-37˚C: Optimum temperature for most bacteria
▪ Superoxide Dismutase (SOD) • PSYCHROPHILE/CRYOPHILE
▪ Catalase ▪ Grows well at 0°C to a maximum of 20°C.

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CLINICAL BACTERIOLOGY|LECTURE(Sir Gelo)
PRELIMS LESSON 4: BACTERIAL METABOLISM AND GROWTH
▪ Examples: Listeria monocytogenes, Yersinia enterocolitica, Pseudomonas ▪ Microorganisms are actively growing and dividing;
syringae Bacterial numbers increased logarithmically –
LOG/ Cellular production is most active during this period.
• MESOPHILE EXPONENTIAL
▪ Grows between 20°-45°C. PHASE ▪ Phase wherein microorganisms are utilized in
(Balance Growth) physiological and biochemical testing
▪ The commonly encountered pathogenic bacteria in the clinical laboratory. ▪ Microorganisms are sensitive to radiation and
• THERMOPHILE/HYPERTHERMOPHILE antimicrobial agents
▪ Grows between 50° to 60°C. ▪ Balance between cell division and dying organisms:
▪ Examples: Geobacillus stearothermophilus, Sulfolobus, Pyrococcus, Number of viable microorganisms remain constant
Pyrodictium, Thermus aquaticus, Alicyclobacillus STATIONARY/ ▪ Phase where metabolic activities of surviving cells
PLATEAU PHASE slow down and nutrients are becoming limited
• EXTREMOPHILE
▪ Prokaryotes that are able to live at unusual conditions like absence of ▪ Phase wherein dead debris are starting to
accumulate
oxygen, increased temperature and below earth’s surface (Bacillus infernus
– Strict Anaerobe). ▪ Cessation of bacterial growth: Number of cell death
DEATH/ DECLINE exceeds number of living microorganisms
• Thermal Death Time: Lowest minimum time required to kill organisms in a PHASE ▪ Loss of nutrients and increased amount of toxic
constant temperature wastes
• Thermal Death Point: Lowest temperature required to kill organism in a
constant time GENERATION: is the doubling of cell number.
ACCORDING TO pH REQUIREMENT GENERATION TIME/ DOUBLING TIME: is the time required for a bacteria to double
• Optimum pH for most pathogenic bacteria is at neutral pH (pH 6.5 to 7.5). its population.
• Diagnostic laboratory media for bacterial isolation are usually adjusted to a final
Notes:
pH between 7.0 & 7.5.
▪ Bacteria have different generation times
• ACIDOPHILE: pH 0 and 5.5 (Sulfolobulus, Picrophilus, Acontium)
▪ In culture, can be as little as 20 minutes for a fast-growing bacterium like E. coli
• NEUTROPHILE: pH 5.5 and 8.0 (Clinically significant pathogenic bacteria) or as long as 24 hours for a slow-growing bacterium like M. tuberculosis.
• ALKALOPHILE: pH 8.5 and 11.5 (Bacillus alcalophilus, Natronabacterium) Calculation of the Final Number of cells
ACCORDING TO HIGH SALT CONCENTRATION (HALOPHILES)
• Requires increased concentration of sodium chloride
• Examples: Staphylococcus aureus, Listeria monocytogenes, Bacillus anthracis
PRESSURE REQUIREMENTS Example: If 5 cells of staphylococci were allowed to divide 9 times, what is the final
• BAROPHILIC: Organisms that grow rapidly in the presence of high pressure number of cells? _
(600 – 1100 atmospheric pressure)
• Examples: Photobacterium, Shewanella, Colwellia
GROWTH FACTORS
• Substances required by fastidious bacteria for growth and multiplication.
• Examples: Amino Acids, Purines, Pyrimidines, Vitamins
REPRODUCTION: Transverse Binary Fission
• Most common asexual reproductive process whereby a single cell divides into
two daughter cells after developing a transverse cell wall.
• COLONY: Aggregation of cells arising from a single parent
BACTERIAL GROWTH
• Length of Generation time: Measure of the growth rate of an organism
• Growth pattern: Exponential

BACTERIAL GROWTH CURVE

▪ Period wherein there is no cell division; no abrupt
LAG PHASE/ increase in cell number
PERIOD OF ▪ Start of biosynthesis although no increase in cell
REJUVENESCENCE mass
▪ Phase of adjusting to new environment

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CLINICAL BACTERIOLOGY|LECTURE
PRELIMS LESSON 5: MICROBIAL GROWTH
TOPIC OUTLINE • Facultative halophiles tolerate high osmotic pressure
 Microbial Growth
• Plasmoptysis (swell and burst of the bacteria when suspended in a hypotonic
1. Requirement for Growth
solution)
2. Classification of Culture Media
3. Reproduction in Prokaryotes • Hypotonic solution (anything that is less than 0.85% NaCl)
4. 4 Stages of Bacterial Growth CHEMICAL REQUIREMENTS
MICROBIAL GROWTH 1. CARBON
• Increase in number of cells, not cell size • Structural organic molecules, energy source.
REQUIREMENTS FOR GROWTH • Chemoheterotrophs use organic carbon sources.
PHYSICAL REQUIREMENTS • Autotrophs use CO2
1. TEMPERATURE AUTOTROPHS
• Minimum growth temperature (Lowest temperature) o Use CO2 as the primary source of carbon such as plants and
• Optimum growth temperature (Most ideal temp. that can support the growth of phytoplankton that is in
the cell) o The utilization of the solar energy (sunlight)
CHEMOHETEROTROPHS
• Maximum growth temperature (highest temp. that will support the microbial
o Require organic carbon sources
growth)
o Ex. fungi
COLD-LOVING
▪ Involve in food spoilage 2. NITROGEN
BACTERIA ▪ PSYCHROPHILES (-5C, 10C, 15C) • Make up amino acids and proteins (component of the bacteria)
▪ PSYCHROTROPHS (0C, 20C, 30C)
• Most bacteria decompose proteins.
HEAT-LOVING ▪ THERMOPHILES (45C, 60C, 80C)
BACTERIA ▪ HYPERTHERMOPHILES (65C, 98C, 110C) • Some bacteria use NH4+ or NO3-.
• A few bacteria use N2 in nitrogen fixation.
▪ Loves the body temperature
3. SULFUR
THERMOPHILES ▪ Pathogenic bacteria
▪ 37C
• In amino acids, thiamine, and biotin
• Bacteria can decompose proteins.
▪ Bacillus cereus • Bacteria use sulfate and hydrogen sulfide.
PSYCHROTROPHS ▪ Grow between 0C to 20-30C 4. PHOSPHORUS
▪ Cause food spoilage
• in DNA, RNA, ATP and membrane
60C-130C ▪ Kills the microbes • Phosphate is a source of phosphorus for bacteria
60C ▪ Slow bacterial growth 5. TRACE ELEMENTS
▪ Lukewarm temperature that can produce • Inorganic elements required in small amounts
20C-50C toxins. • Usually as enzyme cofactors (enzymatic activity can happen within the bacterial
▪ Danger Zone cell)
REFRIGERATOR ▪ BACTERIOSTATIC - retired the growth of 6. OXYGEN
TEMPERATURE bacteria; inhibit microbial growth if contaminated • O2 could be useful or harmful to bacteria
▪ Can endure heat (thermoduric) by protective
spore that can allow survival at high temperature. • Broth: Thioglycollate broth can support various kinds of bacteria even if they
have different oxygen requirements
MESOPHILES ▪ THERMOPHILES (heat-loving organisms)
EXAMPLES:
▪ Ex. Bacillus stearothermophilus (example of
1. OBLIGATE AEROBES
heat-loving organism and considered as an
indicator for the effectiveness of an autoclave) o growth would be on the surface where O2 is present.
2. pH o strictly aerobic
o ex. Mycobacterium tuberculosis
• Most bacteria grow between pH 6.5 to 7.5 (slightly acidic)
2. FACULTATIVE AEROBES
• Molds and yeasts grow between pH 5 and 6 (acidic - acidophiles) o present in any part of the broth
• ACIDOPHILES grow in acidic environments o fundamentally aerobic bacteria but can live in the absence of oxygen.
ACIDOPHILES EXAMPLE: o ex. E. coli
▪ Lactic acid producing bacteria 3. OBLIGATE ANAEROBES
Lactobacillus ▪ Found in vaginal flora, maintains the acidity of o growth would be concentrated at the bottom.
acidophilus the vaginal pH to prevent UTI-causing organisms o strictly anaerobic, has the tendency to die if exposed to oxygen.
from growing 4. AEROTOLERANT ANAEROBES
ALKALOPHILES EXAMPLE: o present in any part of the broth
▪ Grows at a pH level of 8.6 o fundamentally anaerobic bacteria, but can live in the presence of
Vibrio cholerae ▪ Culture medium for the cultivation: TCBS oxygen.
(Thiosulfate Citrate Bile Salt Sucrose) 5. MICRO-AEROPHILES
3. OSMOTIC PRESSURE o growth will be at the center.
o require only small amount of O2.
• Hypertonic environments, increase salt or sugar, cause plasmolysis
o too much O2 could be harmful.
• PLASMOLYSIS (Water will get out from the bacteria, will result to the shrinkage o ex. Yersinia pestis
of bacteria)
• Extreme or obligate halophiles require high osmotic pressure

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CLINICAL BACTERIOLOGY|LECTURE
PRELIMS LESSON 5: MICROBIAL GROWTH
4 TOXIC FORMS OF OXYGEN CAPNOPHILES REQUIRE HIGH CO2:
1. SINGLET OXYGEN: ▪ Candle Jar
▪ O2 boosted to a higher-energy state (toxic for bacteria) ▪ CO2 packet
2. SUPEROXIDE FREE RADICALS: 2. SELECTIVE MEDIA
▪ O2- (To form non-toxic SFR, superoxide dismutase is needed) = Hydrogen ▪ suppress unwanted microbes and encourage desired microbes from
peroxide and oxygen (result) growing
3. PEROXIDE ANION Ex. MAC agar = Crystal Violet (Inhibitory substance - allow Gram Negative
▪ Also toxic for the bacteria bacteria; prevent Gram Positive bacteria from growing)
▪ To form non-toxic, catalase enzyme is needed to be converted into water 3. DIFFERENTIAL MEDIA
and oxygen ▪ make it easy to distinguish colonies of difference microbes
▪ Peroxidase can be added to Peroxide anion to form non-toxic Ex. MAC agar - can classify Gram - bacteria (Lactose fermenters and NLF)
4. HYDROXYL RADICAL 4. ENRICHMENT MEDIA
▪ OH ▪ allows the growth of desired microbe
ENZYME SYSTEM Ex. Selenite F (enrichment broth for Salmonella)
▪ Superoxide dismutase, Catalase and peroxidase (convert toxic form into a ▪ a pure culture contains only one species or strain
non-toxic form to be able to survive) ▪ a colony is a population of cells arising from a single cell or spore or from a
▪ If a bacterium lacks enzyme system, it is considered as ANAEROBIC group of attached cells
BACTERIA ▪ a colony is often called a colony-formingunit (CFU)
▪ If a bacterium has enzyme system, it is considered as AEROBIC
BACTERIA (they are able to survive even in the presence of the toxic forms STREAK PLATE
of oxygen) Multiple interrupted (Common procedure)
7. ORGANIC GROWTH FACTORS • 3rd streak: important because of isolated colonies
• organic compounds obtained from the environment Preserving Bacteria Cultures
• vitamins, amino acids, purines and pyrimidines (nucleotide bases that make up • deep-freezing: -50 to -95C
the DNA) • Lyophilization (freeze-drying): Frozen (-54 to -72C) and dehydrated in a vacuum
CLASSIFICATION OF CULTURE MEDIA (powdered form for long-term preservation)
CULTURE MEDIUM REPRODUCTION IN PROKARYOTES
▪ nutrients prepared for microbial growth 1. BINARY FISSION ▪ simplest method
STERILE ▪ for each generation, the number of cells
▪ no living microbes logarithmically increase
INOCULUM 2. BUDDING
▪ introduction of microbes into medium 3. CONIDIOSPORES (Actinomycetes)
CULTURE 4. FRAGMENTATION OF FILAMENTS
▪ microbes growing in/on culture medium
AGAR 4 STAGES OF BACTERIAL GROWTH
▪ consistency is like a gelatin 1. LAG PHASE ▪ Phase wherein the bacteria are starting to adjust to
the new environment
▪ complex polysaccharide extract from the seaweed 2. EXPONENTIAL ▪ For each generation, the number of cells
▪ Purpose: solidify culture media GROWTH logarithmically increase
▪ used as solidifying agent for culture media in Petri dish, slants and deeps PHASE/ ▪ Best time to perform antibiotic susceptibility testing
(bott or stab) LOGARITHMIC (the tabolytes of the bacteria is produced)
▪ generally, not metabolized by microbes PHASE
▪ liquefies at 100C ▪ The number of live cells is equivalent to the number
3. STATIONARY of dead cells (bacteria have accumulated metabolic
▪ solidifies at 40C PHASE waste and exhaustion of nutrients)
1. CULTURE MEDIA
▪ Plateau phase
▪ Last stage
• Chemically defined media: exact chemical composition is known 4. DEAD PHASE/
• complex media: extracts and digests of yeasts, meat or plants (Nutrient broth or LOGARITHMIC ▪ The number of bacterial population decrease.
DECLINE ▪ If the bacteria have a virulence factor, the dead
Nutrient agar) phase could be prolonged
ANAEROBIC CULTURE METHODS
REDUCING MEDIA
▪ contain chemicals (thioglycollate or oxyrase) that combine O2
▪ heated to drive off O2 (to boil the thioglycollate)
▪ require an environment with no oxygen
ANAEROBIC JAR
▪ anaerobic: methylene blue (indicatorblue: presence of oxygen)
ANAEROBIC CHAMBER
▪ with glove ports and air lock
CAPNOPHILES
▪ bacteria that require high CO2
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CLINICAL BACTERIOLOGY|LECTURE(Sir Gelo and Dean Basit)
PRELIMS LESSON 6: MICROBIAL/ BACTERIAL GENETICS
TOPIC OUTLINE • PYRIMIDINES: Thymine, and Cytosine
 Microbial Genetics
• Backbone is Deoxyribose-phosphate.
1. Requirement for Growth
2. Classification of Culture Media • Strands are held together by hydrogen bonds between AT and CG (Chargaff
3. Reproduction in Prokaryotes rule)
4. 4 Stages of Bacterial Growth • DNA is copied by DNA Polymerase
DIFFINITION OF TERMS • Strands are antiparallel (Leading: 5' to 3’: Lagging: 3' to 5')
GENETICS • DNA replication is semi conservative (1 strand is conserve that is a template to
• Study of what genes are, how they carry information, how information (code) is produce a new strand)
expressed, and how genes are replicated. • DNA is copied by DNA Polymerase
GENE
▪ In the 5’ à 3’ direction
• A segment of DNA that encodes a functional product, usually a protein. ▪ Initiated by an RNA primer
GENOME
▪ LEADING STRAND is synthesized continuously/ continuous 5' to 3'
• All of the genetic material in the cell.
▪ LAGGING STRAND is synthesized discontinuously/ Not continuous 3' to 5'
GENOMICS
▪ Created: Okazaki Fragments
• Molecular study of genomes.
▪ RNA primers are removed, and Okazaki fragments joined by a DNA
GENOTYPE
polymerase and DNA ligase
• Genes of an organism.
• ENZYMES:
• Sequence bases ▪ DNA polymerase (essential for DNA replication; responsible in achieving
PHENOTYPE Chargaff rule)
• Expression of the genes. ▪ DNA helicase (unwinds doublestranded DNA)
• Physically manifested ▪ DNA ligase (seals the gap)
CHROMOSOME MAP ▪ DNA gyrase (relaxes the supercoiling of the DNA)
• kbp (unit) key base pair - no. of nucleotide bases in a pair ▪ Primase - enzyme that synthesizes primer (short strand of RNA) Primer +
Vertical transfer of genes (Daughter cells/Progeny) Lagging strand = It can act on itself

REPLICATION FORK
▪ Placed where it is unzip (first step in the replication)
STABILIZING PROTEINS
FLOW OF GENETIC MUTATION ▪ Prevents the degradation of the replication
OKAZAKI FRAGMENT
▪ Loop that is seen in the lagging strand
▪ RNA primers are removed, and Okazaki fragments seals the gap in the
RNA.
▪ Finishes the gap in the RNA.

DNA (Deoxyribonucleic Acid)

• Polymer of nucleotides bases: Adenine, Thymine, Cytosine, Guanine
• Double helix associated with proteins
• PURINES: Adenine, and Guanine
DNA REPLICATION IS SEMICONSERVATIVE
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CLINICAL BACTERIOLOGY|LECTURE(Sir Gelo and Dean Basit)
PRELIMS LESSON 6: MICROBIAL/ BACTERIAL GENETICS
TRANSCRIPTION STEPS IN TRANSLATION
• Transcription proceeds in the 5’ to 3’ direction
• DNA to RNA
• DNA is transcribed to make RNA (mRNA: forms ribosomes, tRNA: deliver
amino acids to ribosomes, and rRNA: carry information for making specific
proteins from DNA to ribosomes)
• Transcription begins when RNA polymerase binds to the promotor
sequence.
• Transcription stops when it reaches the terminator sequence
RNA TYPE USES 1. Components needed to begin the translation come together.
Ribosomal RNA (rRNA) Form ribosomes
Transfer RNA (tRNA) Deliver amino acids to ribosome
Carry information for making specific proteins from
Messenger RNA (mRNA)
DNA to ribosomes
3 STEPS OF DNA TRANSCRIPTION:
INITIATION ▪ RNA polymerase binds to DNA to a site called Promoter
▪ RNA polymerase moves along the DNA.
▪ How long? depends on the product that is needed
(depends on the molecular weight of a protein)
ELONGATION
▪ As it moves, it ends in the basis to the 3' of the growing
strands.
▪ It stops in the terminator region
▪ Leads to the stopping of the synthesis of RNA 2. On the assembled ribosome, a tRNA carrying the first amino acid (methionine)
▪ Self-Termination (the RNA transcribes itself to the is paired with the start codon on the mRNA. A tRNA carrying the second amino
terminator, the RNA hydrogen bonds with itself forming a acid approaches.
TERMINATION stem-loop structure pulls the RNA polymerase off the
DNA)
▪ Enzyme-dependent Termination (binds to the terminator
and pushes of the RNA off the DNA)

3. The place on the ribosome where the first tRNA sits is called the P site. A site
next to it, the second codon of the mRNA pairs with a tRNA carrying the second
amino acid

TRANSLATION
• mRNA is translated in codons/triplets (Three nucleotides)
4. The first amino acid joins to the second by a peptide bond, and the first tRNA
• Translation of mRNA begins at the start codon: AUG.
is released.
• Translation ends at a stop codon: UAA, UAG, UGA
• Molecule of mRNA to synthesized protein.
• Wobble Effect: 3rd position in codon is not as strict as the first 2 positions
3 STEPS OF mRNA TRANSLATION:
▪ The tRNA will carry the anticodon (complementary
INITIATION nucleotide bases to that of/for the codon)
▪ Assembles the codon and the anticodon
ELONGATION
▪ Enter the A site, the ribosome catalyzes the transfer
▪ 3 sites: A, P, E
TERMINATION

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CLINICAL BACTERIOLOGY|LECTURE(Sir Gelo and Dean Basit)
PRELIMS LESSON 6: MICROBIAL/ BACTERIAL GENETICS
5. The ribosome moves along the mRNA until the second tRNA is in the P site and EXAMPLE OF OPERONS
the process continues. 1. LAC OPERON
▪ function: allow the bacteria to use lactose as an energy source
▪ group of genes with a single promotor
▪ proteins that have a lactose (glucose and galactose) as an energy source
▪ inducible enzyme
▪ structural genes: lacZ, lacY, lacA
▪ If lactose is PRESENT, will convert to ALLOLACTOSE as inducer, binds
and inactivate the repressor proteins (the repressor will not involve with the
operator), the structural genes in the lac operon will be able to produce
mRNA to produce protein or enzymes that can be metabolized to glucose
and galactose)
6. The ribosome continues to move along the mRNA, and new amino acids are ▪ If lactose is NOT PRESENT, the repressor enzyme is active (prevent the
added to the polypeptide. transcription of structural genes to produce enzyme/protein), no need to
use the lac operon since it has no lactose.
2. TRP OPERON
▪ Components: promoter, operator and 5 structural genes
▪ Repressible operon: genes are usually transcribed.
▪ If excess tryptophan is present; it will activate the repressor to bind with the
operator, to prevent structural genes to produce tryptophan.
▪ If excess tryptophan is not present; the repressor is inactive (not involve) in
the promoter and operator to produce more tryptophan.
MUTATION
• A change in the genetic material
• Mutations may be neutral (no effect), beneficial (benefits the organism), or
7. When the ribosome reaches a stop codon, the polypeptide is released. harmful (disease-causing microorganisms)
• MUTAGEN: Agent that causes mutations
• Mutation occurs in nature.
• Error in the DNA replication
TYPES OF MUTATION
• SPONTANEOUS MUTATIONS: occur in the absence of a mutagen.
• BASE SUBSTITUTION (point mutation)
• MISSENSE MUTATION: Change in one base
o Result in change in amino acid
• NONSENSE MUTATION: Results in a nonsense codon
• FRAMESHIFT MUTATION: Insertion or deletion of one or more nucleotide pairs
• IONIZING RADIATION (X rays and gamma rays) causes the formation of ions
8. Finally, the last RNA is released, and the ribosome comes apart. The released that can react with nucleotides and the deoxyribose-phosphate backbone.
polypeptide forms a new protein. o Nucleotide excision repairs mutations.
REGULATION OF BACTERIAL GENE EXPRESSION/GENE REGULATION
• UV RADIATION causes thymine dimers.
• Constitutive enzymes are expressed at a fixed rate. o Light-repair separates thymine dimers.
• Other enzymes are expressed only as needed. TWO CLASSIFICATIONS
• Repressible enzymes 1. BASE-SUBSTITUTION
• Inducible enzymes ▪ only 1 base is substituted.
OPERON 3 SUB-UNITS OF BASE-SUBSTITUTION
SILENT MUTATION MISSENSE MUTATION NONSENSE
• Operon is present in cells.
there's no change in the can be harmful, neutral are harmful that can
• Made up of several structural genes. amino acid sequence; or beneficial in rare leave to the premature
• Arranged by a common promotor and operator. only affects the genotype cases; only 1 amino acid termination of the
• COMPOSITIONS: Promoter, Operator and Structural genes (expressed in a but not the phenotype. is change protein; proteins will no
single strand of mRNA that can produce protein) 1/3 of the mutation is longer reproduce, amino
silent mutation. acid that is produce is a
• Set of adjacent structural genes and adjacent regulatory signals that affect the stop codon
transcription of the structural genes. 2. FRAMESHIFT
TWO ENZYMES (Operator region) 2 SUB-UNITS OF FRAMESHIFT
1. REPRESSOR ENZYME FRAMESHIFT INSERTION FRAMESHIFT DELETION
▪ Always active and transcribe. 1 or more nucleotide bases are added 1 or more bases are removed that
▪ It can be deactivated, once the repressor deactivates it that causes several misreading of the causes misreading of the amino acids.
2. INDUCER ENZYME amino acids.
▪ Not transcribed unless inducer enzyme will activate it
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CLINICAL BACTERIOLOGY|LECTURE(Sir Gelo and Dean Basit)
PRELIMS LESSON 6: MICROBIAL/ BACTERIAL GENETICS
FREQUENCY OF MUTATION • FRAMESHIFT MUTAGENS
• Spontaneous mutation rate = 1 in 109 replicated base pairs or 1 in 106 replicated o Acridine
genes o Insertion or deletion of a newly synthesized DNA
• Mutagens increase to 10–5 or 10–3 per replicated gene. o some chemical mutagens cause small insertions or deletions of
DNA REPAIR ENZYMES nucleotide base pairs.
1. PHOTOLYASES (LIGHT-REPAIR MECHANISM) GENE TRANSFER AND RECOMBINATION
▪ Photolyases uses an energy to break the bond. 1. VERTICAL GENE TRANSFER
2. EXCISION-REPAIR ENZYMES (DARK-REPAIR MECHANISM) • occurs during reproduction between generations of cells
▪ can occur with or without light. • happens during binary fission (progeny/daughter cells would have the
▪ cuts out the damaged DNA, DNA same genetic material from the parent cells)
▪ polymerase can fill the gap. 2. HORIZONTAL GENE TRANSFER
3. MISMATCH-REPAIR ENZYMES • transfer of genes between cells of the same generation
▪ scan newly synthesized DNA
▪ correct the mutated bond.

3 KINDS OF HORIZONTAL GENE TRANSFER:

2 TYPES OF MUTAGENS
1. RADIATION
• IONIZING RADIATION (X-RAYS OR GAMMA RAYS)
o breakage of chromosomes
o x-rays and gamma rays can cause some of the molecules within cells
to lose electrons becoming highly reactive ions and free radicals. Some
of these reactive ions and free radical combine with bases on DNA
resulting in errors in DNA replication and mutation.
• NON-IONIZING (UV LIGHT)
o can produce thymine dimer (can cause apoptosis or cell death)
o in the form of UV light is also mutagenic because it can cause the
adjacent thymine bases to covalent bind to one another producing
thymine dimers. Such dimers cause serious harm or death if not
repaired since this dimer prevent the cell from properly transcribing or
replicating such DNA.
2. CHEMICAL MUTAGENS
• NUCLEOSIDE ANALOGS
o can mimic endogenous nucleosides.
o cause mismatching
o missense mutation
o some chemicals resemble a certain nucleotide basis (5-bromouracil
bases)
o are compounds that are structurally similar to normal nitrogenous
bases but with different base pairing properties. These compounds can
become incorporated into growing DNA during replication replacing
their related base. Once incorporated, the nucleoside analogs can
inhibit further replication or cause mismatching in a future roundup
application.
• NUCLEOTIDE-ALTERING CHEMICALS
o some chemical mutagens can directly alter the structure of the
nitrogenous bases of DNA
1. TRANSFORMATION
• Involves the uptake of free DNA that is released into the environment when
another bacterial cell (donor) dies and undergoes lysis or cell disintegration
caused by a rupture in the cell wall
• Bacteria was able to get the gene from the environment and incorporates the
gene to transform into a more virulent kind of organism.
• Griffith Experiment: (Frederick Griffith)
▪ 1st set of Griffith Experiment:
o He used Strep. pneumoniaea
o Rough strain (doesn't have capsule); Smooth strain (has capsule)
o R strain is injected to the mouse, the mouse survived
o S strain is injected to the mouse, the mouse died
▪ 2nd set of Griffith Experiment:
o He heated the S strain (to kill the S. pneumoniae)
o S strain (heat-killed), the mouse survives
o He combined live R stain + S strain (heatkilled), the mouse died (R
strain was able to get gene from the dead cell to produce capsule =
virulent organism)
2. TRANSDUCTION
• DNA from the chromosome of one cell is transferred to another cell via a
replicating virus a virus that infects bacteria is called a bacterial phage or phage
• Process by which pieces of DNA are broken and recombined to produce new
combinations of alleles.
• Creates genetic diversity at the level of genes that reflects differences in the
DNA sequences of different organisms.
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PRELIMS LESSON 6: MICROBIAL/ BACTERIAL GENETICS
• Facilitated by the presence of a bacteriophage - a virus that can infect the • DNA from two bacteria may come together in one cell exchange of genes
bacteria. between 2 DNA molecules crossing over occurs when two chromosomes and
• The transducing phage will go to a recipient cell; the phage will inject the rejoin.
bacterial DNA. It will incorporate the DNA to its chromosome resulting of transfer TYPES OF TRANSDUCTION
of genes to the recipient cell. It will make the organism more virulent. GENERALIZED ▪ Process in which bacterial DNA in incorporated into
Beginning of generalized transduction in bacteria, a bacterial phage attaches to a TRANSDUCTION: another bacterium, specifically during the lysis of the
bacterial cell wall and inserts its DNA. The phage commandeers the bacterial cellular virulent bacteriophage
machinery to synthesize new phage DNA and produce phage proteins in order to ▪ Process in which a part or a fragment of a bacterial
completely convert the bacterium into an efficient virus producing factory, some of the SPECIALIZED DNA with viral nucleic acid is transferred to another
page proteins break the bacterial DNA up into small pieces. Normally, phage DNA is TRANSDUCTION: bacterium by the temperate bacteriophage during
coated with phage protein to form new virus particles but sometimes pieces of bacterial lysogenic process.
DNA are mistakenly packaged in phage proteins.
When a transducing phage composed of phage proteins and bacterial DNA infects a
new host the phage injects the bacterial DNA into the new bacterial host the affected
bacteria sometimes incorporate this DNA into its own chromosome through a
recombination resulting in the transfer of genes from the donor bacterium to the recipient
bacterium
BACTERIOPHAGE
• Bacteriophage is a virus that can infect the bacteria.
TYPES OF BACTERIOPHAGES
• LYTIC BACTERIOPHAGE – bacteria to burst and lysed, no transduction will
happen.
• TEMPERATE BACTERIOPHAGE – insert its DNA into a bacterial cell. This is
where transduction will happen.
PLASMIDS
• genes that encode for the antibiotic resistance are found here
CONJUGATIVE PLASMID
▪ carries genes for sex pili and transfer of the plasmid.
DISSIMILATION PLASMIDS
▪ encode enzymes for catabolism of unusual compounds.
R FACTORS
▪ encode antibiotic resistance.
3. CONJUGATION TRANSPOSONS
• Transfer of genetic material from a donor cell to a recipient cell • known as the jumping genes.
• Occurs between 2 living cells (cell-to-cell contact) • segments of DNA that can move from one region of DNA to another.
• Requires the mobilization of the donor’s chromosome • contain insertion sequences for cutting and resealing DNA(transposase enzyme)
• Sex Pilus (Donor) establishes a conjugative bridge that serves as the conduit for • complex transposons carry other genes.
DNA transfer to the recipient cell. • transposase can cut transposons from one location of a chromosome and insert
• Plasmid could be transferred by conjugation (horizontal transfer), but not all it to another
plasmid the DNA is transferred from one living bacterial cell to another the cells 2 TYPES OF TRANSPOSONS
must touch one another SIMPLE TRANSPOSONS COMPLEX TRANSPOSONS
• Ex. E. coli contains only the essential elements inhibition to the transposase gene also
needed for transposition. carry other elements.
• Pili - it is where the DNA can be transmitted, structure that will allow the transfer
of one bacterial DNA into another DNA
MECHANISMS OF TRANSPOSONS
• Plasmid (Extrachromosomal DNA) is transferred from F+ to F- negative 1. CUT AND PHASE entire transposons move to another location
• F+ (donor cells); F- (recipient cells), the F- will become a F+ 2. REPLICATIVE transposons are copied to a new location
• 6.5 minutes: The bacteria will transfer the lac operon; it is short because the lac
operon is near to the fertility factor (0 mark is the fertility factor)
• The Trp operon is further from the fertility factor (26mins), the bacteria will
transfer the trp for about 26 mins
• pyrB (93.5mins)
• The further the gene from the fertility factor, the longer it takes for that gene to be
transferred from one bacterial cell to another cell via the process of conjugation.
4. TRANSDUCTION/ GENETIC RECOMBINATION
• Exchange of genes between two DNA molecules
• Crossing over occurs when two chromosomes break and rejoin
• It is the transfer of bacterial genes by a bacteriophage from one cell to another.
o Bacteriophage: virus that infects bacteria
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CLINICAL BACTERIOLOGY|LECTURE(Sir Gelo and Dean Basit)
PRELIMS LESSON 6: MICROBIAL/ BACTERIAL GENETICS
INSERTION SEQUENCES kanamycin resistance, repeated sequence, insertion sequence,
▪ simplest transposons repeated sequence (IS 1)
▪ consist of transposase gene in inverted repeat o Kanamycin resistance gene: it would be advantageous for the part
Ex. IS 1 (1 insertion sequence) of the bacteria
o transposons are being flank - If one original cell would have the kanamycin resistance gene
o Inverted repeat: replication of nucleotides base on the and that particular original cell wants to protect another cell
complementary strand of an inverted strand without the kanamycin resistance gene. The original cell will use
o Transposase gene: codes for the enzymes that facilitate the transposon to transfer the kanamycin resistance gene to
movement of the transposon another cell, the recipient cell would be kanamycin resista
Ex. Tn5 (2 insertion sequence)
o responsible for the kanamycin resistance
o if a bacterial get the gene, the bacteria would be resistant to
kanamycin
o Composition of complex transposons: repeated sequence,
insertion sequence, repeated sequence (IS 1), gene that carries the

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CLINICAL BACTERIOLOGY|LECTURE
PRELIMS LESSON 7: HOST-PARASITE INTERACTION
TOPIC OUTLINE MICROORGANISMS FOUND ON THE SKIN
 Host-Parasite Interaction Common Residents Less Common or Transients
 Disease Transmission Candida spp. Streptococcus spp.
 Micrococcus spp. Acinetobacter spp.
BACKGROUND AND TERMINOLOGY Gram-negative rods
Staphylococcus spp.
FETUS (fermenters and nonfermenters)
• Sterile until birth Propionibacterium spp. Moraxella spp.
Diphtheroids (Corynebacterium spp.)
• Exposure to environment leads to colonization
NORMAL BIOTA OF THE ORAL CAVITY (MOUTH)
COLONIZATION
• Bacterial plaque may develop on teeth
• Refers to the growth of microbiota in or on a body site without causing damage or
• Low oxidation reduction potential
notable symptoms
MICROORGANISM RELATIONSHIPS
▪ Anaerobes grow
• Buccal mucosa and tooth surface
• Symbiosis: two organisms living together
▪ COMMENSALISM: Microorganism benefits while host is not harmed. ▪ Production of acids by microorganisms
▪ Tooth decay
▪ MUTUALISM: Microorganism and host benefit
MICROORGANISMS FOUND IN THE MOUTH
▪ PARASITISM: Microorganism benefits while the host is harmed. Common Residents
CHARACTERISTIC OF INDIGENOUS MICROBIAL BIOTA Staphylococcus epidermidis Actinomyces israelii
INDIGENOUS (NORMAL) FLORA Streptococcus mitis Bacteroides spp.
▪ Microorganisms commonly found on or in healthy persons Streptococcus sanguinis Prevotella/Porphyromonas
RESIDENT FLORA Streptococcus salivarius Bacteroides oralis
▪ Microorganisms that colonize an area for months or years. Streptococcus mutans Treponema denticola
TRANSIENT FLORA Peptostreptococcus spp. Treponema refrigens
▪ Microorganisms temporarily colonizing a host. Veilonella spp.
CARRIER STATE: Less Common or Transients
Staphylococcus aureus
▪ Condition of hosts capable of transmitting the infection Enterococcus spp.
o ACUTE: short term NORMAL BIOTA OF RESPIRATORY TRACT
o CHRONIC: long term • UPPER RESPIRATORY TRACT
COMPOSITION OF USUAL MICROBIAL BIOTA
▪ Mouth, nasopharynx, oropharynx, larynx
• Composition is influenced by specific nutritional and environmental factors • LOWER RESPIRATORY TRACT
• The affinity of microorganisms for specific site depends on the ability of the ▪ Trachea, bronchi, pulmonary parenchyma
organisms to resist the antibacterial effects of bile, lysozyme, fatty acids ▪ Protected by ciliary epithelial cells and mucus
ENVIRONMENTAL FACTORS ▪ Normally considered sterile
• MOIST or DRY – most microorganisms live in moist areas (skin folds) MICROORGANISMS FOUND IN THE NOSE AND NASOPHAYNX
• LOW pH – female genital tract, gastrointestinal (GI) tract of breast-fed infants Common Residents Less Common or Transients
Staphylococcus aureus Strepococcus pneumoniae
• GASEOUS ATMOSPHERE – low oxidation/reduction potential
Staphylococcus epidermidis Moraxella catarrhalis
COMPOSITION OF MICROBIAL BIOTA OF DIFFERENT BODY SITES Diphtheroids (Corynebacterium spp.) Haemophilus influenzae
various strains help to regulate levels of other bacteria Haemophilus parainfluenzae Neisseria meningitidis
in the gut, modulate immune responses to invading Streptococcus spp Moraxella spp.
Bifidobacteria
pathogens, prevent tumor formation and produce
vitamins
MICROORGANISMS FOUND IN THE OROPHARYNX
several types inhabit human gut. They are involved in
Common Residents
Escherichia coli the production of vitamin K2 and help to keep bad
bacteria in check, but some strains can lead to illness Α-Hemolytic and nonhemolytic streptococci
beneficial varieties produce vitamins and nutrients, Diphtheroids (Corynebacterium spp.)
Lactobacilli Staphylococcus aureus Moraxella catarrhalis
boost immunity and protect against carcinogens
C. jejuni and C. coli are the strains most commonly Staphylococcus epidermidis Haemophilus parainfluenzae
Campylobacter associated with human disease. Infection usually Streptococcus pneumoniae Bacteroides spp.
occurs through the ingestion of contaminated food. Streptococcus mutans Prevotella/Porphyromonas
Enterococcus faecalis common cause of post-surgical infections Streptococcus mitis Bacteroides oralis
most harmful following a course of antibiotics when it Streptococcus sanguinis Fusobacerium necrophorum
Clostridium diffiole Streptococcus salivarius
can proliferate.
NORMAL BIOTA OF THE SKIN Less Common or Transients
GENERALLY SUPERFICIAL ORGANISMS Streptococcus pyogenes
Neisseria meningitidis
▪ Skin surface and hair follicles
Haemophilus influenza
APOCRINE SWEAT GLANDS
Gram-negative rods
▪ Secretes substance metabolized by bacteria. NORMAL BIOTA OF THE GI TRACT
▪ Release of odorous amines • GI tract consist of the esophagus, stomach, small intestine and colon
NORMAL FLORA • GI tract is equipped with numerous defenses and effective antimicrobial factors
▪ Colonize skin surface. • Estimated number of possible GI microbial residents: over 35,000
▪ Prevent pathogens from colonizing. • Stomach normally sterile due to acidic pH
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CLINICAL BACTERIOLOGY|LECTURE
PRELIMS LESSON 7: HOST-PARASITE INTERACTION
SOME EXCEPTIONS: MICROBIAL FACTORS CONTRIBUTING TO PATHOGENESIS AND VIRULENCE
▪ Endospores, parasitic cysts, H. pylori PATHOGENICITY
• Other pathogens enter in food particles • Ability of an organism to produce disease
▪ Escape stomach and enter the intestine OPPORTUNISTIC PATHOGENS
▪ Colonize the small and large intestine • Usually do not cause infection
• Antibiotics • May case infection under special circumstances
▪ Can significantly alter the usual flora
COMMON RESIDENTS FOUND IN THE GASTROINTESTINAL TRACT
Bacteroides spp. Lactobacillus spp.
Clostridium spp. Peptostreptococcus spp.
Enterobacteriaceae Peptococcus spp.
Enterococcus spp. Porphyromonas spp.
Eubacterium spp. Prevotella spp.
Fusobacterium spp. Streptococcus spp.
NORMAL BIOTA OF THE GENITOURINARY TRACT
STERILE SITE
▪ Kidneys
▪ Bladder
▪ Fallopian tubes
NONSTERILE SITE
▪ Distal urethra (particularly in women)
▪ Vagina
MICROORGANISMS FOUND IN THE GENITOURINARY TRACT
Common Residents
Lactobacillus spp. Staphylococcus aureus
Bacteroides spp. Staphylococcus epidermidis
Clostridium spp. Enterococcus spp.
Peptostreptococcus spp. Diphtheroids (Corynebacterium spp.)
Less Common or Transients
Group B streptococci
Enterobacteriaceae
Acinetobacter spp.
Candida albicans
ROLE IF MICROBIAL BIOTA IN THE PATHOGENESIS OF INFECTIOUS DISEASE
INSTANCE WHEN GOOD BIOTA TURNS BAD
• Opportunistic infections
▪ Casuse disease when habitat is changed.
▪ May occur due to weakened immune system. PATHOGENESIS
• Trauma TRUE PATHOGENS
• Introduce flora to sterile site. • Organisms that cause disease in healthy immunocompetent hosts
• Immunosuppression ▪ Ex. Y. pestis and B. anthracis
• Immunosuppressive drugs IATROGENIC INFECTIONS
• Chemotherapy • Occur as the result of medical treatment
• Radiation
DISEASE TRANSMISSION
• Immune defects
ROUTES OF TRANSMISSION AND EXIT
• In patients with serious infections associated with
▪ Chronic illness • Airborne
o Ex. Diabetes, severe hepatic disease (cirrhosis) • Transmission by food and water
ROLE OF MICROBIAL BIOTA IN THE HOST DEFENSE AGAINST INFECTIOUS • Close contact
DISEASE ▪ Direct contact
BENEFITS OF MICROBIAL DATA
NORMAL MICROBIAL FLORA
• Cuts and bites (nonarthropod)
• Prime the immune system ▪ Wounds
▪ Axenic animals: germ free • Arthropods
• Sterile environments impair immune development ▪ Bites of insects
MICROENVIRONMENT • Zoonoses
• Microbial flora block colonization of extraneous pathogens ▪ Contact with animals
▪ Antibiotics can alter the indigenous biota

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CLINICAL BACTERIOLOGY|LECTURE
PRELIMS LESSON 7: HOST-PARASITE INTERACTION
FOCAL INFECTION Systemic infection that began as a local infection
BACTEREMIA Bacteria in the blood
SEPTICEMIA Growth of bacteria in the blood
TOXEMIA Toxins in the blood
VIREMIA Viruses in the blood
PRIMARY
Acute infection that causes the initial illness
INFECTION
SECONDARY Opportunistic infection after a primary (predisposing)
INFECTION infection
SUBCLINICAL
No noticeable signs or symptoms (inapparent infection)
DISEASE
PREDISPOSING FACTORS
Make the body more susceptible to disease.
▪ Short urethra in females
▪ Inherited traits such as sickle-cell gene
▪ Climate weather
▪ Fatigue
▪ Age
▪ Lifestyle
▪ Chemotherapy
THE STAGES OF DISEASE

• An infection where the disease is transmissible from vertebrate animals to

humans. The human usually is dead end host for the organism.
▪ RABIES – this virus may be transmitted from a dog, fox, or racoon to
humans.
▪ RINGWORM – this fungal infection that infects skin can be transmitted skin
can be transmitted from cattle or pets to humans.
• A COMMUNICABLE DISEASE is due to specific infectious agent or its toxic
products that is transmitted to a susceptible host. RESERVOIRS OF INFECTION
Reservoirs of infection are continual sources of infection
• TRANSMISSION can occur from an infected person, an infected animal or an
• HUMAN – AIDS, gonorrhea
inanimate reservoir.
• The disease is termed a communicable disease.
▪ Carriers may have inapparent infections or latent diseases
OCCURRENCE OF DISEASE • ANIMAL – Rabies, Lyme disease
Fraction of a population that contracts a disease during a ▪ Some zoonoses may be transmitted to humans
INCIDENCE
specific time • NONLIVING – Botulism, tetanus
PREVALENCE Fraction of a population having a specific disease at a ▪ Soil
given time RESERVOIR
SPORADIC DISEASE Disease that occurs occasionally in a population
▪ is the natural location of the organism. This may be either an animate or
ENDEMIC DISEASE Disease constantly present in population
inanimate location.
EPIDEMIC DISEASE Disease acquired by many hosts in a given area in a short SOURCE
time
PANDEMIC DISEASE Worldwide epidemic ▪ is the immediate location from which the infecting organism has been
transmitted.
HERD IMMUNITY Immunity in most of a population
SEVERITY OF DURATION OF DISEASE The RESERVOIR IS DISTINGUISH FROM THE SOURCE OF INFECTION, the
ACUTE DISEASE Symptoms develop rapidly individual or object from which an infection is actually acquired.
CHRONIC DISEASE Disease develops slowly CARRIERS
SUBACUTE ▪ are hosts that harbour a pathogen without clinical symptoms and transmit
Symptoms between acute and chronic the infection, often unknowingly (HIV, Chlamydia)
DISEASE
LATENT DISEASE
Disease with a period of no symptoms when the patient ▪ The carrier state may be short or long termed.
is inactive The carrier state may occur:
EXTENT OF HOST INVOLVEMENT o During the incubation period before clinical symptoms (Measles)
LOCAL INFECTION Pathogens are limited to a small area of the body o During the convalescent period (Mycoplasma)
SYSTEMIC
An infection throughout the body o By a chronic carrier (Salmonella typhi)
INFECTION
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CLINICAL BACTERIOLOGY|LECTURE
PRELIMS LESSON 7: HOST-PARASITE INTERACTION
TWO TYPES OF TRANSMISSION EMERGING INFECTIOUS DISEASES
• VERTICAL TRANSMISSION: is from mother to child in utero or in breast milk. • Diseases that are new, increasing in incidence or showing a potential to increase
• HORIZONTAL TRANSMISSION: All other transmission is horizontal transmission in the near future.
TRANSMISSION OF DISEASE • CONTRIBUTING FACTORS:
CONTACT ▪ Genetic recombination
• DIRECT: Requires close association between infected and susceptible host o E. coli 0157, Avian influenza (H5N1)
• INDIRECT: Spread by fomites ▪ Evolution of new strains
• DROPLET: Transmission via airborne droplets o V. cholerae 0139
DIRECT PERSONAL CONTACT ▪ Inappropriate use of antibiotics and pesticides
▪ is a major transmission route for the spread of infections in hospitals and other o Antibiotic resistant strains
health care facilities (nosocomial infections–noso disease, komeion to take care ▪ Changes in weather patterns
of)
o Hantavirus
Other forms of direct contact include:
▪ Modern Transportation
▪ Sexual transmission (Herpes, syphilis, HIV)
o West Nile virus
▪ Skin to skin transmission (Scabies, staphylococci) ▪ Ecological disaster, war, and expanding human settlement
INDIRECT CONTACT
o Coccidiodomycosis
• is a common form for transmission of microorganisms from an infected source to
a susceptible host via a contaminated intermediate object. ▪ Animal control measures
SNEEZING AND COUGHING o Lyme disease
• can transmit microorganisms in a fine spray from one host to the respiratory ▪ Public Health failure
passages of a second host. o Diphtheria
AIRBORNE TRANSMISSION
• can occur when microorganisms remain suspended in air or dust particles and
can be carried by air currents to susceptible hosts. This may occur in crowded
rooms (tuberculosis, influenza)
• Airborne transmission can also occur by spread from environmental
reservoirs. (Can extend up to 5 ft)
DROPLET TRANSMISSION
• is a form of contact transmission where the source projects particle droplets
containing infectious agents, from coughing, sneezing or respiratory equipment.
Occurs in the same room, a short distance from the source directly to the new
host.
VEHICLE Transmission by an inanimate reservoir (food, water)
VECTORS Arthropods, especially fleas, ticks, and mosquitoes
MECHANICAL Arthropod carries pathogen on feet
BIOLOGICAL Pathogen reproduces in vector
A VECTOR is a biological or inanimate source that aids in the transmission of infection
from one host to another. This form of transmission is mediated by a variety of
invertebrate and vertebrate sources.
NOSOCOMIAL (HOSPITAL-BASED) INFECTIONS
• Are acquired as a result of a hospital stay
• 5-15% of all hospital patients acquire nosocomial infections

MORBIDITY Incidence of a specific notifiable disease

MORTALITY Deaths from notifiable diseases
MORBIDITY Number of people affected in relation to the total population in a
RATE given time period
MORTALITY Number of deaths from a disease in relation to the population in a
RATE given time.
Common Causes of Nosocomial Infections CENTERS FOR DISEASE CONTROL AND PREVENTION (CDC)
Percentage of Percentage Resistant • Collects and analyzes epidemiological information in the United States.
Nosocomial Infections to Antibiotics
• Publishes Morbidity and Mortality Weekly report (MMWR)
Gram + cocci 51% 29-89%
Gram – rods 30% 3-32% • In the PHILIPPINES, it is the NATIONAL EPIDEMIOLOGY CENTER OF THE
Clostridium difficile 13% DEPARTMENT OF HEALTH (NEC-DOH)
Fungi 6%
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MICROBIAL VIRULENCE FACTORS OF BACTERIA ABILITY TO PRODUCE EXTRACELLULAR TOXINS
ABILITY TO RESIST PHAGOCYTOSIS AND ENZYMES – EXOTOXINS/ ENDOTOXINS
PHAGOCYTES TOXINS
• Major role in clearing bacterial infection. • Poisonous substances secreted by organisms
CAPSULES EXOTOXINS
• Inhibit engulfment. • Secreted by the organism into extracellular environment or are released on lysis
of organism.
• Mask cell structure that are recognized by receptors on the phagocyte cell
• Binding subunit
surface.
PROTEIN A
▪ Allows toxin to enter cell.
• Toxic subunit
• In bacterial cell wall of Staphylococcus aureus
• Interferes with the binding of the host’s antibodies to the surface of the organism ▪ Disrupts or destroys cellular function.
ENDOTOXINS
• Binds Fc portion of immunoglobulin (IgG), preventing opsonization and
• Composed of the lipopolysaccharide (LPS) portion of the outer membrane of
phagocytosis by turning the antibody is turned around on the surface.
gram-negative bacteria (cell wall has two layers)
LEUKOCIDINS
• A staphylococci leucocidin called Panton-Valentine is: • THREE REGIONS:
▪ Lethal to leukocytes ▪ O-specific oligosaccharide
▪ Contributes to organism invasiveness. ▪ Core polysaccharide
INHIBIT CHEMOTAXIS ▪ Inner lipid A (also called endotoxin)
• The movement of white blood cells (WBC) to sites of tissue damage • Lipid A (endotoxin) stimulates the release of proinflammatory cytokines that aid in
• Host is less able to direct polymorphonuclear neutrophils (PMNs) and mounting an innate immune response
macrophages to the site of infection. • These chemicals mediate (cytokines) produce effects of endotoxin resulting in
SURFACE STRUCTURES THAT PROMOTE ADHESION dramatic changes in blood pressure, clotting, body temperature, circulating blood
TO HOST CELLS AND TISSUES cells, metabolism, humoral and cellular immunity and resistance to infection.
• Adhesins are the microbial surface structures that mediate attachment. • ENDOTOXIN EXPOSURE CAUSES:
• Host cells must have receptors for the adhesins ▪ Simulation of the fever centers in the hypothalamus
• Mutation in host or infectious agent that results in surface structure change: ▪ Hypotension
▪ Adhesion does not occur ▪ Septic or endotoxic shock
▪ Virulence of infectious agent is affected ▪ Coagulation initiation
ADHESIVE STRUCTURES ▪ Severe neutropenia
▪ FIMBRIAE (PILI) – main adhesin in bacteria • Immune system disturbance
▪ Surface polysaccharides BACTERIAL EXOTOXINS VERSUS ENDOTOXINS
ENABLE BACTERIA ATTACHMENT TO HOST SURFACE STRUCTURES
▪ Increase ability to colonize
▪ Provide resistance to phagocytosis
SURFACE BACTERIAL STRUCTURES INVOLVEDIN PATHOGENESIS OF
DISEASE

ABILITY TO SURVIVE INTRACELLULARLY AND PROLIFERATE

Mechanisms to prevent organisms from being killed intracellularly:
• SECRETORY ANTIBODY
▪ IgA proteases
▪ Antigenic variation
• LACTOFERRIN: binds free iron.
▪ Meningococci can use lactoferrin for iron.
• LYSOSOMES
▪ Prevent fusion.
▪ Escape phagosome
INVASION: The ability of pathogens to penetrate and grow in tissues:
• LOCALIZED
▪ Few layers or in one body area
• DISSEMINATED
▪ Spread to distant areas and organs.
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PRELIMS LESSON 7: HOST-PARASITE INTERACTION
INTRODUCTION TO IMMUNE SYSTEM
ORGANIZATION OF IMMUNE SYSTEM
LINE OF DEFENSE OF OUR BODY
PHAGOCYTIC CELLS
ENGULFING CELLS
▪ Neutrophils (PMNs)
▪ Macrophages
CHEMOTAXIS
▪ Chemical caused movement to a location
▪ Necessary to mobilize phagocytes to infection
DIAPEDESIS
▪ Movement from blood vessels to tissues
DISTRIBUTION OF MONOCYTES/ MACROPHAGES
STAGES OF PHAGOCYTOSIS
CHEMOTAXIS: Chemical attraction. Movement of an organism or entity in response to
a chemical stimulus.
ADHERENCE: Process by which bacteria stick to the surface of host cells.
OPSONIZATION: Process of facilitating phagocytosis
OPSONINS: Tag foreign pathogens for elimination by phagocytes.
ELIMINATION: After digesting the bacteria, there will be an elimination which the debris
will be eliminated from the cell by exocytosis
• There are bacteria that prevent phagocytosis. If the bacteria has capsule, then
they can prevent phagocytosis. Encapsulated bacteria can prevent
adherence/phagocytosis
• Lysosome known as a suicide bag it has acid hydrolase. It is low in pH (acidic).
Lysosome can digest bacteria. Phagosome fuses with lysosome we will call it
phagolysosome and the bacteria will be digested.
STEPS OF PHAGOCYTOSIS
ATTACHMENT
• Attachment of organism to phagocyte
▪ Facilitated by opsonins.
INGESTION
• Invaginates and engulfs particle
• Enclosed in phagosome
▪ Fuses to lysosome
KILLING
• Increase in metabolic activity
• Causes production of acids and hydrogen peroxide
• Release of enzymes
▪ Bacteriocidal
INTRACELLULAR PATHOGENS
• Circumvent this process
INFLAMMATION
• Chemical mediators increase blood flow causing
• Erythema (redness)
• Edema (swelling)
• Heat
• Pain (due to swelling)
• Increase number of WBCs in tissue
VASODILATION - when blood vessels in your body widen, allowing more blood to flow
through them and lowering your blood pressure.
PHAGOCYTE MIGRATION - to reach the site of infection, phagocytes leave the
bloodstream and enter the affected tissues
TISSUE REPAIR - the restoration of tissue architecture and function following an injury.
FEVER
• Inflammation is a great tool, unless it becomes chronic or non-localized
• Chronic inflammation typically has an underlying cause (e.g. ongoing infection)
• Non-localized inflammatory responses gives rise to body-wide vessel dilation and
leakage, resulting in precipitous drops in blood pressure called Shock.
• Endotoxin signals inflammatory responses and systemic infections with Gram-
negative bacteria can give rise to a very dangerous condition known as Septic
Shock
• Fever is the preferred systemic response to bacterial infection
• Fevers are elevated body temperatures induced either by pathogen molecules or
by body molecules produced in response to pathogen molecules.
• Fevers results in temperatures that, ideally, inhibit micobes while enhancing body
defenses.
IMMUNE RESPONSES
INNATE IMMUNITY
• Natural or nonspecific immunity
▪ Physical barriers
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▪ Chemical barriers ANTIBODY, AKA, IMMUNOGLOBULIN
▪ Phagocytosis
ADAPTIVE OR SPECIFIC IMMUNITY/ ACQUIRED IMMUNITY
• Antibodies = Immunoglobulins
• Lymphocytes
▪ B cells
▪ T cells
o T helper
o Cytotoxic
ANTIGENS
• Antigen stands for Antibody Generator.
• Antigens are molecules that are potentially recognized by adaptive immune-
system molecules
• Recognition specifically means a binding between the antigen and the immune-
IMMUNOGLOBULIN ACTIONS
system molecule, such as between a immunogen and an antibody
• Note, however, that an antigen within its “self” environment will not normally elicit
an immune response
• To describe antigens found in non-self environments, the term Immunogen is more
correct, though we won’t bother making this distinction.
• An antigen consists of a number of recognizable regions known as Antigenic
Determinants aka Epitopes
ANTIBODIES, ANTIGENS, “EPITOPES”

PRIMARY AND SECONDARY IMMUNE RESPONSE

HUMORAL IMMUNE RESPONSE
B CELLS
▪ Aided by helper T cells
IMMUNOGLOBULINS (ANTIBODIES)
▪ IgG-monomer
▪ 70 – 75% of serum immunoglobulin
▪ Opsinizing antibody, crosses placenta
▪ IgM-pentamer
▪ Complement fixation
▪ First antibody produced
“B cells become plasma cells and plasma cells produced antibodies”
CLONAL SELECTION - process wherein B cell is activated and eventually proliferated.
▪ Bacteria is an example of T independent antigen “Memory B cells are important anamnestic response. The role of memory B cells is to
T INDEPENDENT ANTIGEN - Type of antigen that can stimulate B cell to produce remember and encounter previous with an antigen.”
antibodies even without the help of T helper cell IgG vs IgM
T DEPENDENT ANTIGEN - needs the help of T helper cells through antigen
presentation before B cells can proliferate.
▪ IgA – dimer
▪ 15 – 20% of serum immunoglobulin
▪ Secreted at mucous membranes
▪ IgE – receptor bound
▪ Very low serum concentration
▪ Role in clearance of parasites and allergies
▪ IgD – surface bound
▪ Very low serum concentration
▪ Role in signaling of B cell receptors
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PRIMARY AND SECONDARY ANTIBODY RESPONSES
PRIMARY
▪ Rapid appearance of IgM
▪ Peak in 2 – 3 weeks followed by decline
▪ Gradual change over to IgG or IgA antibodies
SECONDARY (ANAMNESTIC IMMUNE RESPONSE)
▪ Rapid increase in IgG antibodies
▪ Higher levels of IgG with prolonged elevation
▪ Higher specificity
- Somatic hypermutation
CELL-MEDIATED IMMUNITY (CMI)
• Protection from intracellular pathogens
• T helper cells
▪ Lymphokines (cytokine)
▪ Signal activation of macrophages and other phagocytes
▪ Cytotoxic T cells
▪ Kill infected cells
• Cytotoxic T cell
▪ Perforin gran system will cause pores and there will be holes, granzyme will
enter the cell and this will cause the destruction of the cell. The infected cell
is destroyed in this manner.
▪ T helper cells - important in both humoral and cell mediated immunity.
▪ Macrophages is an example of antigen precepting cell. Macrophage will
present antigen to T helper cells
CELLS OF THE IMMUNE SYSTEM

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MECHANISMS BY WHICH MICROBES MAY OVERCOME THE HOST DEFENSES
INDUCED IMMUNE TOLERANCE
▪ Not recognized as foreign
IMMUNE SUPPRESSION
▪ Actively destroy, inactivate, or limit the effect of the immune response
ANTIGENIC VARIATION
▪ Intracellular “hiding”

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PRELIMS LESSON 8: MICROBIAL CONTROL AND SAFETY IN MICROBIOLOGY LABORATORY
TOPIC OUTLINE GERMICIDE
 Microbial Control and Safety in Microbiology Laboratory • A germicide is a compound that "kills" microorganisms.
1. Microbial Resistance • It is usually a high-level disinfectant that kills "germs"; bacteria, fungi, parasites
2. Methods of Microbial Control or viruses.
3. Historical Background SANITIZATION
4. Types of Chemicals • A detergent or soap is used to physically remove microorganisms, while ultraviolet
DEFINITION OF TERMS radiation may be used to reduce microbial load in the air.
DECONTAMINATION
• STERILIZATION
• Refers to the removal of infectious agents or toxic products from an
▪ complete removal of the microorganism including the bacterial endospores environment.
• COMMERCIAL STERILIZATION Bacterial population die at a constant logarithmic rate.
▪ clostridium botulinum (canned good bacillus)
• DISINFECTION
▪ removal of pathogenic organism in inanimated objects not necessarily the
bacterial endospores.
• ANTISEPSIS
▪ removal of pathogenic organism in living tissues objects not necessarily
the bacterial endospores.
• DEGERMING
▪ type of antisepsis where in you remove microorganisms in a small are (i.e.
Venipuncture Antisepsis in the antecubital area
• SANITATION
▪ Reduction of microorganisms to a safe public health level (Washing dishes) EFFECTIVENESS OF ANTIMICROBIAL
• GERMICIDE/ BIOCIDE TREATMENT
▪ The use of chemical agents that can kill germs. ▪ DEPENDS ON:
• BACTERIOSTATIC VS. BACTERICIDAL o Number of microbes
o Environment (organic matter, temperature.
▪ BACTERIOSTATIC – stop the growth of microorganism. (Refrigiration)
biofilms)
▪ BACTERICIDAL – kill the bacteria. o Time of exposure
• SEPSIS VS. ASEPSIS o Microbial characteristics
▪ SEPSIS – process of being contaminated. METHODS OF MICROBIAL CONTROL
▪ ASEPSIS – the prevention of contamination. PHYSICAL CHEMICAL
MICROBIAL RESISTANCE ▪ High Temperature ▪ Nonspecific
• Microorganisms vary in their resistance to physical and chemical agents. The
choice of a physical or chemical agent depends on what level of control is required:
▪ Low Temperature − Antiseptics
▪ vegetative organisms only? ▪ Filtration − Disinfectants
▪ tubercle bacilli? AFB ▪ Micturation/ Grinding ▪ Specific
▪ spores? Spore-former ▪ Radiation − Antibiotics
LEVEL OF RESISTANCE ▪ Ultrasonification
MOST RESISTANT MODERATE RESISTANCE LEAST RESISTANCE ▪ Vibration
▪ Bacterial ▪ Fungal spores (reproduction) ▪ Most vegetative bacteria ▪ High/Osmotic Pressure
endospores ▪ Parasite cysts (Infective stage) (bacteria w/o endospores) ▪ Dessication
(i.e., Clostridium tetani)
▪ Mycobacterium ▪ Parasite trophozoites RATE OF MICROBIAL DEATH
Best method of tuberculosis ▪ Enveloped viruses ▪ the number of microbes
(influenza A, SARS-COV-2 )
killing bacterial (Mycolic acid cell wall, waxy) ▪ environmental influences
▪ Naked viruses ▪ Fungi ▪ time of exposure
endospores is
(non-enveloped viruses)
autoclave
▪ Hepatitis B ▪ microbial characteristics
Physical Methods of Microbial Control: HEAT
▪ Polio 1. Moist Heat
STERILIZATION 2. Dry Heat
• Sterilization refers to the complete killing of all microorganisms including TERMS:
vegetative cells bacterial spores and viruses. ▪ Thermal death point (TDP): Lowest temperature at which all cells in a
− Can you sterilize your hands? NO culture are killed in 10 min.
− Can you sterilize your bowel? NO ▪ Thermal death time (TDT): Time to kill all cells in a culture.
− Can you sterilize instruments? YES ▪ Decimal reduction time or D value (DRT): Minutes to kill 90% of a
ANTISEPSIS population at a given temperature.
• Antisepsis is disinfection of animate objects. For instance, disinfectants for the
skin are called antiseptic agents.
− If you use an antiseptic on your skin, do you remove all vegetative
organisms? YES
DISINFECTION
• Disinfection refers to the use of physical or chemical agents to remove
vegetative cells but may not necessarily remove spores.
− Can you disinfect animate objects? No, disinfectants are used on
inanimate objects as the procedure is usually too toxic for animal, cells.

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1. MOIST HEAT ▪ Mechanisms include: incineration burning high temperature ovens and either
• Moist heat works faster than dry heat at the same temperature. gas burners or small incinerators to sterilize bacteriological loops.
• 200 degrees centigrade for 2 1/2 hours at dry heat will accomplish the same killing How is dry heat used?
rate as 15 minutes at 121 degrees centigrade moist heat. Hot air ovens: used to sterilize glassware and metal objects that corrode with moist
heat, powders and oils. Incineration used to dispose of hospital waste of human tissue,
• Mechanism of action: moist heat degrades proteins at much lower temperatures
some needles and other contaminated hospital or animal waste. Used routinely in
than dry heat.
microbiology laboratories to sterilize bacteriological loops, surfaces of glass tubes and
• The proteins coagulate more readily in the presence of moisture. pipettes.
• How is moist heat used? Bolling Autoclave Physical Methods of Microbial Control: FILTRATION
1. BOILING: 100°C, 15mins, • Filtration removes microbes.
2. AUTOCLAVE: 121°C, 15mins (15psi = steam under pressure) • Filtration removes microorganisms in liquid.
3. ARNOLD'S STERILISER/ FRACTIONAL DISTILLATION: 100°C, 30mins,
• Low temperature inhibits microbial growth.
3 consecutive days
4. INSPISSATION: 75°C – 80°C, 1hr, 3 days (thickening thru evaporation) ▪ Refrigeration
5. PASTEURIZATION ▪ Deep freezing
a. LOW TEMP HOLDING (LTH): 63°C, 30mins ▪ Lyophilization
b. HIGH TEMP SHORT TIME (HTST): 72°C, 15secs • High pressure denatures proteins.
c. ULTRA HIGHT TEMPERATURE (UHT): 74-140-74°C < 5 secs • Desiccation prevents metabolism.
MOIST HEAT BY: • Osmotic pressure causes Plasmolysis.
▪ To sterilize instruments that can tolerate water at 100°C.
▪ Most pathogenic organisms are killed within 10min.
BOILING ▪ However, some bacterial endospores (Clostridium tetani
and Clostridium botulinum) may resist boiling for hours
▪ This method is not 100% effective in sterilizing all
microorganisms.
▪ Autoclaves are a form of moist heat control where the
temperature of steam can be raised above the boiling
point by evacuation of air from the autoclave chamber.
▪ Steam entering an autoclave can be raised from 100 C to
121 C by complete evacuation of air from the chamber.
▪ Steam is introduced and the pressure in the autoclave
increases.
AUTOCLAVE
▪ Sterilization is complete at this temperature and at 15 lb.
per square inch pressure in 15. Minutes
▪ Pressure Cooker. A home pressure cooker uses the
same principle, but air is not completely evacuated so that
the steam temperature is not as high FILTRATION
▪ How does this impact on stenlization? The time for • Physical separation of microorganisms
sterilization is increased Heat. • AIR:
▪ Moist Heat: Denatures Proteins ▪ HEPA
▪ Autoclave: Steam Under Pressure • LIQUIDS:
▪ Pasteurization (developed by Louis Pasteur, a French ▪ Membrane filters
microbiologist) is a short exposure to a moderately high ▪ Berkefield
temperature that reduces the number of microorganisms ▪ Chamberland
PASTEURIZATION but does not sterilize the product. ▪ Sietz
▪ Pasteurization of milk at 71.5°C for 15secs. eliminates FILTRATION: FILTER SIZE
Mycobacteriae. Coxiella and Brucella organisms that may • Microbial Filters are made in a variety of materials (cellulose acetate, plastics)
contaminate milk and cause human infection with pore sizes ranging from coarse, 8um, to ultrafine, 0.2um
Why does milk have to be refrigerated if it is pasteurized? ▪ Which size would result in sterilization? 02um
Refrigeration slows the metabolism of these organisms but with time, they will grow and
produce end products of metabolism that may coagulate the milk (the sour in sour
▪ If you can sterilize at 0.2 um, why not use this size for all fitration?
It may clog with proteins large particles and they are expensive
cream) or produce unpalatable acid end products.
2. DRY HEAT
▪ FLAMING
▪ OVEN: 160°C – 170°C for 1hr.
▪ INCINERATION: 300°C – 400°C
▪ CREMATION
• preparation of talcs and powders
• preparation of instruments
• air filtration systems
DRY HEAT-MECHANISMS OF ACTION
▪ Dry heat includes mechanisms to destroy microorganisms by the use of a
flame or heating coil.
▪ Temperatures range from 160°C in hot air ovens to thousand degree
temperatures in incinerators.
VIRUSES: are filterable agents
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FILTRATION: COMMON USES 3. NONCRITICAL • Contact with intact skin.
Where is filtration commonly used? MATERIALS ▪ Intermediate to low level disinfection
▪ Preparation of fluids DEVICE CLASSIFICATION AND METHODS OF EFFECTIVE DISINFECTION
▪ Preparation of vaccines
▪ Preparation of talcs and powders
▪ Air Titration
High Efficiency Particulate Air (HEPA)
▪ 0.45 microns are used in biological cabinet to remove organisms in exhaust
air.
▪ can also be used in high efficiency respiratory mask.
COLD TEMPERATURE
▪ Is bacteriostatic (stop the growth of microorganism)
o REFRIGERATION: decreased chemical reaction and possible
changes in protein CHEMICAL METHODS
o DEEP FREEZING: -50°C to -95°C for foods and drug preservation CHEMICAL METHODS: CHEMOSTERILIZERS
o LYOPHILIZATION: long term preservation (powdered form) KILLING EFFECT BY:
HIGH PRESSURE ▪ Reaction with components of the cytoplasmic membrane
▪ alteration of molecular structure of proteins and carbohydrates o leakage and death
▪ preservation of colors, flavors, nutrient values ▪ Denaturation of cellular proteins
o disrupt metabolism.
▪ fruit juices
DESSICATION ▪ Reaction with thiol groups of enzymes
o inactivation
▪ disruption of metabolism
▪ involves removing of water from microbes. ▪ Damage RNA and DNA
o inhibit replication.
▪ bacteriostatic CHEMICAL AGENTS COMMONLY USED AS DISINFECTANTS AND ANTISEPTICS
▪ food preservation
OSMOTIC PRESSURE
▪ Plasmolysis (suspension of microorganisms in hypertonic solutions, shrinkage)
▪ results in loss of water from microbial cells
▪ food preservation
Radiation damage to DNA
• lonizing radiation (X rays, gamma rays, electron beams)
• Nonionizing radiation (UV)
• (Microwaves kill by heat; not especially antimicrobial)
IONIZING NON-IONIZING HISTORICAL BACKGROUND
▪ destruction of DNA ▪ damage to DNA GERM THEORY
▪ shorter wavelength ▪ longer wavelength ▪ Idea that microorganism|(germs) causes disease.
▪ gamma rays ▪ formation of thymine dimer o Not “evil spirit”
▪ X rays ▪ UV rays ▪ Need to practice asepsis to prevent contamination.
SEMMELWEIS (1816-1865)
▪ high energy electron beams ▪ MICROWAVE
▪ handwashing can prevent disease.
LISTER (1827-1912)
▪ Using chemicals (phenol) to sterile wounds dressing
• Ethyl alcohol and Isopropyl alcohol
▪ Broad spectrum but not sporicidal
o Bactericidal, pseudomonacidal, tuberculocidal,
virucidal
o To remove spores filter through 0.22-um filters
ALCOHOLS
▪ Inactivated by organic material.
▪ Work by denaturing proteins, dehydrating and
dissolving lipids.
o Must be used in 60% to 96% concentration.
E.H. Spauling categories of medical materials o Must be allowed to evaporate from the surface.
• There are three devices classification that determine the ways in which • 37% aqueous solution or as a gas
disinfection and sterilization methods ▪ Carcinogen and irritant
• Those that enter sterile or have vascular FORMALDEHYDE
1. CLINICAL ▪ Nontuberculocidal
system.
MATERIALS ▪ Not recommended on a routine basis
▪ Must be sterile. • Alkylation of RNA and DNA via alkylation of sulfhydryl
o No spores and amino groups ,
• Contact with mucous membranes. • Effective against bacteria, fungi tuberculins and viruses
▪ Require high level disinfection. ▪ 10-minute exposure between 20° C and 30" C:
2. SEMI CRITICAL o Tuberculocidal GLUTARALDEHYDE
germicidal
MATERIALS • Contact with intact skin.
▪ 3-to-10-hour exposure is sporicidal.
▪ Requires intermediate to low level • Does not penetrate organic material well.
disinfection before contact
• Noncorrosive
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• Tinctures
HALOGENS
▪ Alcohol and iodine solution used as antiseptic
• Iodine and neutral polymer carrier that increases slow
release of
▪ lodine Require free iodine, therefore proper dilution is
vital
LODOPHOR • Povidone-iodine (5% to 10%)
▪ Exposure time greater than 30 sec
▪ Disinfectant only not sporicidal
▪ Skin imitant, therefore must be removed from skin
after use
CHLORINE AND CHLORINE COMPOUNDS
• Hypochlorite
▪ Sodium hypochlorite (bleach)
PROS CONS
o Inexpensive o Requires long exposure time for sterility.
and broad- o Corrosive and pH sensitive
spectrum o Inactivation by organic matter
killing power o Rapidly degrades (30 days max)
• Generally used for surface decontamination
▪ 0.5% to 1% solution for surfaces with greater than 3- minute exposure
o Longer if organic material present
▪ 1:10 solution of 5.25% sodium hypochlorite for blood spills
• Cationic, surface active agents
▪ Surfactant reduce surface tension
o Disrupt cell membranes, causing leakage
DETERGENTS: o Reduced effectiveness in hard water and soup
QUATERNARY o Inactivated by excess organic material
AMMONIUM
• Some gram-negative organisms resistant
COMPOUNDS
▪ Pseudomonas spp.
• Generally used on noncritical surfaces
▪ Bench tops and floors
• Molecules of phenol (carbolic acid)
▪ Substituted with halogens, alkyl, phenyl, or benzyl
groups which reduces toxicity and increases
effectiveness
▪ Broad-spectrum activity but not sporicidal
o Additive to detergents to disinfect
▪ Stable and biodegradable
▪ Active in the presence of organic matter
▪ Disrupt cell walls and precipitate proteins
• Used in disinfection of hospital institutional, and
PHENOLICS household environments.
What's a phenolic compound?
A secondary product this contains a phenol group - a hydroxyl
functional group on an aromatic ring.
Phenolics are a chemically diverse group: many different
properties and functions
• Chlorhexidine gluconate (0.5% to 4%)
▪ Disrupts cell membrane
▪ Precipitates cell contents
▪ Broad spectrum and effects can last for 6 hours.
PHENOLICS
CHLOROHEXIDINE ▪ Not generally effective against endospores and
GLUCONATE nonenveloped viruses
▪ Can cause severe skin reactions in infants under 2
months of age
▪ Sensitive to pH
o Optimal range is pH 5.5 to 7.0.
• Hexachlorophene (3%)
▪ Primarily effective against gram-positive bacteria
▪ Interrupts bacterial electron transport
▪ Low concentration; inhibits membrane-bound
PHENOLICS enzymes
HEXACHLOROPHENE
▪ High concentration; ruptures bacterial membrane
▪ Quick effectiveness (15 to 30 sec)
o Longer for gram-negative organisms when
effective
▪ Prescription only due to toxic effects
• Chloroxylenol (0.5% to 4%)
▪ Used primarily against gram- positive bacteria
▪ Primarily in skin applications
PHENOLICS
CHLOROXYLENOL o Handwash and surgical scrub
▪ Unaffected by organics
• Neutralized by nonionic surfactants and polyethylene
glycol (PEG)
• Disrupts cell wall
• Not affected by organics
PHENOLICS
TRICLOSAN • Affected by surfactants, emollients, and pH
• Intermediate reaction time with excellent persistence
• Poor against fungi
Phenolics Triclosan (Cont.)
• Food and Drug Administration (FDA) is re- evaluating safety and efficacy of
triclosan for use as a health care antiseptic for health care personal
handwashes and surgical hand scrub.
• Rarely used due to toxicity and pollution
• Bacteriostatic
HEAVY METALS
▪ Prevent the growth of bacteria
(Ophthalmia
neonatorum)
• Silver nitrate
▪ Used prophylactically for gonococcal conjunctivitis
in newborns
• Ethylene oxide – used for sterilization
▪ Best for plastics and heat sensitive materials
o Explosive hazard
o 450 to 700 mg per liter at 55° C to 60°C for 2
hours
o Humidity best at 30%
GASES
▪ Kills through alkylation of nucleic acids
• Vaporized hydrogen peroxide (HO) and Peracetic acid
▪ Both are bactericidal, fungicidal, tuberculocidal,
virucidal, and sporicidal.
▪ When used together contact time required is
shortened.
EPA REGUALTIONS ON SURFACE DISINFECTANTS
• The Environmental Protection Agency (EPA) regulates use, sale, and distribution
of antimicrobial pesticide products
▪ Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA)
o Require appropriate labels based on laboratory test data
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CLINICAL BACTERIOLOGY|LECTURE
PRELIMS LESSON 8: MICROBIAL CONTROL AND SAFETY IN MICROBIOLOGY LABORATORY
FDA REGULATIONS ON CHEMICAL SKIN ANTISEPTICS
• Antiseptic development options: two options a manufacturer can pursue
▪ NEW DRUG APPLICATION (NDA)
o Recognized as being safe and effective (RASE)
▪ OVER-THE-COUNTER (OTC) DRUG REVIEW
o Also known as the monograph system
o Generally recognized as safe and effective (GRASE)
 CATEGORY I: for claimed therapeutic indication
 CATEGORY II: not GRASE or unacceptable indications
 CATEGORY III: insufficient date to permit final classification
HYGIENIC HANDWASHING/ WATERLESS HAND RUBS
• Goal is to eliminate transient flora (contracted from the environment or other
people).
▪ Also to protect the skin with resident flora
• Handwashing
▪ Remove physical dirt
▪ Before and after patient contact or Objects
• When there is no visible soiling
▪ Use waterless liquid or gel.
o Fast-acting antiseptic
 Small volume, quick acting
SURGICAL HAND SCRUB/WATERLESS SURGICAL HAND RUBS
• Goal is to eliminate transient flora and most resident flora.
▪ Prevent surgical infections
• Broad spectrum, fast acting, persistent
• FDA guidelines
▪ Reduce bacteria on the first day the number of bacteria 2 logo on each
hand within 1 minute after application
▪ The count on each hand does not subsequently exceed baseline within 6
hours.
PRESURGICAL SKIN DISINFECTION
• Goal to degerm an intended surgical site rapidly and provide a high level of
inactivation for up to 6 hours after skin preparation
• Fast acting, broad spectrum, and persistent
• FDA requirements
▪ Reduce bacteria by 2 log per sq. cm on abdomen
▪ Reduce bacteria by 3 log per sq. cm on groin
o Must work within 30 sec and last for 6 hours
▪ Reduce bacteria by 2 log per sq. cm within 30 sec at dry skin test sites
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CLINICAL BACTERIOLOGY|LECTURE
FINALS LESSON 9: MECHANISM OF ANTIMICROBIAL RESISTANCE
TOPIC OUTLINE o The bacteria remain embedded within the EPS which protects them.
 Mechanism of Antimicrobial Resistance MATURATION – The matrix would go much thicker.
DEFINITION OF TERMS DISPERSION – The only time microorganisms can be detached from the biofilm.
INTRINSIC RESISTANCE – naturally occurring.
▪ Naturally found in bacteria (chromosal)
▪ Transmitted to progeny vertically.
▪ Predictable once the organism is identified.
ACQUIRED RESISTANCE
▪ Acquired from exogenous (came from other bacterial cell) DNA (plasmid,
conjugation, transposons, bacteriophage, etc. )
▪ Extrachromosomal circular DNA in the form of plasmid
▪ Plasmid can be transferred from one cell to another by conjugation.
▪ CONJUGATION – two bacterial cells must be adjacent to one another.
▪ TRANSPOSONS – jumping genes.
▪ BACTERIOPHAGE – particularly lysogenic, responsible for transduction.
▪ TRANSDUCTION - The viral DNA could come from other bacteria and could
be transmitted to another bacteria.
ORIGINS OF ANTIBIOTIC RESISTANCE
• Some resistance is 2000 years old.
▪ Canadian Glaciers.
• Some resistance in deep sea areas is 10,000 years old.
▪ Near Papua New Guinea
• Evolution of Resistance
▪ May have occurred as a way to prevent autotoxicity.
THE EVOLUTION OF BACTERIA ON A “MEGA-PLATE”
• Because of some conditions, the bacteria are able to develop resistance and
adapt.
• Antibiotics should not be taken discriminately.
• Culture and Sensitivity Test - a sensitivity test checks to see what kind of
medicine, such as an antibiotic, will work best to treat the illness or infection. IMPERMEABILITY AND EFFLUX
• Empirical Treatment IMPERMEABILITY
▪ giving antibiotics to patients without culture and sensitivity. • Antibiotics are unable to penetrate the bacteria cell wall.
▪ Giving prescriptions based on medical history and information of the patient. EFFLUX – active transport mechanism
INTRINSIC MECHANISMS
• Involves efflux pumps
• Innate ability of a bacterial species to resist the activity of a particular antimicrobial ▪ Proteins located in the bacterial cell membrane that transport molecules out
agent of the cell.
• Possible causes ▪ Transports molecules out of the cell
▪ Lack of affinity of the drug for the bacterial target • Increase secretion of drug
▪ Inability of the drug to enter the bacterial cell. ▪ Five major superfamilies based on amino acid sequence and energy source
▪ Removal of the drug by chromosomally encoded efflux pumps (ATP) used to export their substrates.
▪ Innate production of enzymes that inactivate the drug.
BIOFILMS
ANALOGY: sand na nakakapit sa semento, yung sand is yung bacteria, yung semento
yung matrix.
• SESSILE – layers of bacteria. Bacterial communities. If they are in communities,
they are high in number.
• Pag mataas yung population they can do quorum sensing.
• If they can do quorum sensing, they can join forces to communicate.
• Sessile bacterial communities that are irreversibly attached to a solid surface and
are embedded in an exopolysaccharide matrix. (output of quorum sensing)
• The matrix makes them tenacious, it makes them difficult to remove,.
• Biofilms are highly resistant to antimicrobial agents.
• Resistance is not attributed to typically acquired genetic mechanisms but instead
is determined by chemical and physical characteristics of biofilm formation.
• Inside biofilms, bacteria can communicate, live, feed and grow. Bacteria are
typically 200 times harder to kill with antibiotics or disinfectants inside a biofilm,
and whilst they’re alive they remain a threat to patients. Enzymatic Inactivation
• The bacteria will continue to grow until the biofilm is ‘disturbed’ or free bacteria are • Produce enzymes that destroy the drug before they are able to reach their targets
released, transferring onto other surfaces via hands, gloves, cleaning cloths or • Example B-lactamases
other materials. ▪ many antibiotics have B-lactamases.
HOW IS BIOFILM FORMED
ADHESION – Microorganism adhere to a solid surface.
▪ Production of Extracellular Polymeric Substance (EPS)
o a sticky type of glue-like material.
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CLINICAL BACTERIOLOGY|LECTURE
FINALS LESSON 9: MECHANISM OF ANTIMICROBIAL RESISTANCE
ß-Lactams: Mechanisms of Action and Resistance YOUTUBE VIDEO
− Peptidoglycan, the most important component of the cell wall, is a polymer made
of N-acetyl muramic acid alternating with N-acetyl glucosamine which are cross-
linked by chains of four amino acids.
− The function of the bacterial cell wall is to maintain the characteristic shape of the
organism and to prevent the bacterium from bursting when fluid flows into the
organism by osmosis.
− Synthesis of peptidoglycan and ultimately the bacterial cell wall occurs in a number
of stages. One of the first stages is the addition of 5 amino acids to N-acetyl
muramic acid.
− Next, N-acetyl glucosamine is added to the N-acetyl muramic acid to form a
precursor of peptidoglycan. This peptidoglycan precursor is then transported
across the cell membrane to a cell wall acceptor in the periplasm.
− Once in the periplasm, the peptidoglycan precursors bind to cell wall acceptors,
and undergo extensive crosslinking.
− Two major enzymes are involved in crosslinking: transpeptidase and D-alanyl
carboxypeptidase.
− These enzymes are also known as penicillin binding proteins because of their
ability to bind penicillins and cephalosporins.
− Eventually, several layers of peptidoglycan are formed all of which are crosslinked
to create the cell wall. Gram positive bacteria have many more layers than gram
negative bacteria and thus have a much thicker cell wall.
− Beta-lactam antibiotics include all penicillins and cephalosporins that contain a
chemical structure called a beta-lactam ring. This structure is capable of binding
to the enzymes that cross-link peptidoglycans.
− Beta-lactams interfere with cross-linking by binding to transpeptidase and D-alanyl
carboxypeptidase enzymes, thus preventing bacterial cell wall synthesis.
− By inhibiting cell wall synthesis, the bacterial cell is damaged. Gram positive
bacteria have a high internal osmotic pressure.
− Without a normal, rigid cell wall, these cells burst when subjected to the low
osmotic pressure of their surrounding environment. As well, the antibiotic-penicillin
binding protein complex stimulates the release of autolysins that are capable of
digesting the existing cell wall. Beta-lactam antibiotics are therefore considered
bactericidal agents.
− Bacterial resistance to beta-lactam antibiotics may be acquired by several routes.
− One of the most important mechanisms is through a process known as
transformation. During transformation, chromosomal genes are transferred from
one bacterium to another.
− When a bacterium containing a resistance gene dies, naked DNA is released into
the surrounding environment.
− If a bacterium of sufficient similarity to the dead one is in the vicinity, it will be able
to uptake the naked DNA containing the resistance gene.
− Once inside the bacterium, the resistance gene may be transferred from the naked
DNA to the chromosome of the host bacteria by a process known as homologous
transformation.
− Over time, the bacterium may acquire enough of these resistance genes to result
in a remodelling of the segment of the host DNA.
− If this remodelled DNA segment codes for cross-linking enzymes (i.e. penicillin
binding proteins), the result is the production of altered penicillin binding
proteins.
− These altered penicillin binding proteins can still cross-link the peptidoglycan
layers of the cell wall but have a reduced affinity for beta-lactam antibiotics thus
rendering the bacterium resistant to the effects of penicillin and other beta-lactam
agents.
− This transfer process has resulted in penicillin-resistant S. pneumoniae through
the acquisition of genes from other naturally occurring penicillin-resistant
Streptococcus species.
− A second important mechanism by which bacteria become resistant to beta-lactam
antibiotics is by the production of enzymes capable of inactivating or
modifying the drug before it has a chance to exert its effect on the bacteria
depending on the bacterial species.
− The gene coding for this enzymes may be found as part of the host DNA or on
plasmids which are small self-replication units of genetic material.
− Bacteria are capable of passing these resistance plasmid to each other by
conjugation, when two bacteria come into close contact with each other, a small
channel is created between them which allows one of the bacteria to pass a copy
of the resistance plasmid to the other.
− If the plasmid is transcribed and translated, the bacteria will begin to produce in
activation enzymes. These enzymes capable of destroying beta-lactam antibiotics
are known as beta-lactam aizaz,
− In gram positive bacteria, the beta lactamase enzyme is generally inducible
resulting in a large amount of enzyme being produces in the presence of the drug
in the gram-negative bacteria.
− The beta lactam enzymes are produced constitutively even when antiobiotic is not
present.
− Gram positive bacteria released the beta-lactamase enzyme from the cell into the
extracellular environment where it inactivates the drug before it enters the bacterial
cell.
− In contrast, gram-negative bacteria retain the beta lactamase enzyme within the
periplasmic space resulting in a more efficient mechanism than gram positive
bacteria.
− Ultimately, the destruction of the beta-lactam ring of the antibiotic renders it,
incapable of binding to the penicillin binding protein and thus the bacteria become
resistant to that drug or class of drugs.
DEAN
− Result in alteration in binding site of penicillin.
− There is a conformational change in the penicillin binding protein.
− Drugs as penicillin are not able to bind.
− Destruction of Beta Lactam ring because of the enzyme beta lactamases
−
ACQUIRED
• Whenever a certain cell received genes from the environment or from another
bacterial cell
• Horizontal Transfer of genes
EFFLUX PUMPS
▪ Increase secretion of drug
▪ Antibiotic cells are actively being pumped out of the bacterial cell.
Target site modification
▪ Usually occurs by chromosomal mutation
▪ Mutations that reduce effectiveness of drug to act on its target
▪ Enzymatic alteration of antibiotic target sites
o Reduce effectiveness of drug to act on its target
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CLINICAL BACTERIOLOGY|LECTURE
FINALS LESSON 9: MECHANISM OF ANTIMICROBIAL RESISTANCE
MOST COMMON IN THE HOSPITAL: • Used in combination with specific antibiotics
ESPL – Extended Spectrum Beta Lactmase producing microorganism.
• Increased hydrolysis of different kinds of antibiotcs that contains oxyimino-beta
lactams. Hence, bacteria would be resistance to strong antibiotics.
CHROMOSOMAL AND RIBOSOMAL MUTATIONS
• CHROMOSOMAL MUTATION
▪ Targets DNA gyrase and topoisomerase IV
▪ Inhibits DNA synthesis
▪ Generally localized to the amino terminal domains of GyrA and ParC
• RIBOSOMAL MUTATION
▪ Amino acid changes
ENZYMATIC TARGET SITE ALTERATION AND ACQUISITION OF NEW TARGETS
• Enzymatic target site alteration DISSEMINATION OF RESISTANT DETERMINANTS
▪ Enzymes alter antibiotic targets resulting in reduced affinity and • LGT and Mechanisms of Transfer Lateral gene transfer (LGT)
effectiveness. - Also known as Horizontal Gene Transfer
• Acquisition of new targets TWO PROCESSES
▪ Microorganisms acquire new cellular targets with reduced affinity to ▪ Physical movement
antibiotic. ▪ Incorporation into genome
▪ Transfer of mobile genetic elements MECHANISMS OF TRANSFER
• Target site substitution ▪ Conjugation – involves the pili, transfer of plasmid.
▪ Acquisition of a new enzyme that is unaffected by drugs or creates a new ▪ Transformation – needs competent cells i.e. S. Pneoumiae
pathway ▪ Transduction – lysogenic bacteriophage
ANTIBIOTIC RESISTANCE PLASMIDS
EFFLUX • Conjugative and self-transmissible
▪ Fluoroquinolones • Nonconjugative, requiring mobilization by conjugative plasmids
▪ Aminoglycosides INTEGRONS
▪ Tetracyclines • Genetic elements that capture mobile gene cassettes by site-specific
▪ B-lactams recombination composed of
▪ Marcolids • A gene (int/) that encodes a specific recombinase (Intl) – would allow the genes
IMMUNITY AND BYPASS to the cassette to be inserted
▪ Tetracyclines • An adjacent primary recombination site (attl) – dictate where the cassette is
▪ Trimethoprim inserted
▪ Sulfonamides • Gene cassettes can be integrated into this site.
▪ Vancomycin INSERTION SEQUENCES
TARGET MODIFICATION • Short DNA sequences that act as simple transposable elements
▪ Fluoroquinolones • Two major characteristics: relatively small and only code for proteins implicated in
▪ Aminoglycosides the transposition activity
▪ Vancomycin NANOTECHNOLOGY TO DELIVER THERAPEUTIC AGENTS
▪ Penicillin • Nanoparticles and Components Nanoparticles
▪ Macrolids • Less than 100 nm in size Unique biological, physical, and chemical properties
INACTIVATING ENZYMES Nanomaterial can be loaded with drugs. biomolecule diagnostic tools. contrasting
▪ B-lactams agents
▪ Aminoglycosides TECHNOLOGY POTENTIAL AND DRUG DELIVERY MECHANISMS
▪ Macrolides Technology potential
▪ Rifamycins ▪ New ways to deliver drugs Improve circulation time
ENZYMATIC INACTIVATION OF ANTIBIOTICS ▪ Improve drug localization within the body Improve solubility and
pharmacokinetic profile
• Enzymes produced by the microorganisms inactivate antibiotics directly. ▪ Drug delivery mechanisms
• Destroy antibiotic ▪ Liposomes
• Modification rendering it ineffective ▪ Nanoshells + Cochleates
B LACTAMASE INHIBITORS Antimicrobial Drugs Terms
• Prevent the degradation of ß-lactam antibiotics ▪ Antibiotics
▪ Drug Fast
• Extends the range of bacteria the ß-lactam antibiotics are effective against ▪ Combination Therapy
• Important in treating gram-negative bacterial infections ▪ Synergism
• In the case of S. aureus (gram-positive pathogen), the cause of antibiotic ▪ Antagonism Broad spectrum
resistance is mostly due to variant PBPS. ▪ Narrow spectrum
• BLIs have little antimicrobial activity of their own. ORIGINS OF ANTIBIOTICS
HERBAL REMEDIES CHEMOTHERAPY OF MICROBIAL INFECTIONS IS NOT
NEW.
▪ South American Indians used the bark of the chinchona tree to extract quinine to
control malaria and mercury was known to cure syphilis in the late 1400's.
▪ In fact, many native herbal remedies are now being reexamined for potential
sources of new antimicrobial agents.
PAUL EHRLICH
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FINALS LESSON 9: MECHANISM OF ANTIMICROBIAL RESISTANCE
▪ The "dawn" of modern antimicrobial therapy began with the physician chemist Paul − Viral coded enzymes e.g., AZT interferes with the virus coded enzyme
Ehrlich (1854-1915). reverse transcriptase.
▪ He knew that arsenic could kill the syphilis organism, but it was toxic to humans. Could you target the ribosome?
He systematically tried many combinations of arsenics with organics until in the − No, as viruses use the host ribosomes.
early 1900's the effective compound #606 called Salvarson was synthesized. Could you target the membrane?
▪ He coined the term selective toxicity, where a compound could be targeted against
a microorganism but be less toxic to the host cells
− No, as viruses do not have membranes.
Could the target be the receptor site?
SULFONAMIDE
▪ The next major breakthrough did not occur until the 1930's. − Yes, could be a target but no drugs available yet.
▪ Gerhardt Domagk found that a red dye, prontosil, was effective against infections The Ideal Antimicrobial - ANTIPARASITIC ACTION
in animals despite a lack of activity in the test tube. What targets would you suggest for antiparasitic action?
▪ The product made in vivo was sulfonamide and it was not long before this drug ▪ Nucleic acid synthesis: quinolones, sulfa drugs for protozoa.
was manufactured on a wide scale. ▪ Carbohydrate metabolism: mebendazole for helminths.
▪ How can the dye prontosil act in the animal but not in the test tube? The dye ▪ Neuromuscular function: avermectins, praziquantel
prontosil was converted by the animal (in vivo) to the active agent sulfonamide. The ideal Antimicrobial - ANTIFUNGAL ACTION
▪ Sulfa drugs are now manufactured by chemical synthesis to provide the active What targets would you suggest for antifungal action?
drug. ▪ Cell membrane:
SULFA DRUGS ▪ polyene agents: e.g., amphotericin B and nystatin azoles: e.g.,
▪ "Sulfa" drugs were introduced into field hospitals during the Second World War ketoconazole, fluconazole etc.
and save countless lives from surgical wound infections. ▪ Nucleic acid synthesis: analogues 5-fluorocytosine.
SIR ALEXANDER FLEMING ACTION OF ANTIBACTERIAL DRUGS
▪ The next breakthrough came by accident. • Inhibition of cell wall synthesis
▪ In 1928, Sir Alexander Fleming was working with Staphylococcus aureus cultures • Inhibition of protein synthesis
in his London laboratory.
▪ The plate became contaminated with a mold, Penicillium.
• Injury or destruction of plasma membrane
▪ Where you or I may have discarded the plate, Fleming noted inhibition of the • Inhibition of nucleic acid synthesis
bacterial colonies near the mold colony • Inhibiting the synthesis of esential metabolites
▪ Fleming extracted a compound from the mold that he called penicillin. INHIBITION OF CELL WALL SYNTHESIS
▪ High mortality from infections during WWII provided the incentive for both British • Penicillin inhibits the synthesis of peptidoglycan thereby making the cell wall weak
and then American scientists to develop methods for large scale fermentation and
extraction of penicillin from the fungus.
• Natural penicillin: Penicillin G,V
▪ The miracle drug was introduced on a large scale during the 1940's. • Semisynthetic Penicillin: oxacillin, ampicillin, amoxicillin, Azteronam, Imipenem
NEW ANTIBIOTICS • Cephalosporins: Cephalotin, Cefixime
▪ The rush for new antibiotics was on. Thousands of cultures from sources across • Polypeptide antibiotics: Bacitracin, Vancomycin
the world were screened for antibacterial activity.
▪ Streptomycin came from the soil actinomycete Streptomyces, Lincomycin came
• Anti-TB: Isoniazid, Ethambutol
AGENTS
from an isolate in the soil in Lincoln Nebraska, and bacitracin from an isolate of
Examples of Agents
Bacillus species isolated from the wound of a girl named Tracy
REPRESENTATIVE SOURCES OF SOME ANTIBIOTICS • PENICILLINS
▪ Bacillus subtilis – bacitracin o e.g., penicillin, ampicillin, cloxacillin, piperacillin
▪ Bacillus polymyxa - polymyxin • CEPHALOSPORINS
▪ Streptomyces nodosus - Amphotericin B o 1st generation: e.g., cephalexin, cefazolin
▪ S. venezuelae - Chloramphenicol o 2nd generation: e.g., cefuroxime, cefoxitin
▪ S. aureofaciens - Tetracycline/Cotetracy o 3rd generation: e.g., ceftriaxone, ceftazidime
▪ S. fradiae neomycin –
▪ S. griseus - streptomycin • GLYCOPEPTIDES
▪ S. micromonospora - gentamicin o vancomycin
▪ Cephalosporium - cephalotin PENICILLINS
▪ Penicillium griseofulvum - griseofulvin ▪ The penicillins are derivatives of a basic structure known as aminopenicillanic acid.
▪ P. notatum - penicillin CEPHALOSPORINS
The Ideal Antimicrobial – WHAT CHARACTERISTICS SHOULD THE IDEAL DRUG ▪ The cephalosporins are closely related to the penicillins
HAVE? ▪ They also have a beta lactam ring as part of the basic structure (cephalosporanic
▪ Selective acid).
▪ Toxicity ▪ The various cephalosporins differ from each other in their side chains (R groups)
▪ Microcidal VANCOMYCIN
▪ Stable ▪ Vancomycin is not a beta lactam antibiotic. However, like the beta lactams, it
▪ Complementary to Host Defense interferes with cell wall synthesis leading to osmotically fragile organisms.
▪ Extensive Tissue Distribution ▪ It is a bactericidal antimicrobial and is effective only against Gram positive
▪ Remains Active in the Presence of Organic Compounds organisms.
▪ Vancomycin does not bind to the penicillin binding proteins (PBP) but to the D-
Alanyl-D-Alanine termini of peptidoglycan precursors. This interference with
The Ideal Antimicrobial - TARGET SITE OF ACTION elongation and cross- linking of the peptidoglycan weakens the cell wall and the
▪ The most convenient way to classify antimicrobial agents is by the target site of organisms lyse
action. INHIBITION OF PROTEIN SYNTHESIS
Nucleic acids • Chloramphenicol: binds with 50s portion of ribosomes and inhibits the formation
o If you were going to target a bacterial cell to find a selective target that works of peptide bonds
on the procaryote but not the eucaryote host, what would you select? • Erythromycin: binds to 50s, prevent translocation and movement of ribosomes
The Ideal Antimicrobial - ANTIVIRAL ACTION along mRNA
If you were to target a virus what would you select?
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CLINICAL BACTERIOLOGY|LECTURE
FINALS LESSON 9: MECHANISM OF ANTIMICROBIAL RESISTANCE
• Tetracylcine: Interfere w/ attachment of tRNA to mRNA-ribisome complex QUINOLONES
• Streptomycin: changes the shape of 30s portion, causes code of mRNA to be read • The newer quinolones proved to be substantially more potent in vitro and broader
incorrectly in antibacterial spectrum than nalidixic acid.
Protein Synthesis Inhibitors • These agents interfere with the action of bacterial gyrase, one group of enzymes
These drugs include: that helps to control the supercoiling of the DNA molecule.
▪ aminoglycosides (gentamicin, tobramycin, netilmycin, amikacin) • The bacterial chromosome must be supercoiled so that it can fit in the bacterial
▪ tetracyclines cell (remember, bacteria do not have a nucleus).
▪ chloramphenicol
▪ macrolides (e.g., erythromycin, azithromycin, clarithromycin)
• In the presence of quinolones, this supercoiling does not occur and the bacterial
cells elongate and die
Where do they act?
RIFAMPIN
Either directly on the ribosome to change its shape and prevent translation or on the
interaction between mRNA and the ribosome. • Rifampin selectively inactivates the cells DNA dependent RNA polymerase.
SPECTRUM OF ACTIVITY • By inactivating the polymerase, mRNA synthesis is inhibited.
AMINOGLYCOSIDES COMPETITIVE INHIBITORS OF THE SYNTHESIS OF ESSENTIAL METABOLITES
• These agents are bactericidal and are most effective against Gram negative • organisms can be competitively inhibited by a substance (antimetabolite)
organisms. • PABA(paraaminobenzoic acid) impt for (1) synthesis of folic acid (2) synthesis of
• Side effects can include ototoxity (ear) and nephrotoxicity (kidney). Uptake of the nucleic acid
agents may be by an active transport process • sulfanilamide can combine with PABA ▸ Trimetophrim-sulfamethoxazole
Why bactericidal? AGENTS AFFECTING ENZYMES
▪ The binding of the drug is irreversible, so that even on dilution the effect SULFONAMIDES and TRIMETHOPRIM
remains on the ribosome.
Why is uptake important?
• Sulfonamides and Trimethoprim are synthetic chemotherapeutic agents and are
not produced naturally by other microorganisms.
▪ Since the uptake is dependent of oxidative phosphorylation, the drug is not
Mechanisms or Action – SULFONAMIDES
active in anaerobes or facultative anaerobes such as Streptococcus
pneumoniae. • These agents have a chemical core that resembles para-aminobenzoic acid
TETRACYCLINE (PABA).
These agents are broad spectrum and bacteriostatic. • Sulfa drugs compete for the enzyme that converts PABA during the synthesis of
▪ They are used for a wide variety of infections including bacteria such as folic acid.
Chlamydia, Mycoplasma, Yersinia, Legionella. • If the microbe cannot synthesize folic acid, it cannot make the precursors for
Why bacteriostatic? nucleic acid synthesis which is Dihydrofloric acid.
▪ Because the binding of the drug to the ribosome is reversible. As the drug is
diluted the effect is no longer active.
• Sulfanilamide has almost the same chemical structure with para-aminobenzoic
acid (PABA).
CHLORAMPHENICOL
SULFONAMIDES
• This agent is broad spectrum and bacteriostatic. Why do sulfa drugs not compete for the synthesis of folic acid in humans?
• It has good penetration into the cerebrospinal fluid but its use has been restricted ▪ This step in folic acid synthesis is performed only by microbes.
because of a side effect, aplastic anemia following or during treatment ▪ We cannot synthesize folic acid and require it in our diet.
MACROLIDES Mechanisms of Action – TRIMETHOPRIM
• The initial macrolide, erythromycin, has been recently modified to what are now • This synthetic agent also acts on the enzyme system involved in nucleic acid
referred to as the extended spectrum macrolides (e.g., azithromycin / synthesis
clarithromycin) • Trimethoprim prevent the conversion of Dihydrofolic acid to tetrahydrofolic acid
• These macrolides have: longer half lives and therefore require less frequent by inhibiting the Dihydrofolic reductase
dosing TRIMETHOPRIM
• less side effects (reduced gastrointestinal disturbances) Why doesn't Trimethoprim interfere with mammalian dihydrofolate reductase and
DESTRUCTION OF PLASMA MEMBRANE impair our nucleic acid synthesis?
▪ Our DHFA enzyme does not recognize Trimethoprim as a potential substrate
• polypeptide antibiotics bring about changes in the permeability of the plasma
and thus does not interfere with THFA synthesis.
membrane.
Test to Guide Chemotherapy
• loss of important metabolites from the microbial cell 1. Diffusion methods
• Polymxyin B a. Kirby Bauer
INHIBITION OF NUCLEIC ACID SYNTHESIS b. Agar Diffusion
• interfere with the process of DNA replication and transcription c. E Test
2. Broth Dilution test -MIC / MBC
• Rifampin: inhibits synthesis of mRNA 3. Automated Method
Quinolones: inhibit DNA synthesis
- Nalidixic acid
TERMINOLOGY
• Nofloxacin – Ciprofloxacin
Susceptible (Sensitive)
MECHANISMS OF ACTION
FLUOROQUINOLONES • The term susceptible is used to refer to organisms susceptible to concentrations
of antibiotic that are achievable in the blood of patients with normal dosing
• A number of antimicrobial agents which specifically interfere with the structure and schedules.
function of DNA have been developed.
• The diameter to be considered susceptible is called breakpoints. Clinical
• However, few have shown selective toxicity for bacterial DNA while not affecting Laboratory Standards Institute revise the breakpoint each year.
eucaryotic (host) DNA.
• The breakpoint to be considered as susceptible is increasing meaning many
• In the late 1980's a group of compounds known as the fluoroquinolones came onto bacteria had become resistant to antibiotic.
the market.
Resistant
• These agents are analogues of the earlier developed nalidixic acid which had been • The term resistant is used to refer to organisms that have an MIC higher than the
used for several decades in the limited treatment of urinary tract infections.
level of drug that is achievable in the blood of patients with a normal dosing regime.
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FINALS LESSON 9: MECHANISM OF ANTIMICROBIAL RESISTANCE
Intermediate
• The term intermediate refers to organisms that have a MIC to a drug that is
borderline between sensitive and resistant.
When would this be a useful drug?
▪ A drug that has an intermediate susceptibility to an organism may be useful
if high drug levels can be delivered to a body site either by safely increasing
the dose, or, if the drug is concentrated in particular organs. If the drug is
concentrated in the kidneys and excreted in the urine, the drug may be useful
in the treatment of urinary tract infection
DISK DIFFUSION TESTING
Disk Diffusion Testing - KB Testing
• This procedure was introduced by Drs. Kirby and Bauer in the 1960's and is
referred to as KB testing.
• Paper disks are impregnated with a given concentration of antibiotic and placed
on a lawn of bacteria.
• The drug diffuses out from the disk. If the drug inhibits the growth of the organism,
a zone of inhibition is seen.
Factors affecting Kirby Bauer Technique
▪ diffusibility of the antibiotics – the antibiotic must be able to diffuse from the
paper disk
▪ thickness of the agar – only using 20ml agar
▪ amount of inoculum
▪ growth rate of the microorganisms
▪ suitability of the medium used – for fastigious organ exams blood agar plates
are used
▪ discrepancy in inoculum and incubation
▪ pH of the medium
Disk Diffusion Testing - Standards
• The width of the zone is measured and compared to standards to determine
whether the organism is sensitive or resistant to the drug.
• For example, with ampicillin, a zone size of < 13mm indicates resistance when
testing Gram negative bacilli and a zone size of > 17 indicates a susceptible strain
• Between 13 and 17 it is called intermediate.
• Breakpoint to be considered as susceptible is more than 17. The breakpoint of
being resistant is less than 13 zone of infection.
• To check for the lowest concentration of the drug that kills all the inoculum, aliquots
are taken from the clear tubes and plated on media without the antimicrobial to
determine the lowest concentration of antimicrobial that kills all the organisms
BROTH DILUTION MICROBROTH DILUTION
• There are a number of commercial companies that use small volumes of broth
(microbroth dilution) with standardized antimicrobial concentrations. Due to the
amount of labor required to set up the traditional tube MIC's, commercial
companies have developed microbroth MIC systems. Both automated and semi-
automated instruments are available to read and record growth patterns.
Minimal Bacteriocidal Concentration (MBC)
• Bactericidal antimicrobials have a MBC no more than 2-4 times the MIC while
bacteriostatic antibiotics have MBC's much higher than the MIC (if at all).
Why?
▪ With bactericidal drugs the effect is irreversible and diluting out the
antibiotic does not reduce drug activity. Remember the
aminoglycosides?
AUTOMATED SUSCEPTIBILITY TESTING
• The MIC method of antimicrobial susceptibility performed in test tubes is far too
cumbersome for large scale testing.
• The method can be performed in a 'micro' method where the process has been
adopted to microtiter plates or small plastic cards which contain antibiotics in the
appropriate dilution. Bacteria are added and then the cards/ trays can be read by
an instrument.
• This method of susceptibility testing is widely used in diagnostic microbiology
laboratories.

• This corresponds to a MIC of 8ug/ml. The antibiotic disks, inoculum, medium and
incubation are rigidly controlled to ensure reproducible and accurate results.
E Test (Gradient Diffusion Susceptibility Testing
• uses a strip
• Exact MIC can be determined
• Less time consuming than broth dilution
• More Expensive
Broth Dilution Procedure
• For this test, a series of tubes containing dilutions of an antimicrobial are
inoculated with a given concentration of bacteria (fungus, parasite) and incubated
overnight The tube that has the lowest concentration of drug that remains clear
(no growth) determines the minimum inhibitory concentration for that drug.
• This result is compared to standards to determine whether this concentration
indicates that the organism is susceptible or resistant.
• For example, a MIC of < 8ug/ml would be called sensitive for Gram negative bacilli
against ampicillin while 32 ug/ml would be considered resistant for that bug/drug
combination.
TERMINOLOGY
Minimal Inhibitory Concentration (MIC)
• To determine the susceptibility of an organism, the antimicrobial is diluted out and
a given inoculum of bacteria is added.
• The lowest concentration of antimicrobial that is capable of preventing growth is
the minimal inhibitory concentration.
Minimal Bacteriocidal Concentration (MBC)
• This term is used to denote the concentration of antimicrobial that kills all the
bacteria. For example, organisms in the clear tubes may be inhibited at that
concentration but still viable when the antimicrobial is removed.

BSMLS | MMLS 3-5 (2023) | 47