CLINICAL BACTERIOLOGY BSMLS-3D

Prof. Keith Ivan Jemuel Luis, RMT

Topic Outline
• His results were a serious blow to the long-held
I. Historical Development belief that large forms of life could arise from
II. Microbiology Taxonomy nonlife.
III. Structure of Bacterial Cells
IV. Bacterial Growth and Genetics Conclusion:
V. Medical Bacterial Implications
VI. Normal Flora • Fly eggs and maggots (life) do not spontaneously
generate from decaying meat (non-living matter).
• Maggots could only form when flies were allowed
to lay eggs in the meat, and that the maggots were
HISTORICAL DEVELOPMENT the offspring of flies, not the product of
ROBERT HOOKE spontaneous generation.

JOHN NEEDHAM (1748)

• Coined the term Cell Theory
→ All living things are composed of cells • Concept:
• A renaissance man in the 17th century → Had poor experimental setup and incorrectly
• Volume of documenting experiments believed that briefly boiling a broth would
(mycophagy) which he made by the use of sterilize it.
microscope in the year 1665 → He observed that boiled mutton broth
• Coined the term Cell while discussing a cork w/ eventually became cloudy with
the use of microscope which is made by microorganisms after pouring it into a flask
Cristopher Cock and sealed tightly.
→ He proposed that organic matter possessed
ANTON VAN LEEUWENHOOK
a “vital force” that could give rise to life.
• Animalcules- microorganisms
Sterilization– process of killing all microbes in sample.
• Spontaneous generation (Abiogenesis) w/ John
Needham • Results:
→ A living thing arises from a non-living thing. → after briefly boiling a flask of broth, allowing it
→ If a person has a disease, it is a punishment to cool and sealing it with a cork, Needham
from the Gods. saw microbial growth.
FRANCISCO REDI
• Conclusion:
• Concept: → Needham incorrectly concluded that his
→ Italian scientist that became the first to “sterile” flask of broth spontaneously
disprove the idea of spontaneous generated microbes.
generation.
LAZZARO SPALLANZI
→ In 1668, he demonstrated that fly eggs and
maggots do not arise spontaneously from • Concept:
decaying meat → Italian physiologist who performed
experiments that contradicted Needham’s
• First piece of meat was left opened to allow to flies results.
to enter and lay their eggs on the meat. → He proposed that Needham’s experiment was
• Second piece of meat was covered with fine gauze flawed and that organisms do not
to prevent flies from entering and laying their eggs. spontaneously generate.
• Third piece of meat was covered with cork-sealed → Spallanzani sealed flask by melting it closed
container to prevent flies from entering and laying
and then boiled broth for a longer period of
their eggs.
time.

He boiled broth in two bottles, left one bottle open and

one closed,

Results: the microorganisms entered the bottle through the air;

they were not generated spontaneously in the broth
• Fly eggs and maggots appeared on meat in
uncovered jar but did not appear on meat in the • Results:
covered jar. → Spallanzani never saw microbial growth in
sealed flasks; only saw growth in flasks that
were cracked.
 CLINICAL BACTERIOLOGY
Prof. Keith Ivan Jemuel Luis, RMT
• Conclusion:
→ microbes do not spontaneously generate;
cracked flasks allowed microbes to enter form
the air.
→ Opposers claimed Spallanzani’s sealed flasks
excluded a “vital source” that was needed for
spontaneous generation
RUDOLF VIRCHOW
• First person to challenge spontaneous
generation
• Proposed Theory: Biogenesis
• “Father of Modern Pathology.”
• German Physician
LOUIS PASTEUR
• Father of Immunology
• It is not the air that creates microbes but instead
there are real microbes
ROBERT KOCH
• KOCH’S POSTULATE
→ The microorganisms must be present in
every case of disease but absent in a
healthy host
→ Suspected microorganisms is isolated
from a diseased host & grown in pure
culture
→ Grown microorganism in a culture
medium, when inoculated in a healthy
individual, same disease must be present
from the first host
→ Same organism must be isolated again
from the diseased host
• He was the first to culture bacteria on boiled
potatoes, gelatin and used meat extracts and
protein digests for cultivation
• He developed culture media for observing growth
of bacteria isolated from the human body.
• Pathogenesis- A specific disease is caused by a
specific organism.
BSMLS-3D
MICROBIOLOGY TAXONOMY
• Greek: Taxis (Arrangement) Nomos (Law)
• Carolus Linnaeus
• Taxonomy is the area of biologic science
comprising three distinct (but highly interrelated)
disciplines IDENTIFICATION,
CLASSIFICATION, and NOMENCLATURE,
• Taxonomy provides a consistent means to
classify, name, and identify organisms.
• Importance is not only realized in phylogeny but
also in virtually every other discipline of biology.
• In diagnostic microbiology: classification,
nomenclature, and identification of
microorganisms play a central role in providing
accurate and timely diagnosis of infectious
diseases.
CLASSIFICATION
• is the organization of microorganisms that share
similar morphologic, physiologic, and genetic
traits into specific groups, or taxa.
→ Species
→ Genus (composed of similar species)
→ Family (composed of similar genera)
→ Order (composed of similar families)
→ Class (composed of similar orders)
→ Division (composed of similar classes)
→ Kingdom (composed of similar divisions)
NOMENCLATURE
• is the naming of microorganisms according to
established rules and guidelines.
• provides the accepted labels by which
organisms are universally recognized.
• the discussion of rules governing microbial
nomenclature is limited to these two taxon
designations.
 CLINICAL BACTERIOLOGY BSMLS-3D
Prof. Keith Ivan Jemuel Luis, RMT

• binomial system of nomenclature every

organism is assigned a genus and species name
of Latin or Greek derivation.
• every organism has a scientific "label" consisting
of two parts:
→ the genus designation, which is always
capitalized
→ the species designation, which is never
capitalized.
• Both components are always used simultaneously
and are printed in italics, or underlined in script.
WHAT IS MICROBIOLOGY?
IDENTIFICATION
• Study of microorganisms/microbes
• is the process by which a microorganism's key • Includes the study of nonliving entities as well
features are delineated. as certain living organisms collectively called
• a wide variety of methods and criteria are used to microbes that cause infectious diseases.
establish a microorganism's identity. → Bacteria - Prokaryote
• genotypic characteristics and phenotypic → Fungi (yeast and molds) - Protist
characteristics. → Protozoa-Protist
Role of Taxonomy in Diagnostic Microbiology → Helminths-Animal kingdom
• Establishes and maintains records of key → Viruses-non cellular or cellular
characteristics of clinically relevant → Protozoa and Helminths – parasites
microorganisms
• Facilitates communication among IMPORTANT FEATURES OF MICROBES
technologists, microbiologists, physicians, and
scientists by assigning universal names to
clinically relevant microorganisms. This is
essential for:
• Establishing an association of particular
diseases or syndromes with specific
microorganisms
• Epidemiology and tracking outbreaks
• Accumulating knowledge regarding the
management and outcome of diseases CHARACTERISTICS OF PROKARYOTIC AND
associated with specific microorganisms EUKARYOTIC CELLS
• Establishing patterns of resistance to
antimicrobial agents and recognition of
changing microbial resistance patterns
• Understanding the mechanisms of
antimicrobial resistance and detecting new
resistance mechanisms exhibited by
microorganisms
• Recognizing new and emerging pathogenic
microorganisms
• Recognizing changes in the types of
infections or diseases caused by
characteristic microorganisms
• Revising and updating available CELL MEMBRANE VS. CELL WALL
technologies for the development of new Cell Membrane Cell Wall
methods to optimize the detection and
Cell membrane is found Cell wall is absent in
identification of infectious agents and the
in all living cells animal cells
detection of resistance to antiinfective agents
A thin delicate structure. A thick rigid structure 20
(microbial, viral, fungal, and parasitic)
5-10 nm wide um wide
• Developing new antiinfective therapies
Can be observed under Can be observed under
(microbial, viral, fungal, and parasitic)
electron microscope the light microscope
Outermost layer of Outermost layer of
animal cells bacteria, archaea, fungi,
and plant cells
 CLINICAL BACTERIOLOGY BSMLS-3D
Prof. Keith Ivan Jemuel Luis, RMT

Functions as protective Functions as a protective

covering to the covering to the cell
protoplasm and membrane and
maintains a constant maintains the shape of
environment in the cell
protoplasm
Provides a round flexible Provides a fixed shape to
shape to the cell the cell
Made up of lipids, Made up of
proteins, and peptidoglycan in
carbohydrates bacteria, chitin in fungi
and cellulose in plants
Selective permeable Completely permeable
Alive and metabolically Non-living and • The shape of the bacterium will determine how
active metabolically inactive rigid their cell wall is.
• 0.2 or 0.5 under a microscope
• Smallest bacteria- Mycoplasma
EUBACTERIA VS. ARCHAEBACTERIA (KINDOM
MONERA) STRUCTURE OF BACTERIAL WALL

ARCHAEBACTERIA

- Single celled that lives in an extreme

environment. (Thermophiles, Halophiles,
Methanogens)
- They evolved as the first life in the world.
- They have a role in our carbon and nitrogen
cycle.
- They are not pathogenic.

Mycoplasma do not have a cell wall rather they have a

cell membrane.

STRUCTURE OF BACTERIAL CELLS

SHAPE AND SIZE OF BACTERIA

Gram negative have somatic antigens.

Weakly acid-fast bacteria- Nocardia asteroides

• They have weaker solution affinity to

hydrochloric acid.
 CLINICAL BACTERIOLOGY
Prof. Keith Ivan Jemuel Luis, RMT
Capsule- murein layer of gram-positive and outer
membrane of Gram negative.
- They protect the bacteria so that it will not be
easily killed by our immune system.
Fimbre
- Enables the bacteria to properly attach itself
to the cell membrane.
Pilus
- Hairlike structure that extends in the external
environment of the bacteria.
Flagellum
- Complex structure which contains a flagellin
that are intricately connected to each other.
Teichoic acid can have glycerol teichoic acid.
Glycerol teichoic acid
• This helps in the penetration to the
peptidoglycan layers and is covalently link to
the cytoplasmic membrane
Teichoic acid medical importance
• Has the ability to reduce or lower the
inflammation or septic shock that is happening
to a person that has this.
Gram Stain
• Hans Christian Gram (1884)
• Gram stain is the most important staining
procedure.
• Organic solvent that is used in Gram staining-
Alcohol or Acetone.
• Gram- positive bacteria stain purple, whereas
gram-negative bacteria stain pink. This difference
is due to the ability of gram-positive bacteria to
retain the crystal violet-iodine complex in the
presence of a lipid solvent, usually acetone-
alcohol.
BSMLS-3D
• Gram-negative bacteria, because they have an
outer lipid-containing mem- brane and thin
peptidoglycan, lose the purple dye when treated
with acetone-alcohol. They become colorless and
then stain pink when exposed to a red dye such as
safranin.
• Not all bacteria can be visualized using Gram
stain. Some Important human pathogens, such as
the bacteria that cause tuberculosis and syphilis,
cannot be seen using this stain.
2 IMPORTANT USES OF GRAM STAIN
1. To identify the bacterias (First line)
2. To choose what antibiotic to give.
Gram positive
- More susceptible to penicillin.
ALL COCCI ARE GRAM POSITIVE EXCEPT
Neisseria
Moraxella
Veilonella
ALL BACILLI ARE GRAM NEGATIVE EXCEPT
Corynebacterium
Mycobacterium
Bacillus
Clostridium
Lactobacillus
Listeria
Erysipilothrix
Bifidobacterium
Nocardia
Streptomyces
Actinomyces
CYTOPLASMIC MEMBRANE
• Chemically similar but Eukaryotes contain
STEROLS, Prokaryotes do not except for
Mycoplasma
Functions:
→ Active transport of molecules into the cell
→ Energy generation by oxidative phosphorylation
→ Synthesis of precursors of the cell wall
→ Secretion of enzymes an toxins
 CLINICAL BACTERIOLOGY BSMLS-3D
Prof. Keith Ivan Jemuel Luis, RMT

• Cytoplasm contains
→ matrix - ribosomes, nutrient granules,
metabolites and plasmids
→ Nucleoid region-composed of DNA

MESOSOME - Invaginations of cytoplasmic membrane

→ Results in Cell division

RIBOSOMES - site of protein synthesis in eukaryotic

cells

→ Bacterial ribosomes are 70s in size, 50s and

30s subunits
→ Eukaryotic ribosomes are 80s in size, 60s
and 40s subunits

The difference of ribosomal RNA and Proteins is that it

constitutes generative actions which inhibits the
bacteria which is not done by the human protein system.

GRANULES -Storage areas for nutrients and stain

characteristically with certain dyes

Ex: Volutin granules- reserve energy source or stored

energy in the human body in the form of polymerized
metaphosphate in which it appears as metachromatic
granules is it is stained with methylene blue dye.

NUCLEOID- area where DNA is located

Prokaryotes

→ DNA is single circular molecule; no nuclear

membrane, no mitotic apparatus, no histones

PLASMIDS extrachromosomal, double stranded,

circular DNA molecules capable of self-replicating
independently of the bacterial chromosome

Can be seen in both Gram + and Gram – Bacterias

Transmissible plasmids- it can be transmitted from

one cell to another.

Non-transmissible plasmids- it cannot be transmitted

from one cell to another.

Carry the genes for the following functions:

BACTERIAL GROWTH
→ Antibiotic resistance
→ Resistance to heavy metals (mercury, silver) BACTERIAL CYCLE OF GROWTH
→ Resistance to ultraviolet light (mediated by DNA 1. BINARRY FISSION
repair enzymes) • One parent cell divides to form 2 progeny
→ Pili (fimbriae) mediate the adherence of cells
bacteria to epithelial cells • One cell gives rise to 2 progeny cells,
→ Exotoxins bacteria undergo exponential (logarithmic
growth)
 CLINICAL BACTERIOLOGY BSMLS-3D
Prof. Keith Ivan Jemuel Luis, RMT

2. MITOSIS (Eukaryotes) Classification of Bacteria according to Oxygen

PHASES OF BACTERIAL GROWTH CYCLE Requirement

Obligate Aerobes - require oxygen to grow (M.

tuberculosis)

Facultative Aerobes - oxygen is utilized if present but

can use the fermentation pathway to synthesize ATP in
the absence of sufficient oxygen (E. coli)

Obligate Anaerobes - cannot grow in the presence of

REQUIREMENTS OF BACTERIAL GROWTH oxygen because they lack either superoxide dismutase,
catalase or both (Clostridium tetani)
PHYSICAL GROWTH FACTORS

• Temperature - mesophilic
→ Psychophiles (0-10C)
→ Mesophiles (20-40C) - most human pathogens
→ Thermophiles (50-60C)
• pH-6.5-7.5 (Neutrophiles)

CHEMICAL GROWTH FACTORS

• Carbon
• Nitrogen, Sulfur, Phosphorus, Amino acids, trace
elements
• Oxygen

CULTURE MEDIA FOR MICROBES

Obligate intracellular pathogens (viruses, rickettsias,

chlamydias, etc.): Charcoal Yeast Extract Agar
Pasteurella
• Tissue cultures (cell cultures), eggs, animals, or not Legionella
at all Prevotella
Francisella
Facultative intracellular or extracellular organisms:
Thayer Martin
• Inert lab media (broths and agars) Neisseria

Selective medium (S): A medium that selects for

certain bacteria by inclusion of special nutrients and/or IRON
antibiotics.
• Ferric ion (Iron), an essential component of
Differential medium (D): A medium on which different cytochrome and other enzymes
bacteria can be distinguished by differences in colonial • Required for bacterial growth
morphology or color • Bacteria produce SIDEROPHORES
• Iron-binding compounds (Chelation)
AEROBIC AND ANAEROBIC GROWTH
• Bacteria that are in need of Iron- E. coli
• An adequate supply of OXYGEN enhances • E. coli- produces siderophores (enterobactin) and
metabolism and growth is excreted and will chelate Iron for it to be its
→ Acts as Hydrogen acceptor in the final steps specific receptor of its bacterial surface to which
of energy production Iron will be used in other components.
• Use of Oxygen generates 2 toxic molecules
→ Hydrogen Peroxide (H2O2)
→ Free Radical Superoxide (02)
• Bacteria requires two enzymes to utilize Oxygen
→ Superoxide Dismutase
→ Catalase
 CLINICAL BACTERIOLOGY BSMLS-3D
Prof. Keith Ivan Jemuel Luis, RMT

→ Chemicals act in several different ways:

o Nitrous acid and alkylating agents- they
will alter the existing bases that will form a
hydrogen bond.
o 5-bromouracil- it copies bases; less
hydrogen binding
o Benzpyrene- can be seen in tobacco
smokers. DNA will have frameshift
mutation.
→ Xrays and UV lights- lower energy leading to
cross-linking of Thymine to someone Thymine
is not suppose to bind.
→ Bacterial Mu (mutator bacteriophage)

WITHIN BACTERIAL CELL

1. Movement of Transposons
- Small pieces of DNA that move to different
sites cause it to be heat-sensitized or
resistant to an antibiotic.
BACTERIAL GENETICS

• BACTERIA are haploid 2. Programmed Rearrangements

→ They have only ONE copy of their genome DNA - It is programmed to be rearranged. Since
→ DNA is circular there is rearrangement, there will be a new
protein or new antigen for the bacteria.
→ Any gene that has mutated and is not
expressed results in a cell that has lost its trait
• EUKARYOTES are diploid BETWEEN BACTERIAL CELLS
→ Have TWO copies of their genome DNA (can be
dominant or recessive) • Conjugation
→ DNA is linear -
Direct transfer from one bacteria.
→ One copy of a gene (allele) may be expressed • Transduction
(dominant) while another may not be expressed - It is transferred from one bacteria to another
(recessive) viral bacteria. Indirectly.
• Transformation
BACTERIAL GENETIC MUTATIONS - It is transformed. From a naked DNA to
another dying naked DNA or purified DNA.
• a change in the base sequence of DNA that
usually results in insertion of a different
→ amino acid into a protein and the appearance
of an altered phenotype
• Results from 3 types of molecular changes
→ Base substitution one base is inserted in
place of another
o MISSENSE MUTATION results in a codon
that causes a different amino acid to be
inserted
o NONSENSE MUTATION results in a
termination codon that stops protein
synthesis prematurely
→ Frameshift mutation one or more base pairs
are added or deleted
o Shifts the reading frame on the ribosome
resulting in incorporation of wrong amino
acid producing inactive protein
→ Transposon or insertion sequences are
integrated into the DNA
• Mutations can be caused by chemicals,
radiation, or viruses.
 CLINICAL BACTERIOLOGY BSMLS-3D
Prof. Keith Ivan Jemuel Luis, RMT

NORMAL FLORA - There is reactivation; recurrence of

symptoms
7. Chronic carrier stage
- Continued growth; Continued symptoms
- Ex. Typhoid Mary
8. Normal flora
- Normally resides to a certain part of the body.
9. Colonization
- Presence of new organism. Its either normal
flora or pathogenic.

PATHOGEN- microorganism capable of causing

disease

OPPORTUNISTIC PATHOGEN- those that rarely

cause disease in immunocompetent people but can
cause serious infection in immunocompromised
patients

VIRULENCE- A quantitative measure of pathogenicity

and is measured by the number of organisms required
to cause disease

• 50% Lethal Dose (LD50) -number of

organisms needed to kill half the hosts
• 50% Infectious Dose (ID50) - number needed
to cause infection in half the hosts
• ORGANISMS WITH LOWER LD50 are more
virulent because fewer organisms are needed to
cause death or disease

PARASITE- refers to the parasitic relationship of the

bacteria to the host cells (i.e., the presence of the
bacteria is detrimental to the host cells).

obligate intracellular parasites (e.g., Chlamydia and

Rickettsia can grow only within host cells

facultative parasites can grow within cells, outside

cells, or on bacteriologic media

PATHOGENECITY

DEFINITION OF TERMS

1. Communicable
- Spreads from host to host
- Can be contagious
2. Epidemic FACTORS AFFECTING PATHOGENESIS
- Most frequently appears than usual
3. Pandemic • Organism/Agent
- Worldwide - The greater the number of organism the
4. Endemic greater chance that we can acquire an
- Specific population infection.
5. Inapparent or subclinical • Host
- Isolating microorganism form the host - Innate, Adaptive, or Acquired
6. Latent Stage
 CLINICAL BACTERIOLOGY BSMLS-3D
Prof. Keith Ivan Jemuel Luis, RMT

BACTERIAL INFECTIONS

• An organism has infected the person (the

presence of microbes in the body).
• Infection does not have to be equated with disease
• Two mechanisms
→ Toxin productions
→ Invasion and inflammation

STAGES OF BACTERIAL PATHOGENESIS

1. Transmission from an external source into the

portal of entry.
2. Evasion of primary host defenses such as skin
or stomach acid.
3. Adherence to mucous membranes, usually by
bacterial pili.
4. Colonization by growth of the bacteria at the
site of adherence.
5. Disease symptoms caused by toxin production
or invasion accompanied by inflammation
6. Host responses, both nonspecific and specific
(immunity)
7. Progression or resolution of the disease.

DETERMINANTS OF BACTERIAL PATHOGENESIS

• Transmission
• Adherence to Cell Surface – because of pili and
appendages
• Invasion, Inflammation and Intracellular Survival-
because of the capsule
Some Nasty Killers Have Some Capsular
Protection
1. Streptococcus pneumoniae/ S. Agalactiae 2.
Neisseria meningitidis
3. Klebsiella pneumoniae
4. Haemophilus influenzae Type B
5. Salmonella typhi
6. Cryptococcus neoformans (FUNGI)
7. Pseudomonas aeruginosa (PAE)
8. ** Some strains of Escherichia coli (K1 antigen)
• Toxin Production
• Immunopathogenesis
CLINICAL BACTERIOLOGY BSMLS-3D
Prof. Keith Ivan Jemuel Luis, RMT
 CLINICAL BACTERIOLOGY BSMLS-3D
Prof. Keith Ivan Jemuel Luis, RMT

HOST DEFENSES The recovery period is the time during which

symptoms resolve and health is restored.
• Innate (Nonspecific) Defenses → Protect against
microorganisms in general, nonspecific After the recovery period, some people become
→ Physical barriers - intact skin and mucous chronic carriers of the organism and in others latent
membranes infections develop
→ Phagocytic cells - neutrophils, macrophages,
Some people have subclinical infections during which
natural killer ells
they remain asymptomatic. The presence of antibodies
→ Proteins-complement, lysozyme and
reveals that a prior infection has occurred
interferon
→ Inflammation- redness, swelling, warmth and TYPICAL STAGES OF AN INFECTIOUS DISEASE
pain
o Redness, swelling, warmth - result of • The incubation period is the time between the
increased blood flow and vascular moment the person is exposed to the microbe (or
permeability caused by mediators such as toxin) and the appearance of symptoms.
Histamine, Prostaglandin and Leukotrienes • The prodrome period is the time during which
o Bradykinin mediates pain; Complement (C3a, nonspecific symptoms occur.
C5a) increase vascular permeability • The specific-iliness period is the time during
o Predominant phagocytic cells of inflammation which the characteristic features of the disease
are Neutrophils (pyogenic infections like occur.
Staph) and Macrophages (granulomatous • The recovery period is the time during which
inflammatory response Mycobacterium symptoms resolve and health is restored.
tuberculosis) • After the recovery period, some people become
o Severe, recurrent pyogenic infections occur if chronic carriers of the organism and in others
neutrophils are inadequate (<500/ ul) latent infections develop
• Some people have subclinical infections during
ACQUIRED IMMUNITY which they remain asymptomatic.
a. Passive Immunity - protection based on the • The presence of antibodies reveals that a prior
transfer of preformed antibody from one person infection has occurred.
to another person
• Provides immediate but short-lived
protection (few months)
• i.e., transplacental passage of IgG from
mother to fetus; IgA from mother to
newborn through breastmilk
b. Active Immunity - protection based on the
formation of both antibodies and cell- mediated
immunity after exposure to the microbe (with or
without disease) or through vaccination
• Provides long term protection not
immediately effective for days after
exposure to the microbe
• Primary response-antibody appears in 7-
10days
• Secondary response - appears in
approximately 3 days (anamnestic
response)

TYPICAL STAGES OF AN INFECTIOUS DISEASE

The Incubation period is the time between the moment

the person is exposed to the microbe (or toxin) and the
appearance of symptoms...

The prodrome period is the time during which

nonspecific symptoms occur.

The specific-iliness period is the time during which the

characteristic features of the disease occur.