TOPIC 1: MICROBIAL TAXONOMY, PHYSIOLOGY, METABOLISM AND GENETICS Species Example of Taxonomic ranks:

Taxonomy –the vocabulary of Medical Microbiology - the most basic taxonomic group defined as a collection of bacterial strains that Formal rank example
What is Taxonomy? share many common physiologic and genetic features and as a group differ Kingdom Prokaryotae
- It comes from a Greek word, ‘Taxon’ meaning arrangements. notable from other bacterial species. Division Gracilicutes
- It is defined as the classification of organisms in an ordered system that indicates Subspecies- taxonomic subgroups within a species. Class Scotobacteria
a natural relationship. biotype, serotype, or genotype are given to groups below the subspecies levels
- Order Eubacteriales
- Allows all biologists to use a common label for every organism they study within that share specific but relatively minor, characteristics. These subgroups are still Family Enterobacteriaceae
their particular disciplines. taxonomically important but their use in diagnostic microbiology is limited. Genus Escherichia
- In diagnostic microbiology, classification, nomenclature, and identification of Genus – is the next higher taxon and comprises different species that have several Species coli
microorganisms play a central role in providing an accurate and timely important features in common but differ sufficiently to still maintain their status as Subtype Escherichia coli O157: H7
diagnosis of infectious diseases. individual species.
Role of Taxonomy: NOMENCLATURE – refers to the naming of an organism by international rules IDENTIFICATION – it is the process in which a microorganism’s key features are
Clinically, taxonomy facilitates communication among technologists, physicians, (established by a recognized group of medical professionals) according to its delineated. Once those features are established the profile is compared with those
microbiologists and scientists by assigning universally useful names to clinically relevant characteristics. Genus and species are the groups of most concern to microbiologists. other previously characterized microorganisms so that the organism in question can
microorganisms. This is essential for: The discussion of rules governing microbial nomenclature is limited to these two taxons be classified within the most appropriate taxa (classification) and can be assigned an
● Recognizing new and emerging pathogenic microorganisms. designations. appropriate genus and species (nomenclature).
● Recognizing the changes in the types of infections or diseases caused by familiar - In this Binomial (two-name) system of nomenclature: It is practical use of a classification scheme to:
microorganisms ● Every organism is assigned a genus or species name of Latin or Greek derivation 1. isolate and distinguish desirable organisms from undesirable ones
● Understanding the mechanism of antimicrobial resistance and detecting new ● The scientific label consists of two parts: 2. verify the authenticity or special properties of a culture in a clinical setting, and
resistance mechanisms exhibited by a particular microorganism. a. The Genus designation – which is always capitalized 3. isolate and identify the causative agent of a disease.
● There are three areas that make up taxonomy in relation to diagnostic b. the species designation – which is never capitalized Identification Methods:
microbiology: ● Both components should be in Italics or underlined in script. For example: - A wide variety of methods and criteria are used to establish a microorganism’s
a. Classification Streptococcus pneumoniae identity. These methods can be separated into two categories:
b. Nomenclature Notes: A. Genotypic Characteristics –relate to the organism’s genetic makeup, including the
c. Identification 1. The name may be abbreviated using the upper-case form of the first letter of nature of the organism’s genes and constituent nucleic acids.
CLASSIFICATION – is the organization of microorganisms that share similar the genus designation followed by a period (.) and the full species name which B. Phenotypic characteristics are based on the features beyond the genetic level and
morphologic, physiologic and genetic traits into specific groups or taxa. is NEVER abbreviated. For example: Streptococcus pneumoniae = S. include readily observable characteristics and those characteristics that may require
Classification Hierarchy: pneumonia extensive analytic procedures to be detected.
Kingdom 2. Frequently an informal designation for example: streptococci, staphylococci;
Division may be used to label a particular group of organisms but such designations Identification Criteria and Characteristics for Microbial Classification:
Class are not capitalized or italicized. Phenotypic Criteria Examples:
Order ● As more information is gained regarding organism classification and Macroscopic Characteristics of microbial growth patterns on artificial media as
Family identification, a particular species may be moved to a different genus or Morphology observed when inspected with the unaided eye. Examples: size,
Genus assigned to a new genus. texture and pigmentation of bacterial colonies
Species ● The such changes are documented in the International Journal for Systematic
Microscopic Size, shape and intracellular inclusions, cellular appendages, and
Bacteriology.
1. Kingdom-comprised of similar divisions Morphology arrangement of cells when observed with the aid of the
● In diagnostic laboratory, changes in the nomenclature are phased in gradually microscope magnification.
2. Division- composed of similar classes
so that physicians and laboratorians have ample opportunity to recognize the
3. Class- composed of similar order Staining Ability of the organism to reproducibly stain with a particular color
changes in the naming.
4. Order- composed of similar families Characteristics with the application of specific dyes and reagents. Staining is
- This is usually accomplished by using the new genus designation while
5. Family-composed of similar genera usually used in conjunction with microscopic morphology.
continuing to provide the previous designation in parenthesis:
6. Genus- composed of similar species Environmental Ability of the organism to grow at various temperature, in the
Stenotrophomonas (Xanthomonas) maltophilia
7. Species requirements presence of oxygen and other gases , at various pH levels, or in
- Classification of bacteria requires experimental and observational techniques; the presence of ions and salts.
this is because biochemical, physiologic, genetic, and morphologic properties Nutritional Ability of the organism to utilize various carbon and nitrogen
are often necessary for an adequate description of a taxon. requirements sources as nutritional substrates when grown under specific
environmental conditions.
Resistance Exhibition of a characteristic inherent resistance to specific
Profiles antibiotics, heavy metals, or toxins by certain microorganisms
 Antigenic Establishment of profiles of microorganisms by various serologic
Properties and immunologic methods that are useful for determining the
relatedness among various microbial groups
Subcellular Establishment of the molecular constituents of the cell that are
properties 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 a microbial cell.
Genotypic Criteria Examples
DNA base DNA comprises four bases (guanine, cytosine, adenine,
composition ratio thymine). The extent to which DNA from two organisms is
made up of cytosine and guanine relative to their total
base content can be used as indicator of relatedness, or
lack thereof
Nucleic acid The order of the bases along a strand of DNA or RNA are
(DNA and RNA known as the base sequence and the extent to which the
base sequence sequences are similar (homologous) between two
analysis 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.
BACTERIAL GENETICS, METABOLISM AND STRUCTURE:
Knowledge regarding genetic, metabolic, and structural characteristics of
microorganisms provides the basis for understanding almost every aspect of
diagnostic microbiology, including:
● The mechanism by which microorganisms cause disease
● Developing, implementing optimum techniques for microbial detection,
cultivation, identification, and characterization
● Understanding microbial action and resistance
● Developing and implementing tests for antimicrobial resistance detection
● Designing strategies for disease therapy and control.
BACTERIAL GENETICS:
Genetics is the process of heredity and variation and is the starting point from all other
cellular pathways, functions and structures originate. The ability of the microorganisms
to maintain viability, adapt, multiply and cause disease is founded by genetics.
Three major aspects of microbial genetics include:
a. Structure and organization of genetic material
b. Replication and expression of genetic information
c. The mechanisms by which genetic information is changed and exchanged
among bacteria.
NUCLEIC ACID STRUCTURE AND ORGANIZATIONS:
- DNA is most common form macromolecule that encodes genetic information
in bacteria
- RNA is most common in viruses
- DNA consists of deoxyribose sugars connected by phosphodiester bonds
- the bases are covalently linked to each deoxyribose sugar are the key to the
genetic code within the DNA molecule
- four bases:
1. Two Purines: Adenine and Guanine
2. Two Pyrimidines: Cytosine and Thymine
- In RNA, Uracil replaces Thymine.
- When taken together, the sugar, the phosphate and a base form a single unit
referred to as nucleotide.
- DNA and RNA nucleotide polymers and the order of bases along a DNA or RNA
strand is known as the base sequence. This sequence provides the information
that specifies the proteins that will be synthesized by microbial cells. The
sequence is the genetic code.
DNA MOLECULAR STRUCTURE:
- is composed of two nucleotide polymers. Each strand has a 5’ and a 3’ hydroxyl
terminus. The two strands run anti-parallel, with the 5’ terminus of one strand
opposed to the 3’ terminal of the other
- The strands are bound via two hydrogen bonds.
Adenine bound to Thymine (bound to two hydrogen bonds)
Guanine bound to Cytosine (bound to three hydrogen bonds)
MECHANISMS OF GENE TRANSFER:
Three mechanisms by which bacteria physically exchange DNA include
Transformation, Transduction and Conjugation.
TRANSFORMATION – donor bacterial cell dies and undergoes lysis recipient bacterial
cell receives the free DNA.
- The free DNA are seen as fragments in the environment. Certain bacteria is able
to uptake the free DNA and then undergoes the transformation.
- bacteria is usually competent on this manner.
- examples: Neisseria, Haemophilus and Streptococcus genera.
Process:
1. Once the donor DNA, usually singular strand, gains access to the interior of the
recipient cell, recombination with the recipient’s homologous DNA can occur.
2. The mixing of DNA between bacteria via transformation and recombination
plays a major role in antibiotic resistance and in the dissemination of genes that
encode factors essential to the organism’s ability to cause disease.
TRANSDUCTION – a mechanism wherein DNA from two bacteria may come together in
one cell, thus allowing for recombination. This process is mediated by viruses that infect
bacteria (bacteriophages)
Process:
The viruses integrate their DNA into the bacterial cell’s chromosome, where viral
replication and expression is directed
1. When the production of viral products is completed, viral DNA is excised/ cut
from the bacterial chromosome and packaged within protein coats.
2. The viruses are then released when the infected bacterial cell lyses.
3. In transduction, the virus not only packages its own DNA but may also package
a portion of a bacterium’s DNA. When the viruses infect another bacterial cell,
they release all their DNA contents.
4. Therefore, the newly infected cell is the recipient of donor DNA introduced by
infecting bacteriophage and recombination between DNA from two different
cells may occur.
CONJUGATION – this process occurs between two living cells, involves cell-to-cell
contact, and requires mobilization of the donor bacterium’s chromosome
1. The contact is mediated by the sex pilus. The sex pilus from the donor bacterial
cell established a conjugative bridge that serves as the conduit for DNA transfer
from donor to recipient cell.
2. With intracellular contact established, chromosomal mobilization is undertaken
and involves DNA synthesis. One DNA strand is produced by the donor and is
passed to the recipient. Where a strand complimentary to the donor strand is
synthesized.
3. The amount of DNA transferred depends on how long the cells are able to
maintain contact, but usually the portions of the chromosome are transferred. In
any case the new DNA is then available to recombine with the recipient’s
chromosome.
- in addition to chromosomal DNA, genes encoded in non-chromosomal genetic
elements, such as plasmids and transposons may be transferred by conjugation
as well.
Transposition – is the process in which these genetic elements excise from one genomic
location and insert into another.
- Transposons do not exist independently within the cell. They are referred to as
the “jumping genes”, because their ability to change location within and even
between the genomes of bacterial cells.
- Transposons carry genes whose products help mediate transpositional process
as well as genes that encode for antimicrobial resistance.
- Plasmids and transposons play a key role in genetic diversity and dissemination
of genetic information among bacteria.
(Please put picture about bacterial transport or video of each processes)
BACTERIAL METABOLISM:
Bacteria uses various strategies for obtaining essential nutrients from the environment,
but the common goal is to transport these substances from the external environment
to the cell’s interior.
Microbial metabolism consists of the biochemical reaction’s bacteria use to break
down organic compounds and the reactions they use to synthesize new bacterial parts
from the resulting carbon skeletons. Energy for the new constructions is generated
during the metabolic breakdown of the substrate.
The occurrence of all biochemical reactions in the cell depends on the presence and
activity of specific enzymes. Thus, metabolism can be regulated in the cell either by:
● regulating the production of an enzyme itself (a genetic type of regulation, in
which production of the enzyme can be induced or suppressed by molecules
present in the cell) or
● by regulating the activity of the enzyme (via feedback inhibition, in which the
products of the enzymatic reaction or a succeeding enzymatic reaction inhibit
the activity of the enzyme).
Diagnostic schemes analyze each unknown microorganism for:
1. utilization of various substrates as a carbon source,
2. production of specific end products from various substrates, and
3. production of an acid or alkaline pH in the test medium.
Knowledge of the biochemistry and metabolism of bacteria is important in the clinical
laboratory.
For nutrients to be internalized they must cross the bacterial cell envelope, a complex
cell structure that helps protect the cells from environmental insults, maintains
intracellular equilibrium and transports substances into and out of the cell.
● Water, carbon and oxygen enter the cell through passive diffusion across the
envelope
● Other nutrients require energy and selectivity by the cell envelope:
Active Transport is among the most common methods used for uptake of amino acids,
organic and inorganic acids.
- The mechanism, which is driven by an energy-dependent pump, involves a
carrier molecule embedded in the membrane portion of the cell envelope.
These carriers combine with the nutrients, transports them across the membrane
and releases them within the cell.
FERMENTATION AND RESPIRATION:
Bacteria use biochemical pathways to catabolize (break down) carbohydrates and
produce energy by two mechanisms—fermentation and respiration (commonly
referred to as oxidation).
Fermentation is an anaerobic process carried out by both obligate and facultative
anaerobes. In fermentation, the electron acceptor is an organic compound.
- Fermentation is less efficient in energy generation than respiration (oxidation)
because the beginning substrate is not completely reduced; therefore, all the
energy in the substrate is not released.
- When fermentation occurs, a mixture of end products (e.g., lactate, butyrate,
ethanol, and acetoin) accumulates in the medium. Analysis of these end
products is particularly useful for the identification of anaerobic bacteria.
- End-product determination is also used in the Voges-Proskauer (VP) and methyl
red tests, two important diagnostic tests used in the identification of the
Enterobacteriaceae.
- (The term fermentation is often used loosely in the diagnostic microbiology
laboratory to indicate any type of utilization—fermentative or oxidative—of a
carbohydrate—sugar—with the resulting production of an acid pH.)
Respiration (not an act of breathing) is an efficient energy generating process in which
molecular oxygen is the final electron acceptor.
- Obligate aerobes and facultative anaerobes carry out aerobic respiration, in
which oxygen is the final electron acceptor.
- Certain anaerobes can carry out anaerobic respiration,in which inorganic forms
of oxygen, such as nitrate and sulfate, act as the final electron acceptors.
BIOCHEMICAL PATHWAYS FROM GLUCOSE TO PYRUVIC ACID
The starting carbohydrate for bacterial fermentations or oxidation is glucose. When
bacteria use other sugars as a carbon source, they first convert the sugar to glucose,
which is processed by one of three pathways. These pathways are designed to
generate pyruvic acid, a key three-carbon intermediate.
The three major biochemical pathways bacteria use to break down glucose to pyruvic
acid are:
1. Embden-Meyerhof-Parnas (EMP) glycolytic pathway
2. pentose phosphate pathway and
3. Entner-Doudoroff pathway
- Pyruvate can be further processed either fermentatively
or oxidatively.
EMP GLYCOLYTIC PATHWAY:
● Major pathway in conversion of glucose to pyruvate
● Generates reducing power in the form of NADH2
● Generates energy in the form of ATP
● Anaerobic; does not require oxygen
● Used by many bacteria, including all members of Enterobacteriaceae
PENTOSE PHOSPHATE (PHOSPHOGLUCONATE) PATHWAY
● Alternative to EMP pathway for carbohydrate metabolism
● Conversion of glucose to ribulose-5-phosphate, which is rearranged into other 3-
, 4-, 5-, 6-, and 7-carbon sugars
● Provides pentoses for nucleotide synthesis
● Produces glyceraldehyde-3-phosphate, which can be converted to pyruvate
● Generates NADPH, which provides reducing power for biosynthetic reactions
● May be used to generate ATP (yield is less than with EMP pathway)
● Used by heterolactic fermenting bacteria, such as lactobacilli, and by Brucella
abortus, which lacks some of the enzymes required in the EMP pathway
ERTNER-DOUDOROFF PATHWAY:
● Converts glucose-6-phosphate (rather than glucose) to pyruvate and
glyceraldehyde phosphate, which can be funneled into other pathways
● Generates one NADPH per molecule of glucose but uses one ATP
● Aerobic process used by Pseudomonas, Alcaligenes, Enterococcus faecalis,
and other bacteria lacking certain glycolytic enzymes
Anaerobic Utilization of Pyruvic Acid (Fermentation)
- Pyruvic acid is a key metabolic intermediate. Bacteria process pyruvic acid
further using various fermentation pathways. Each pathway yields different end
products, which can be analyzed and used as phenotypic markers Some
fermentation pathways used by the microbes that inhabit the human body are
as follows:
● Alcoholic fermentation: The major end product is ethanol. This is the pathway
used by yeasts when they ferment glucose to produce ethanol.
● Homolactic fermentation: The end product is almost exclusively lactic acid. All
members of the Streptococcus genus and many members of the Lactobacillus
genus ferment pyruvate using this pathway.
● Heterolactic fermentation: Some lactobacilli use this mixed fermentation
pathway, of which, in addition to lactic acid, the end products include carbon
dioxide, alcohols, formic acid, and acetic acid.
● Propionic acid fermentation: Propionic acid is the major end product of
fermentations carried out by Propionibacterium acnes and some anaerobic
non–spore-forming, gram-positive bacilli.
● Mixed acid fermentation: Members of the genera Escherichia, Salmonella, and
Shigella within the Enterobacteriaceae use this pathway for sugar fermentation
and produce a number of acids as end products—lactic, acetic, succinic, and
formic acids. The strong acid produced is the basis for the positive reaction on
the methyl red test exhibited by these organisms.
● Butanediol fermentation: Members of the genera Klebsiella, Enterobacter, and
Serratia within the Enterobacteriaceae use this pathway for sugar fermentation.
The end products are acetoin (acetyl methyl carbinol) and 2,3-butanediol.
Detection of acetoin is the basis for the positive VP reaction characteristic of these
microorganisms. Little acid is produced by this pathway. Thus, organisms that have a
positive VP reaction usually have a negative reaction on the methyl red test, and vice
versa.
● Butyric acid fermentation: Certain obligate anaerobes, including many
Clostridium species, Fusobacterium, and Eubacterium, produce butyric acid as
their primary end product along with acetic acid, carbon dioxide, and
hydrogen.
Aerobic Utilization of Pyruvate (Oxidation)
- The most important pathway for the complete oxidation of a substrate under
aerobic conditions is the Krebs or tricarboxylic acid (TCA) cycle. In this cycle,
pyruvate is oxidized, carbon skeletons for biosynthetic reactions are created,
and the electrons donated by pyruvate are passed through an electron
transport chain and used to generate energy in the form of ATP. This cycle results
in the production of acid and the evolution of carbon dioxide
Carbohydrate Utilization and Lactose Fermentation
- The ability of microorganisms to use various sugars (carbohydrates)for growth is
an integral part of most diagnostic identification schemes.
- The fermentation of the sugar is usually detected by acid production and a
concomitant change of color resulting from a pH indicator present in the culture
medium.
- Bacteria generally ferment glucose preferentially over other sugars, so glucose
must not be present if the ability to ferment another sugar is being tested.
- An important step in classifying members of the Enterobacteriaceae family is the
determination of the microorganism’s ability to ferment lactose.
- These bacteria are classified as either lactose fermenters or lactose
nonfermenters. Lactose is a disaccharide consisting of one molecule of glucose
and one molecule of galactose linked together by a galactoside bond.
- Two steps are involved in the utilization of lactose by a bacterium.
1. The first step requires an enzyme, β-galactoside permease, for the transport
of lactose across the cell wall into the bacterial cytoplasm.
The second step occurs inside the cell and requires the enzymeβ-galactosidase to
break the galactoside bond, releasing glucose, which can be fermented. Thus, all
organisms that can ferment lactose can also ferment glucose.
TOPIC 2: METHODS OF STUDYING ORGANISMS
MICROSCOPIC EXAMINATION OF ORGANISMS:
o 1880 – Robert Koch had published his direct observations of cluster-forming
cocci in purulence from human disease. He named these
cocci Staphylococcus.
o 1884 – Christian Gram developed the Gram stain, which toda yallows us to
examine a pus specimen directly for the gram -positive cocci Staphylococcus.
b. Iodine – MORDANT - It fluoresces when exposed to long-wavelength ultraviolet and short
c. Alcohol – DECOLORIZER wavelength visible light. Special filters are required for optimal use.
d. Safranin – COUNTERSTAIN o APPLICATION:
o APPLICATION: - Calcofluor white may be used as a specific stain for rapid screening of clinical
- The Gram stain is used routinely and as requested in the clinical microbiology specimens for fungal elements. This stain may be useful when morphology is
laboratory for the primary microscopic examination of specimens submitted for ambiguous and the nonspecific staining of other techniques such as Grocott-
smear and culture. Gomori methenamine–silver nitrate gives confusing results.
- It is ideally suited for specimen types in which bacterial infections are strongly o RESULT:

DIRECT MICROSCOPIC EXAMINATION: suspected, but it may be used to characterize any specimen. - Yeast cells, pseudohyphae, and hyphae display a bright apple-green or blue-
- Cerebrospinal fluid, sterile fluids, expectorated sputum or bronchoalveolar white fluorescence.
o It can be used to determine the quality of the specimen. Sputum specimens
lavages, and wounds and exudates are routinely stained directly.
that represent saliva rather than lower respiratory secretions can be RAPID MODIFIED WRIGHT-GIEMSA STAIN:
- Urine and stool may not be routinely stained directly. Samples sent for focused
determined by the quantitation of WBCs or epithelial cells. o PRINCIPLE:
screening cultures usually are not stained.
o The routine culture workup can be guided by the results of the smear. The - The Wright-Giemsa stain is available in a modification that requires only 1 to 3
- The Gram stain is regularly used to characterize bacteria growing on culture
technologist can correlate the bacterial isolates with the types detected in the minutes. This neutral dye is a combination of basic thiazine dyes and acid eosin
media.
smear. that attach to oppositely charged sites on proteins. The results are
o RESULTS:
o It can give the microbiology technologist and the physician an indication of metachromatic.
- Gram-positive bacteria stain dark blue to blue.
the infectious process involved. o APPLICATION:
- Among the gram-negative bacteria stain bright red; the enterics have strong
o It can dictate the need for non-routine or additional testing. - Wright-Giemsa (modified) is a rapid stain for smears and imprints to stain fully
avidity.
STAINING TECHNIQUES: o NOTE: Always check the quality of the stain before moving to interpretation. background materials and cells and a wide variety of microorganisms.
o RESULTS:
- There are many types of stains, each with specific applications. Stains can be ACID FAST STAINING:
- Blood cells stain as with Wright stain. The cytoplasm is basophilic. The chromatin
categorized as simple stains, differential stains, and probe-mediated stains.
o PRINCIPLE: of white cells is purple. Bacteria are blue. Parasitic protozoan
1. Simple stains are directed toward coloring the forms and shapes present. - The primary stain binds to mycolic acid in the cell walls of the mycobacteria
STAINS USED FOR GENERAL MORPHOLOGY:
and is retained after the decolorizing step with acid alcohol. The counterstain
2. Differential stains are directed toward coloring specific components of the Stains: Applications (specimens)
does not penetrate the mycobacteria to affect the color of the primary stain.
elements present. Wright Giemsa - Bronchoalveolar lavages
o APPLICATION:
- Tzanck’s preparations
3. Diagnostic antibody or DNA probe–mediated stains are directed - The direct smear examination is a valuable diagnostic procedure for the
- Samples with complex cellular backgrounds (visualizes
specifically at identification of an organism. detection of mycobacteria in clinical specimens.
bacteria, yeast, parasites, and viral inclusions)
GRAM STAIN TECHNIQUE: o RESULTS:
● Fluorescent Stain: Mycobacteria stain bright orange. Count the number of
o PRINCIPLE:
acid-fast bacilli seen on the smear and report as follows:
- The following Gram staining method was developed empirically by the Danish STAINS USED FOR SELECTED MORPHOLOGY:
- No. Acid-Fast Bacilli: Report:
bacteriologist Christian Gram in 1884. Stains: Applications:
- 1-20 Number seen
- The sequential steps provide for crystal violet (hexamethyl-p-rosanaline Leifson Flagella
- 21-80 Few
chloride) to color all cells and background material a deep blue and for Methylene Blue Metachromatic granules of Corynebacterium diptheria
- 81-300 Moderate
Gram’s iodine to provide the larger iodine element to replace the smaller Acid-fast stains:
- >300 Numerous
chloride in the stain molecule. 1. Ziehl-Neelsen Sediments for mycobacteria (concentrated smears)
● Kinyoun and Ziehl-Neelsen Stains: Mycobacteria stain red, whereas the
- Bacteria with thick cell walls containing teichoic acid and peptidoglycan layer Partial acid-fastness of Nocardia spp.
background material and non– acid-fast bacteria stain blue.
retain the crystal violet–iodine complex dye after decolorization and appear 2. Fluorochrome Sediments for mycobacteria (concentrated smears,
o REAGENTS:
deep blue; they are gram-positive bacteria. auramine, and rhodamine)
- CARBOLFUCSHIN – acts as a PRIMARY STAIN
- Other bacteria with thinner walls containing lipopolysaccharides do not retain
- METHYLENE BLUE –acts as a COUNTERSTAIN
the dye complex; they are gram-negative bacteria. 3. Kinyoun Acid-fast stain modification of Ziehl-Neelsen method for
- ALCOHOL- acts as DECOLORIZER
- The alcohol-acetone decolorizer damages these thin lipid walls and allows the Cryptosporidium and Cyclospora parasites in stool
- WATER – required in washing step
stain complex to wash out. specimen

- All unstained elements are subsequently counterstained red by safranin dye. Calcofluor white stain Bronchoalveolar fungi and some parasitic cysts
- The differential ability of the Gram stain makes it useful in microbial taxonomy. Differentiates them from background materials of similar
The quickness and ease with which the method can be performed make it an morphology.
CALCOFLOUR WHITE STAIN:
ideal choice for the clinical laboratory setting. Gram stain:
o REAGENTS: o PRINCIPLE: 1. Traditional Routine stain for diagnostic area
a. Crystal violet – PRIMARY STAIN - Calcofluor white is a colorless dye that binds to cellulose and chitin. Yeast differentiated from all other organisms.
 2. Enhanced Provides same differential staining but enhances red-
negative organisms by staining the background material
green to gray-green.
GENUS SPECIFIC STAINS:
Stains: Applications:
Antibody or DNA probe Used for specific identification of selected
stains pathogens, such as Chlamydia trachomatis,
Bordetella pertussis, Legionella pneumophila,
herpes simplex virus, varicella-zoster virus,
cytomegalovirus, adenovirus, and respiratory
viruses
QUICK RECALL!!
o Bacteria vary in size from 0.4 to 2 μm. They occur in three basic shapes:
● Cocci = spherical
● Bacilli = rod-shaped
● Spirochetes = spiral
o Individual bacteria may form characteristic groupings:
- Cocci-(plural of coccus) may occur singly or in pairs (diplococci), in chains
(streptococci), or in clusters (staphylococci).
o Bacilli- (plural of bacillus) may vary greatly in size and length from very short
coccobacilli to long filamentous rods. The ends may be square or rounded.
Bacilli with tapered, pointed ends are termed fusiform. Some bacilli are
curved.
o When a species varies in size and shape within a pure culture, the bacterium is
pleomorphic.
o Bacilli may occur as single rods or in chains or may align themselves side by
side and is called palisading. Spirochetes vary in length and in the number of
helical turns (not all helical bacteria are called spirochetes).
IMPORTANCE OF COLONIAL MORPHOLOGY AS A DIAGNOSTIC TOOL:
- In many ways, the usefulness of colonial morphology extends the capabilities
of the microbiologist and, ultimately, the clinical laboratory. The ability to
provide a presumptive identification by colonial morphology may include the
following:
o Provide a presumptive identification to the physician.
Types of Culture Media:
o Enhance the quality of patient care through rapid reporting of results and by
increasing the cost-effectiveness of laboratory testing.
o Play a significant role in quality control, especially of automated procedures
and other commercially available identification systems.
Colony Morphology:
o Bacteria grow on solid media as colonies. A colony is defined as a visible mass
of microorganisms originating from a single mother cell. Key features of these
bacterial colonies serve as important criteria for their identification.
o Colony morphology can sometimes be useful in bacterial identification.
Colonies are described on the basis of size, shape, texture, elevation,
pigmentation, and effect on growth medium.
● Colony Shape: It includes form, elevation, and margin of the bacterial Classification of Culture Media
colony. Based on chemical Based on physical Based on functions:
● Form of the bacterial colony: The form refers to the shape of the colony. composition: nature:
These forms represent the most common colony shapes you are likely to 1. Simple/basal 1. Liquid media 1. Supportive media
encounter. e.g., circular, irregular, filamentous, rhizoid, etc. media
● Elevation of the bacterial colony: It gives information about, how much
2. Defined 2. Semi-solid media 2. Enriched media
does the colony rise above the agar. This describes the “side view” of a
(synthetic) media
colony. These are the most common elevations; e.g., flat, raised, umbonate
(having a knobby protuberance), crateriform, convex, pulvinate (cushion- 3. Complex media 3. Solid media 3. Selective/enrichment
shaped). media
● Margin of bacterial colony: The margin or edge of a colony may be an
4. Biphasic media 4. Differential media
important characteristic in identifying organisms. Common examples are
5. Indicator media
entire (smooth), irregular, undulate (wavy), lobate, curled, filiform, etc.
● Colonies that are irregular in shape and/or have irregular margins are likely 6. Transport media
to be motile organisms. Highly motile organisms swarmed over the culture
7. Anaerobic media
media, such as Proteus spp.
● Size of the bacterial colony: The size of the colony can be a useful
characteristic for identification. The diameter of a representative colony
Let me give you a heads up:
may be measured in millimeters or described in relative terms such as
Type: Purpose:
pinpoint, small, medium, large. Tiny colonies are also referred to
Chemically defined Growth of Chemoautotrophs, photoautotrophs, and
as punctiform (pin-point). Colonies larger than about 5 mm are likely to be
microbiological assays
motile organisms.
Complex Growth of most Chemoheterotrophic organisms
● Appearance of the colony surface: Bacterial colonies are frequently shiny
Reducing Growth of obligate anaerobes
and smooth in appearance. Other surface descriptions might be: dull
Selective Surpression of unwanted microboes; encouraging desired
(opposite of glistening), veined, rough, wrinkled (or shriveled), glistening.
microbes
● Consistency/Texture: Several terms that may be appropriate for describing
Differential Differentiation of colonies of desired microbes from others
the texture or consistency of bacterial growth are: dry, moist, viscid (sticks to
Enrichment Similar to selective media but designed to increase
loop, hard to get off), brittle/friable (dry, breaks apart), mucoid (sticky,
numbers of desired microbes to detectable levels.
mucus-like)
● Color of the colonies (pigmentation): Some bacteria produce pigment
when they grow in the medium e.g., green pigment produces Nonselective Media:
by Pseudomonas aeruginosa, buff-colored colonies of Mycobacterium
- Blood agar and chocolate agar are examples of complex, nonselective
tuberculosis in L.J medium, red-colored colonies of Serratia marcescens.
media, which support the growth of many different bacteria. These media are
● Opacity of the bacterial colony: Is the colony transparent (clear), opaque
intended to cultivate as many species as possible, thus giving rise to numerous
(not transparent or clear), translucent (almost clear, but distorted vision–like
types of bacterial colonies.
looking through frosted glass), or iridescent (changing colors in reflected
light)? Selective Media:
- Because of the diversity of microorganisms that typically reside at some
sampling sites (eg, the skin, respiratory tract, intestines, vagina), selective
- In contrast to viruses and most parasites, many bacterial pathogens can be
media are used to eliminate (or reduce) the large numbers of irrelevant
isolated on solid agar-containing media. The general cultivation of most
bacteria in these specimens.
bacteria requires media rich in metabolic nutrients. These media generally
- The basis for selective media is the incorporation of an inhibitory agent that
include agar, a carbon source, and an acid hydrolysate or enzymatically
specifically selects against the growth of irrelevant bacteria. Examples of such
degraded source of biologic material (eg, casein). Because of the undefined
agents are:
composition of the latter, these types of media are referred to as complex
● Sodium azide—selects for gram-positive bacteria over gram-negative
media.
bacteria
- Culture media may be divided into categories defined by the ability to support
● Bile salts (sodium deoxycholate)—select for gram-negative enteric
bacterial growth.
bacteria and inhibit gram-negative mucosal and most gram-positive
bacteria
 ● Colistin and nalidixic acid—inhibit the growth of many gram-negative because the organisms produce haemolysis or clearance
bacteria around their colonies on BA plates.
- Examples: MacConkey agar, Lowenstein-Jensen media, Tellurite media
Chocolate Chocolate agar (CA) plate like BA is used for the culture of some
(Tellurite inhibits the growth of most of the throat organisms except diphtheria
agar place fastidious bacteria such as Neisseria gonorrhoeae,
bacilli). Antibiotic may be added Differential Media:
Streptococcus pneumoniae and Haemophilus influenzae. It is
Media: Classification: Selective and Differential Type of Organisms prepared from nutrient agar base by adding defibrinated horse - Differential media allow grouping of microbes based on different
Agent (s): Isolated: or sheep blood to the medium after sterilization in the autoclave characteristics demonstrated on the medium. Media may be differential and
Chocolate Enriched 1% hemoglobin and Most fastidious and cooling to 80°C. The blood-laden medium is then heated nonselective (e.g., sheep blood agar is nonselective but differentiates
agar supplements pathogens such as over Bunsen burner flame until it turns chocolate-brown in colour organisms on the basis of hemolysis). Media can be differential and selective
Neisseria and to form CA or heated blood agar. (e.g., MacConkey agar inhibits gram-positive organisms and differentiates
Haemophilus gram-negative bacilli on the basis of lactose fermentation).
Nutrient agar Nutrient agar (NA) plate is a general-purpose medium that
Blood agar Enriched and 5% defibrinated sheep Almost all - Upon culture, some bacteria produce characteristic pigments, and others can
plate supports the growth of a wide variety of bacteria. It is used for
plates (BAP) differential blood bacteria; be differentiated on the basis of their complement of extracellular enzymes;
subculturing organisms to get pure cultures and for conducting
differential for the activity of these enzymes often can be detected as zones of clearing
susceptibility studies in the microbiology laboratory. NA is a basal
hemolytic surrounding colonies grown in the presence of insoluble substrates (e.g., zones
medium required for the preparation of other bacteriological
organisms of hemolysis in agar medium containing red blood cells).
media including CA and BA. And the liquid medium of NA (i.e.
Mannitol salt Selective and 7.5% NaCl and mannitol Staphylococci - Examples: Blood agar and MacConkey agar are indicator or differential media
nutrient broth) is generally used to spur the growth of bacteria
sugar (MSA) differential for isolation and and micrococci - Many of the members of the Enterobacteriaceae can be differentiated on the
present in clinical specimens and environmental samples prior to
identification of most S. basis of their ability to metabolize lactose. For example, whereas pathogenic
their subculture onto other solid media.
aureus strains salmonellae and shigellae do not ferment lactose on a MacConkey plate form
MacConkey Selective and Lactose, bile salts, neutral Gram-negative CLED plate Cystein lactose electrolyte deficient (CLED) medium is a
white colony, lactose-fermenting members of the
agar differential red, and crystal violet enteric bacilli bacteriological medium that is lacking in electrolyte (e.g. sodium
- Enterobacteriaceae (e.g., E coli) form red or pink colonies.
Eosin Selective and Lactose, eosin Y, and Gram-negative chloride). CLED is usually used for the cultivation of
Enriched Media:
methylene differential methylene blue enteric bacilli uropathogens (i.e. bacteria that cause urinary tract infections)

blue agar from clinically important urine specimens. The absence of - Enriched media contain growth enhancers that are added to nonselective

(EMB) electrolyte (i.e. NaCl) in CLED media prevents the growth of agar to allow fastidious organisms to flourish. Chocolate agar is an enriched
swarming bacteria (e.g. Proteus species) which may medium.
Hektoen Selective and Lactose, sucrose, bile salts, Salmonella and
contaminate the culture media. - The media are usually enriched usually by adding blood, serum or egg.
enteric agar differential ferric ammonium sulfate, Shigella species
(HE) sodium thiosulfate, (enteric - Examples: Blood agar, Lowenstein Jensen media
MacConkey MacConkey agar (MAC) plate is a differential media which is
bromthymol blue, acid pathogens) ● Enrichment Broth
agar plate mainly used to culture Gram-negative bacteria especially
fuchsin ▪ Enrichment broth is a liquid medium designed to encourage the growth
members of bacteria in the family Enterobacteriaceae. MAC
Phenylethyl Selective Phenylethyl alcohol Gram-positive of small numbers of a particular organism while suppressing other flora
contains substances such as lactose, bile salt and indicator dyes
alcohol agar (inhibits gram negatives) bacteria present.
which aid to different lactose fermenters (eg. Escherichia coli)
(PEA) ▪ Enrichment broths are incubated for a certain period and then must be
from nonlactose fermenters (e.g. Salmonella and Shigella
Colistin Selective Colistin and nalidixic acid Gram-positive subcultured to isolate the particular organism. Lim broth (Todd Hewitt
species). It is an ideal medium for the presumptive isolation of
nalidixic acid (inhibit gram negatives) bacteria with CNA) is used to enhance the growth of groupB streptococci.
lactose fermenters; and MAC can also serve as a selective
sugar (CNA) ▪ Examples: Selenite Broth: an enrichment medium used for Salmonella
medium since it encourage the growth of particular microbes.
and Shigella.
Modified selective Hemoglobin, growth Pathogenic
SDA plate Sabouraud dextrose agar (SDA) plate is used for the culture and Sodium selenite-inhibits the growth of Gram positive and many Gram-
Thayer-Martin factors, and antimicrobial Neisseria species
isolation of fungi from samples emanating from the environment negative including Enterococci and coliforms and Salmonella is
agar (MTM) agents
or hospital. SDA is usually prepared with the incorporation of affected.
chloramphenicol and cycloheximide which inhibit the growth of Tetratrionate Broth: an enrichment medium used to isolate Salmonella
Culture media: Function: bacteria and saprophytic fungi (i.e. non-pathogenic fungi). It is
Broth Media:
Blood agar Blood agar (BA) is an agar-based culture medium that contains the idea 1 medium for mycological investigation in the
plate defibrinated blood (especially horse or sheep blood). BA is microbiology laboratory, and SDA is generally inhibitory to the - Broth media can be used as a supplement to agar plates to detect small
basically used for the culture of fastidious organisms such as growth of bacteria. numbers of most aerobes, anaerobes, and microaerophiles.
Streptococcus pyogenes that lyses RBCs. For haemolytic - Examples: Thioglycollate broth (THIO) is an example of a supplemental broth
CLED = Cystein lactose electrolyte deficient medium. SDA = Sabouraud dextrose
organisms such as pathogenic Staphylococci and Streptococci, media. It is made in oxygen gradient. It demonstrates the ability of the
agar.
BA can help in their presumptive identification in the laboratory organism to live in the presence of molecular oxygen. The oxygen gradient
 separates bacteria based on their ability to survive in different concentrations
of oxygen.
Thioglycollate media:
o Anaerobic Environment: conditions with Low Oxygen Concentration (examples:
Human Gastrointestinal Tract)
o Aerobic Environment: conditions with High Oxygen Concentration. (Examples:
Atmosphere)
o Facultative anaerobes: are able to grow either with or without free oxygen.
o Obligate anaerobes: can live only in the absence of oxygen.
o Obligate aerobes: organisms that require oxygen to grow and survive.
o Aerotolerant anaerobes: refers to an organism that is anaerobic as it does not
need oxygen to survive and thrive. An organism has an optimum oxygen
concentration range in which it can achieve its maximum growth. However, for
an obligate anaerobe, the optimum oxygen concentration is zero.
Routine Plating Media Order:
1. Nonselective agar plate
2. Enriched medium for fastidious organisms for normally sterile body fluids or a
site in which fastidious organisms are expected
3. Selective and differential medium for enteric gram-negative bacilli for most
routine bacterial cultures
4. Selective medium for gram-positive organisms for specimens in which mixed
gram-positive and gram-negative bacteria are found
5. Additional selective media or enrichment broths for specific pathogens as
needed
6. Broth medium may be used as a supplement with specimens from sterile body
fluids, tissues, lesions, wounds, and abscesses
BIOCHEMICAL TESTS:
- Biochemical tests are the tests used for the identification of bacterial species
based on the differences in the biochemical activities of different bacteria.
Bacterial physiology differs from one type of organism to another.
- Biochemical test is classified as:
1. Enzymatic Tests
2. Metabolic Pathway Tests
● Carbohydrate Oxidation and Fermentation Tests
● Amino Acid Degradation Tests
● Single Substrate Utilization Tests
3. Antibiotic Inhibition Tests
4. Specific Tests
Catalase Production:
- The enzyme catalase catalyzes the conversion of hydrogen peroxide to water
and oxygen. When a colony is placed in hydrogen peroxide, liberation of
oxygen as gas bubbles can be seen. The test is particularly useful in
differentiation of staphylococci (positive) from streptococci (negative), but
also has taxonomic application to Gram-negative bacteria.
Coagulase Test:
- The enzyme coagulase acts with a plasma factor to convert fibrinogen to a
fibrin clot. It is used to differentiate Staphylococcus aureus from other, less
pathogenic staphylococci.
Oxidase Tests:
- The oxidase tests detect the c component of the cytochrome–oxidase
complex. The reagents used change from clear to colored when converted
from the reduced to the oxidized state. The oxidase reaction is commonly
demonstrated in a spot test, which can be done quickly from isolated colonies.
Proteinase Production:
- . Proteolytic activity is detected by growing the organism in the presence of
substrates such as gelatin or coagulated egg. The use of gelatin in the culture
medium provided us a qualitative assay, a simple, inexpensive, straight forward
method to assess the presence of proteolytic activity of a given colony.
Urease Production:
- Urease hydrolyzes urea to yield two molecules of ammonia and one of CO2.
This reaction can be detected by the increase in medium pH caused by
ammonia production. Urease-positive species vary in the amount of enzyme
produced; bacteria can thus be designated as positive, weakly positive, or
negative.
Carbohydrate Breakdown:
- The ability to produce acidic metabolic products, fermentatively or oxidatively,
from a range of carbohydrates (eg, glucose, sucrose, and lactose) has been
applied to the identification of most groups of bacteria. Such tests are crude
and imperfect in defining mechanisms, but have proved useful for taxonomic
purposes
O-Nitrophenyl-β-d-galactoside (ONPG) breakdown:
- The ONPG test is related to lactose fermentation. Organisms that possess the β-
galactoside necessary for lactose fermentation but lack a permease
necessary for lactose to enter the cell are ONPG-positive and lactose-
negative.
Decarboxylation or Deamination:
- The decarboxylation or deamination of the amino acids lysine, ornithine, and
arginine is detected by the effect of the amino products on the pH of the
reaction mixture or by the formation of colored products. These tests are used
primarily with Gram-negative rods.
Citrate Utilization:
- An agar medium that contains sodium citrate as the sole carbon source may
be used to determine ability to use citrate. Bacteria that grow on this medium
are termed citrate-positive.
Hydrogen Sulfide Production:
- The ability of some bacteria to produce H2S from amino acids or other sulfur-
containing compounds is helpful in taxonomic classification. The black color of
the sulfide salts formed with heavy metals such as iron is the usual means of
detection.
Indole Production Tests:
- The indole reaction tests the ability of the organism to produce indole, a
benzopyrrole, from tryptophan. Indole is detected by the formation of a red
dye after addition of a benzaldehyde reagent. A spot test can be done in
seconds using isolated colonies.
Voges-Proskauer Test:
- The Voges–Proskauer test detects acetylmethylcarbinol (acetoin), an
intermediate product in thebutene glycol pathway of glucose fermentation.
IMMUNODIAGNOSIS ON INFECTIOUS DISEASES:
Serologic Testing of Syphilis:
- Sexually transmitted diseases (STDs) are among the most common infectious
diseases in the United States. The most common of these diseases are genital
warts, chlamydia, and genital herpes; however, syphilis is one of the most
dangerous if left untreated. Serologic testing for syphilis is the most common
diagnosis
- T. pallidum subsp. pallidum, the spirochete that causes syphilis, may be
identified from a lesion or chancre.
- Two forms of testing exist for syphilis: screening tests and confirmatory tests.
● Confirmatory Test Assays: use treponemal antigens and are sensitive and
very specific but can't be used to monitor therapy.
1. FTA-ABS test
2. Microhemeagglutination Assay
● Screening Tests: Nontreponemal antigen tests are technically easier and
more rapid to perform, and they are the tests of choice for
syphilis screening.
1. Venereal Disease Research Laboratory (VDRL)
▪ named after the laboratory that developed it, is a microscopic
flocculation test
▪ using heat-inactivated serum and performed on glass slides. The
test detects reaginic antibodies (reagin) that bind to an
alcoholic solution of cardiolipin-lecithin-cholesterol particles. It
is be used to test CSF for diagnosing neurosyphilis
2. The RPR test uses the same antigen as the VDRL test; however,
carbon particles have been added so that the flocculation reaction
can be seen more clearly.
Serologic testing for Streptococcal Infections:
- The ASO antibody test is used to demonstrate serologic response to
Streptococcus pyogenes. One method to detect ASO is to measure the ability
of the patient’s serum to neutralize the erythrocyte lysing (hemolytic) ability of
the streptococcal enzyme (streptolysin O).
- The patient’s serum is mixed with a standard concentration of streptolysin O.
- If antibody to streptolysin O is present, it binds to the hemolysin neutralizing it.
When red blood cells (RBCs) are added, the RBCs are not lysed.
TOPIC 3: MICROBIAL CONTROL
- If antibody to streptolysin O is not present in the serum sample, the hemolysin is
not neutralized, and the RBCs are lysed.
APPLICATIONS ON NUCLEIC ACID METHODS:
- Categories for the application of molecular diagnostic microbiology methods
are the same as those for conventional phenotype based- methods:
● Direct detection of microorganisms in patient specimens.
● Identification of microorganisms grown in culture.
● Freezing is the typical storage method for nucleic acid amplification assays.
However, specimens should be refrigerated rather than frozen if labile
viruses (eg, varicella-zoster virus, influenza virus, HIV-2) are suspected or if
viral cultures are also to be done (frozen specimens may not be usable for
standard cultures).
- Safety in the laboratory cannot be overemphasized. Quantization of the risk of
working with an infectious agent is difficult.
- Risk to an individual increases with the frequency and type of organism and
level of contact with the agent.
- Each laboratory must develop and institute a plan that effectively minimizes
exposure to infectious agents.
● Nature of surface to be disinfected: Certain medical instruments are
manufactured of biomaterials that exclude the use of certain disinfection or
sterilization methods because of possible damage to the instruments.
● Temperature: For example, glutaraldehyde: can be used as a disinfectant or a
sterilant, with the difference being the amount of time the glutaraldehyde is in
contact with the contaminated object.
- The contact time is much longer than when it is used as a disinfectant.
- Alcohol and iodine preparations (e.g., Betadine) must be in contact with an
object for at least 1 to 2 minutes for them to kill microorganisms.
- The spores of both bacteria and fungi must be in the presence of disinfectants
or sterilants for a much longer time than their vegetative counterpart before
they are killed.
● pH
● Biofilms: can be considered as a community of bacteria or other
microorganisms. These communities are generally layers of microorganisms that
often have a protective material over them that shields them from outside

● Characterization of microorganisms beyond identification HISTORY: environmental factors.

- Nucleic acid–based (molecular) identification has become commonplace in ● Compatibility of disinfectants and sterilants
- The scientific use of disinfection and sterilization methods originated more than
clinical settings; the resulting rapid identification allows the patient to be 100 years ago when Joseph Lister introduced the concept of aseptic surgery. METHODS OF DISINFECTION AND STERILIZATION:
placed on specific antimicrobial therapy and avoid prolonged management
STERILIZATION VERSUS DISINFECTION: - Spaulding categorized medical materials into three device classifications:
on empiric, potentially inappropriate drugs.
1. Critical materials: Critical materials are materials that invade sterile tissues
● Sterilization refers to the destruction of all forms of life, including bacterial
Nucleic Acid Amplification: or enter the vascular system. These materials are most likely to produce
spores.
- Nucleic acid amplification techniques take tiny amounts of DNA or RNA, infection if contaminated and require sterilization.
● Chemical or physical methods may be used to accomplish this form of
replicate them many times, and thus can detect minute traces of an organism 2. Semicritical materials: semicritical materials come into contact with
microbial destruction.
in a specimen, avoiding the need for culture. These techniques are particularly mucous membranes, they require high-level disinfection agents.
● Disinfection refers to a process that eliminates a defined scope of
useful for organisms that are difficult to culture or identify using other methods 3. Noncritical materials: require intermediate-level to low-level disinfection
microorganisms, including some spores.
(eg, viruses, obligate intracellular pathogens, fungi, mycobacteria, some other before contact with intact skin.
● Physical or chemical methods may be used, but most disinfectants are
bacteria) or that are present in low numbers. chemical agents applied to inanimate objects. PHYSICAL METHODS:
- These tests may involve: ● A substance applied to the skin for the purposeof eliminating or reducing the
● HEAT
● Target amplification (eg, polymerase chain reaction [PCR], reverse number of bacteria present is referred to as an antiseptic. Antiseptics do not kill
transcriptase–PCR [RT-PCR], strand displacement amplification, transcription spores and cannot be used as disinfectants. Method Temperature (in Time Applications:
amplification) Celsius) required:
FACTORS THAT INFLUENCE THE DEGREE OF KILLING:
● Signal amplification (eg, branched DNA assays, hybrid capture)
● Probe amplification (eg, ligase chain reaction, cleavase-invader, cycling ● Types of organisms: Organisms vary greatly in their ability to withstand Boiling Water 100 15 min Kills microbial vegetative forms;
probes) chemical and physical treatment. (Steam) endospores survive.
● Postamplification analysis (eg, sequencing of the amplified product, - Cell walls of mycobacteria are rich in lipids, which may account for their Autoclave 121.6 15 min Sterilizes and kills Endospores
microarray analysis, and melting curve analysis, as is done in real-time PCR) resistance to chemical and environmental stresses, particularly desiccation (steam under at
- Appropriate specimen collection and storage before arrival at the molecular - For example, spores have coats rich in proteins, lipids, and carbohydrates pressure) 15 psi
diagnostic laboratory are critical. Because amplification methods are so aswell as cores rich in dipicolinic acid and calcium, all of which protect the Pasteurization: 63 30 min Disinfects and kills milk-borne
sensitive, false-positive results from trace contamination of the specimen or spores. 1. Batch pathogens and vegetable forms;
equipment can easily occur. - In contrast, viruses containing lipid-rich envelopes are more susceptible to the method endospores survive.
- Despite high sensitivity, false-negative results sometimes occur even when a effects of detergents and wetting agents.
2. Fish 72 15 s Same, but shorter time at higher
patient is symptomatic (eg, in West Nile virus infection). False-negative results - The organisms known today to be the most resistant to the actions of heat,
method temperature.
can be minimized by the following: chemicals, and radiation are prions.
● Avoiding use of swabs with wooden shafts or cotton tips (the swab that has ● Presence of organic material: Organic material, such as blood, mucus, and Over (dry heat) 160-180 1.5-3 h Sterilizes; keeps materials dry.
been validated for the amplification assay must be used) pus, affects killing activity by inactivating the disinfecting agent.
● Transporting specimens rapidly ● Number of organisms: Another factor to consider is the total number of
● FILTRATION
● Freezing or refrigerating specimens if transport is likely to take > 2 hours organisms present, referred to as the microbial load (bioburden).
● Concentration of disinfecting agent
 ▪ Filtration methods may be used with both liquid and air. Filtration of liquids is Phenolics Phenol, carbolic Denature proteins; Disinfectants at - The microbiology technologist does not usually perform this preanalytical
accomplished through the use of thin membrane filters composed of acid, Lysol, disrupt cell high portion of the laboratory testing process, and yet it directly affects the
plastic polymers or cellulose esters containing pores of a certain size. Hexachlorophene Membranes concentrations; outcome.
▪ Filtration of air is accomplished with the use of high-efficiency particulate air used in soaps at - The following basic principles of specimen collection are fundamental to
(HEPA) filters. HEPA filters are able to remove microorganisms larger than 0.3 low concentration ensuring appropriate specimen management:
μm and are used in laboratory hoods and in rooms of immunocompromised Sterilization of ● If possible, collect the specimen in the acute phase of the infection and
patients. heat-sensitive before antibiotics are administered.
● RADIATION objects ● Select the correct anatomic site for collection of the specimen.
- Radiation may be used in two forms—ionizing and non-ionizing. Gases Ethylene oxide Alkylating agent ● Collect the specimen using the proper technique and supplies with minimal
▪ Ionizing radiation: in the form of gamma rays or electron beams, is of short contamination from normal biota (normal flora).
wavelength and high energy. ● Collect the appropriate quantity of specimen.
DISINFECTANTS VERSUS ANTISEPTICS:
▪ This method of sterilization is used by the medical field for the sterilization of ● Package the specimen in a container or transport medium designed to
disposable supplies such as syringes, catheters, and gloves. Nonionizing - The germ theory of disease also contributed to the development of maintain the viability of the organisms and avoid hazards that result from
radiation in the form of ultraviolet rays is of long wavelength and low antimicrobial chemotherapeutics. leakage.
energy. - Ignatz Semmelweis (1816-1865) and Joseph Lister (1827- 1912) are considered ● Label the specimen accurately with the specific anatomic site and the
to be important pioneers for the promotion of asepsis. patient information—patient’s name and a unique identification number.
CHEMICAL METHODS:
● Transport the specimen to the laboratory promptly or make provisions to
Storytime!!
- Just as physical methods are used mainly to achieve sterilization, chemical store the specimen in an environment that will not degrade the suspected
More than 100 years ago, Semmelweis demonstrated that routine handwashing
agents are used mainly as disinfectants. Chemical agents exert their killing organism(s).
can prevent the spread of disease.
effect by the following mechanisms: ● Notify the laboratory in advance if unusual pathogens or agents of
Semmelweis worked in a hospital in Vienna where maternity patients were dying
● Reaction with components of the cytoplasmic membrane: Damage to the bioterrorism are suspected.
at an alarming rate. Most of the maternity patients who died had been treated
integrity of the cytoplasmic membrane causes the cytoplasm and its contents
by medical students who worked on cadavers during an anatomy class before PATIENT COLLECTED SPECIMEN:
to leak out, resulting in cell death
beginning their rounds in the maternity ward.
● Denaturation of cellular proteins: effectively disrupts the metabolism of the - Medical personnel should provide patients with thorough instructions on how to
Because the students did not wash their hands between touching the dead and
cells. collect the sample. It should not be assumed that the patient knows how to
the living (handwashing was an unrecognized hygienic practice at the time),
● Reaction with the thiol (–SH) groups of enzymes through inactivation collect a particular type of specimen.
pathogenic bacteria from the cadavers were transmitted by the hands of the
● Damage of RNA and DNA: inhibits the replication of the organism - The most effective method is to provide verbal and written instructions!
students to the mothers.
● Urine
Type: Chemical Agents: Actions: Applications and
The result was a death rate five times higher for mothers who delivered in the
▪ First morning specimen is preferred because it provides a more
Precautions: hospital in contrast tothe mothers who delivered at home. Of women who
concentrated sample.
Alcohols (50%- Ethanol, isopropanol, Denature proteins; Skin antiseptics deliveredtheir babies in hospitals, 25% died of childbed fever (puerperalsepsis),
- The patient collects this specimen following cleansing of the external genitalia
70%) benzyl alcohol; make lipids soluble Disinfectants; kill later found to be caused by infection with Streptococcus pyogenes.
to reduce the presence of indigenous flora.
Aldehydes (in Formaldehyde (8%), React with NH2, – endospores; toxic After the death of Semmelweis, Lister, an academic surgeon, benefited by
- Patients are asked to void without collecting the first portion of the urine flow
solution glutaraldehyde SH, and –COOH to humans reading Pasteur’s works about bacteria as causes of infection before he ventured
and instead to collect the middle portion.
(2%) groups into studies of antisepsis.
- The first portion of the urine flow washes contaminants from the urethra, and
Halogens Tincture of iodine (2% Inactivates Skin disinfectants In 1867, Lister introduced handwashing and the use of phenol as an antimicrobial
the midstream portion is more representative of the urine in the bladder.
in 70% proteins Used to disinfect agent for surgical wound dressings to British surgery.
▪ Personnel who collect catheterized specimens should also use this
Alcohol Reacts with water drinking water; His principles were gradually, although reluctantly, adopted in Britain, and the
technique to eliminate organisms carried up the urethra during
Chlorine and to form surface mortality rate for amputation decreased from 45% to 15%. The Listerian technique
catheterization.
chlorine compounds hypochlorous disinfectants was approved in the United States at the first official meeting of the American
● Sputum
acid (HClO); Surgical Association in 1883, 20 years after Semmelweis’ initial publications.
▪ Sputum specimens are often collected for the diagnosis of bacterial
oxidizing agent This was the beginning of infection control.
pneumonia. Lower respiratory tract specimens are among the most difficult
Heavy metals Silver nitrate (AgNO3) Precipitates Eye drop (1%
specimens to collect adequately because they are contaminated with
proteins solution)
SPECIMEN COLLECTION AND PROCESSING: oropharyngeal flora.
Reacts with –SH Disinfectant: toxic
▪ Other specimens, such as blood or a bronchoalveolar lavage (BAL), may
groups; lyses cell at high - The major goal of the microbiology laboratory is to aid in the diagnosis of
be more accurate in detecting the etiologic agent (i.e., the microorganism
membrane Concentrations infectious diseases. Appropriate specimen selection, collection, and
causing the disease).
transportation are critical if laboratory results are used to provide information
▪ Collection of a quality sputum sample requires thorough patient education
Detergents Quaternary Disrupt cell Skin antiseptics; that establishes a diagnosis and successful treatment.
and medical personnel oversight of the process.
ammonium membranes disinfectants
▪ The first early morning specimen is preferred.
compounds
 ▪ The patient needs to understand the difference between sputum, saliva,
and nasal secretions. The patient should rinse the mouth with water and
expectorate with the aid of a deep cough directly into a sterile container
(expectorated sputum).
▪ A single specimen should be adequate for detection of bacterial lower
respiratory
▪ tract infection. If fungal or mycobacterial infections are suspected, three
separate early morning specimens are appropriate.
▪ Respiratory therapy technicians may assist patients who are unable to
expectorate a respiratory specimen.
▪ These specimens may be collected through aerosol induction, in which the
patient breathes aerosolized droplets of a solution that stimulates cough
reflex (induced sputum).
● Stool:
▪ The specimen of choice for the detection of gastrointestinal pathogens is
stool.
▪ A rectal swab can be submitted for bacterial culture as long as fecal
material is visible on the swab.
▪ A single specimen that has yielded a negative result is not usually sufficient
to exclude bacteria or parasites:
- If a bacterial infection is suspected, three specimens should be collected—one
a day for 3 days.
- If parasites are suspected, three specimens collected within 10 days should be
sufficient for microscopic detection of ova and parasites.
▪ Specimens should never be taken from the toilet and should not be
contaminated with urine.
▪ The appropriate ratio of stool to preservative is 1 : 3, and the patient must
understand that if this ratio is not met, the test will be invalid.
LABELING AND REQUISITIONS:
- It is important that correct patient identification be put on the specimen and
the requisition. The specimen label must contain sufficient information for the
specimen and requisition to be matched up when received in the laboratory.
- Proper identification of each specimen includes a label firmly attached to the
container with the following information:
● Name
● Identification number
● Room number
● Physician
● Culture site
● Date of collection
● Time of collection
- Some specimens that will not be transported or processed immediately can be
maintained by being stored under certain conditions.
- Some specimens, such as urine, stool, sputum, swabs (not for anaerobes),
foreign devices such as catheters, and viral specimens can be maintained at
refrigerator temperature (4° C) for 24 hours.
- Pathogens that are cold sensitive may be found in other specimens, and those
specimens should be kept at room temperature if culture is to be performed.
- This includes samples that might contain anaerobic bacteria as well as most
other sterile body fluids, genital specimens, and ear and eye swabs.
SPECIMEN STORAGE:
- Some specimens that will not be transported or processed immediately can be
maintained by being stored under certain conditions.
- Some specimens, such as urine, stool, sputum, swabs (not for anaerobes),
foreign devices such as catheters, and viral specimens can be maintained at
refrigerator temperature (4° C) for 24 hours.
- Pathogens that are cold sensitive may be found in other specimens, and those
specimens should be kept at room temperature if culture is to be performed.
- This includes samples that might contain anaerobic bacteria as well as most
other sterile body fluids, genital specimens, and ear and eye swabs.
- If cerebrospinal fluid is not processed immediately, it can be stored in a 35° C
incubator for 6 hours
ANTIMICROBIAL SUSCEPTIBILITY TESTING:
- Antimicrobial susceptibility testing is performed on bacteria isolated from
clinical specimens to determine which antimicrobial agents might be effective
in treating infections caused by the bacteria.
- In clinical laboratories, susceptibility testing is usually performed by a disk
diffusion or dilution (minimal inhibitory concentration [MIC]) method. Standards
that describe these methods are published and frequently updated by the
Clinical and Laboratory Standards Institute (CLSI),
Reasons and Indications for Performing Antimicrobial Susceptibility Testing:
● Antimicrobial susceptibility testing should be performed on a bacterial isolate
from a clinical specimen if the isolate is determined to be a probable cause of
the patient’s infection and the susceptibility of the isolate to particular
antimicrobial agents cannot be reliably predicted based on previous
experience with the bacteria.
● Susceptibility testing of isolates can also provide information on decreases in
the susceptibility of bacteria to antimicrobials.
Selecting Antimicrobial Agents for Testing and Reporting:
● Approximately 50 antimicrobial agents are presently used for treating bacterial
infections, and many of these have comparable clinical efficacy.
● Each laboratory must determine which agents are appropriate for routine
testing against various organisms (or organism groups) in its particular setting.
Bacteria: Drug: Result:
Escherichia coli Ampicillin S bacterial suspension containing approximately 1.5 × 108 CFU/mL.
(source: urine Cephalothin S ▪
Gentamicin S stable alternative to barium sulfate to achieve turbidity comparable with
Nitrofurantoin S that of the McFarland standard.
Trimethoprim/ sulfamethoxazole S TRADITIONAL ANTIMICROBIAL SUSCEPTIBILITY TEST METHODS:
E. coli (source: Ampicillin R
urine) Ampicillin-sulbactam R
Cefoxitin S
Cephalothin R
Ciprofloxacin S
Gentamicin S
Nitrofurantoin S
Trimethoprim/ sulfamethoxazole R
Reporting of Susceptibility Test Results:
● As noted, reporting protocols should be developed following discussion with
infectious disease clinicians, pharmacists, and others who have clinical
experience with the antimicrobial therapy practices of the particular institution.
Inoculum Preparation and Use of McFarland Standards:
● Inoculum Preparation:
▪ It is one of the most critical steps in susceptibility testing. Inocula are
prepared by adding cells from four to five isolated colonies of similar colony
morphology growing on a non-inhibitory agar medium to a broth medium
and then allowing them to grow to the log phase.
▪ Inoculum can also be prepared directly by suspending colonies grown
overnight on an agar plate directly in broth or saline.
▪ This direct inoculum suspension preparation technique is preferred for
bacteria that grow unpredictably in broth (e.g., fastidious bacteria).
Because it does not rely on growth in an inoculum broth, the use of fresh
(16- to 24-hour) colonies is imperative.
● McFarland Turbidity Standards:
▪ The inoculum concentration of bacteria to be tested must be
standardized.
▪ The most widely used method of inoculum standardization involves
comparing the turbidity of the inoculum preparation to McFarland turbidity
standards.
▪ McFarland standards can be prepared by adding specific volumes of 1%
sulfuric acid and 1.175% barium chloride to obtain a barium sulfate solution
with a specific optical density.
▪ The most commonly used is the McFarland 0.5 standard: 99.5 mL of 1%
sulfuric acid and 0.5 mL of 1.175% barium chloride.
▪ This solution is dispensed into tubes comparable with those used for
inoculum preparation, which are sealed tightly and stored in the dark at
room temperature.
▪ The McFarland 0.5 standard provides turbidity comparable with that of a
Recently, suspensions of latex particles have been used as a simpler, more
● Dilution Susceptibility Testing Methods:
▪ Principle: Dilution antimicrobial susceptibility test methods are used to
determine the MIC ((Minimum Inhibitory Concentration) , or the lowest
concentration of antimicrobial agent required to inhibit the growth of the
bacterium.
 ▪ Varying concentrations of an antimicrobial agent are added to broth or
agar media. Generally serial twofold-dilution concentrations are tested
(expressed in µg or mcg/mL).
▪ Once the MIC is determined, the organism is interpreted as non-susceptible,
susceptible, intermediate, or resistant to each agent with the use of a table
provided in the CLSI dilution testing document or in the FDA-approved
package insert for the antimicrobial.
▪ For each antimicrobial agent, the MIC breakpoint separates susceptible
from resistant results.
▪ For each antimicrobial agent, the MIC breakpoint separates susceptible
from resistant results.
● Kirby Bauer Disk Diffusion Testing:
▪ Principle: The disk diffusion test, also commonly known as the KirbyBauer
test.
▪ Briefly, a McFarland 0.5 standardized suspension of bacteria is swabbed
over the surface of a Mueller-Hinton agar plate, and paper disks containing
specific concentrations of antimicrobial agent are placed onto the
inoculated surface.
▪ After overnight incubation, the diameters of the zones produced by
antimicrobial inhibition of bacterial growth are measured, and the result is
interpreted as non-susceptible, susceptible, intermediate, or resistant to a
particular drug according to preset criteria.
QUALITY ASSURANCE:
- Laboratories have always taken measures to control the testing performed on
patient specimens. This effort has been termed quality control (QC).
- It is defined as the measures designed to ensure the medical reliability of
laboratory data.
- Examples are checking media and reagents with specific organisms to
determine whether expected results are obtained and documenting that the
instrumentation meets all operating parameters before it is used on patient
samples.
General Guidelines for Establishing Quality Control:
● All QC activities that take place must be recorded to prove their existence. All
record sheets must list tolerance limits, when applicable, so that the person
recording the results will always know whether the value being recorded is
acceptable. Corrective action must also be recorded when any measurement
falls outside a tolerance limit.
● A QC program for a laboratory must include procedures for control of the
following items: temperature, equipment, media, reagents, susceptibility
testing, and personnel.
THREE STAGES OF ACTIVITIES THAT AFFECT LABORATORY TESTING ACTIVITY OUTCOMES:
Stage: Activities:
Preanalytic Phase Test ordering
Order transcription
Specimen collection
Specimen identification
Specimen transport
Analytic Phase Sample testing
Postanalytic Phase Result delivery Result review Action taken on basis of result.
TOPIC 4: GRAM POSITIVE COCCI: STAPHYLOCOCCI AND STREPTOCOCCI
GRAM POSITIVE COCCI: STAPHYLOCOCCI
o General Characteristics:
- The genus name, Staphylococcus, is derived from the Greek term staphle,
meaning “bunches of grapes.”
- Although the Gram stain can be characteristic of staphylococci,
microscopy alone cannot differentiate staphylococci from other gram-
positive cocci.
- Staphylococci are members of the newly formed family
Staphylococcaceae.
o Clinically Significant Species:
● Staphylococcus aureus
▪ S. aureus is the most clinically significant species of staphylococci.
▪ It is responsible for numerous infections ranging from relatively mild to
life-threatening. S. aureus can be recovered from almost any clinical
specimen and is an important cause of nosocomial infections.
▪ S. aureus continues to increase in importance as a community-acquired
pathogen and increasing drug resistance continues to be a concern
with this common isolate.
o What is a virulence factor?
- Virulence is described as an ability of an organism to infect the host and
cause a disease. Virulence factors are the molecules that assist the
bacterium colonize the host at the cellular level
Virulence Factor: Description:
Enterotoxins - Staphylococcal enterotoxins are heat-stable exotoxins
that cause various symptoms, including diarrhea and
vomiting.
- Nine serologically distinct enterotoxins have been
identified that fall into groups A through E and G
through J.
- Because the enterotoxins are stable at 100° C for 30
minutes, reheating contaminated food does not
prevent disease.
- Staphylococcal food poisoning is most commonly
caused by enterotoxins A, B, and D. Enterotoxins B and
C and sometimes G and I are associated with TSS.
- Enterotoxins B and C and sometimes G and I are
associated with TSS.
- Enterotoxin B has been linked to
staphylococcal pseudomembranous enterocolitis
Toxic Shock - TSST-1 causes nearly all cases of menstruating-
Syndrome Toxin-1 associated TSS.
- TSST-1 is a superantigen stimulating T-cell proliferation
and the subsequent production of a large amount of
cytokines that are responsible for the symptoms.
- TSST-1 is absorbed through
- the vaginal mucosa leading to the systemic effects
seen in TSS associated with tampon use.
Exfoliative Toxin - It is also known as epidermolytic toxin.
- It causes the epidermal layer of the skin to slough off
and is known to cause staphylococcal SSS, sometimes
referred to as Ritter disease.
- This toxin has also been implicated in bullous impetigo.
Cytolytic Toxins - S. aureus produces other extracellular
- proteins that affect red blood cells and leukocytes.
Enzymes: - Staphylocoagulase is produced mainly by S. aureus but
coagulase, the pathogenicity is uncertain.
protease, - Hyaluronidase This enzyme hydrolyzes hyaluronic acid
hyaluronidase, present in the intracellular ground substance that
and lipase makes up connective tissues, permitting the spread of
- are capable of bacteria during infection.
destroying tissue - Lipases are produced by both coagulase-positive and
and may facilitate CoNS. Lipases act on lipids present on the surface of
the spread of the skin, particularly fats and oil secreted by the
infection to sebaceous glands.
adjoining tissues
Protein A - Protein A is one of several cellular components that
have been identified in the cell wall of S. aureus.
- Probably the most significant role of protein A in
infections caused by S. aureus is its ability to bind the Fc
portion of immunoglobulin G (IgG). Binding IgG in this
manner can block phagocytosis and negate the
protective effect of IgG.
o Clinical Infections:
● Skin and Wound Infections
▪ Infections caused by S. aureus are suppurative. Typically, the abscess is
filled with pus and surrounded by necrotic tissues and damaged
leukocytes.
▪ Some common skin infections caused by S. aureus are folliculitis,
furuncles, carbuncles, and bullous impetigo. These opportunistic
 infections usually occur as a result of previous skin injuries, such as cuts, ▪ The initial clinical presentation of TSS consists of high fever, rash, and ▪ S. saprophyticus has been associated with UTIs in young women. This
burns, and surgical incisions. signs of dehydration, particularly if the patient has had watery diarrhea species adheres more effectively to the epithelial cells lining the urogenital
and vomiting for several days. In extreme cases, patients may be tract than other coagulase negative species.
FOLLICULITIS FURUNCLES
severely hypotensive and in shock. The rash is found predominantly on ⮚ Staphylococcus lugdunensis
It is a relatively mild inflammation of a Also known as boils. It can be an
the trunk but can spread over the entire body. ▪ S. lugdunensis can cause both community-associated and hospital
hair follicle or oil gland; the infected extension of folliculitis, are large, raised,
● Toxic Epidermal Necrolysis (Ten): acquired infections.
area is raised and red. superficial abscesses.
▪ It is a clinical manifestation with multiple causes; it is most commonly ▪ This organism can be more virulent and can clinically mimic S. aureus
CARBUNCLES BULLOUS IMPETIGO
drug induced, but some cases have been linked to infections and infections
Carbuncles occur when larger, more Bullous impetigo caused by S. aureus
vaccines. ▪ It is an important pathogen in infective endocarditis, septicemia, meningitis,
invasive lesions develop from multiple differs from streptococcal non-bullous
▪ The cause is unknown, but symptom appear to be due to a skin and soft tissue infections, UTIs, and septic shock. Endocarditis caused by
furuncles, which can progress into impetigo in that staphylococcal pustules
hypersensitivity reaction. Although it has a very similar initial presentation S. lugdunensis is particularly aggressive, frequently requiring valve
deeper tissues. are larger and surrounded by a small
to that of SSS, treatments differ. TEN can be resolved by the replacement, and infections have a high mortality rate.
In contrast to patients with furuncles, zone of erythema.
administration of steroids early in the initial stages of presentation, o Laboratory Diagnosis:
patients with carbuncles often present Bullous impetigo is a highly contagious
whereas steroids aggravate SSS. ● Specimen collection:
with fever and chills, indicating systemic infection that is easily spread by direct
● Food Poisoning: - Although the recovery of staphylococci requires no special procedures,
spread of the bacteria. contact, fomites, or autoinoculation.
▪ S. aureus enterotoxins, most commonly A (78%), D (38%), and B (10%), specimens should be taken from the site of infection after appropriate
have been associated with gastrointestinal disturbances. cleansing of the surrounding area to avoid contamination by the skin
▪ The mode of transmission: infected food handler. microbiota.
● Scalded Skin Syndrome: ▪ Disease occurs when food becomes contaminated with enterotoxin- - Specimen of choice: purulent exudates, joint fluids, aspirated secretions,
▪ SSS is a bullous exfoliative dermatitis that occurs primarily in newborns producing strains of S. aureus by improper handling and is then and other body fluids
and previously healthy improperly stored, which allows growth of the bacteria and resulting ● Microscopic Examination:
▪ young children. This syndrome is caused by staphylococcal exfoliative toxin production. - Numerous gram-positive cocci, along with polymorphonuclear cells in
or epidermolytic toxin produced by S. aureus, which is probably present ▪ Foods that are often incriminated in staphylococcal food poisoning purulent exudates, joint fluids, aspirated secretions, and other body
at a lesion distant from the site of exfoliation. include salads, especially salads containing mayonnaise and eggs; fluids, are easily seen when these sites are infected with staphylococci.
▪ The severity of the disease varies from a localized skin lesion in the form meat or meat products; poultry; egg products; bakery products with - Take note: A culture should be done regardless of the results of the
of a few blisters, pemphigus neonatorum, to a more extensive cream fillings; sandwich fillings; and dairy products. microscopic examination because the genus or species cannot be
generalized condition affecting 90% of the body, known as Ritter ▪ Foods kept at room temperature are especially susceptible to higher appropriately identified by microscopic morphology alone.
disease. levels of toxin production!! - An aspirate is the best sample, whereas a single swab would be less
▪ This lesion can progress to the generalized form, which is characterized ▪ The enterotoxins do not cause any detectable odor or change in the satisfactory for both culture and smear results. Optimally, clinicians
by cutaneous erythema followed by profuse peeling of the epidermal appearance or taste of the food. should send two swabs when requesting a Gram stain and culture.
layer of the skin. The typical pattern in which the erythema occurs is ▪ Symptoms appear rapidly (approximately 2 to 8 hours after ingestion of o Cultural Characteristics:
origination from the face, neck, axillae, and groin and then extension to the food) and resolve within 24 to 48 hours. Although no fever is ● On Sheep Blood Agar:
the trunk and extremities. associated with this condition, nausea, vomiting, abdominal pain, and ▪ On Sheep Blood Agar:
▪ The duration of the disease is brief, about 2 to 4 days. The incidence of severe cramping are common. Diarrhea and headaches can also ▪ S. aureus produce round, smooth, white, creamy colonies after 18 to 24
spontaneous recovery among children is high. The toxin is metabolized occur. hours of incubation at 35° C to 37° C. S. aureus can produce hemolytic
and excreted by the kidneys zones around the colonies.
OTHER STAPHYLOCOCCUS SPECIES:
▪ A profuse peeling of the epidermal layer of the skin of a newborn with ▪ S. epidermidis colonies are usually small- to medium-sized, nonhemolytic,
Scalded Skin Syndrome. ⮚ Staphylococcus epidermidis gray-to-white colonies
● Toxic Shock Syndrome: ▪ Infections caused by S. epidermidis are predominantly hospital acquired. ▪ S. haemolyticus produces medium-sized colonies, with moderate or
▪ TSS is a rare but potentially fatal, multisystem disease characterized by a ▪ Some predisposing factors are instrumentation procedures such as weak hemolysis and variable pigment production.
sudden onset of fever, chills, vomiting, diarrhea, muscle aches, and rash, catheterization, medical implantation, and immunosuppressive therapy. S. ▪ Colonies of S. lugdunensis are often hemolytic and medium sized,
which can quickly progress to hypotension and shock. epidermidis is a common cause of health care-acquired UTIs. Prosthetic although small colony variants can occur.
▪ It was first described by Todd in 1978 and was associated with use of valve endocarditis is most commonly caused by S. epidermidis o Identification Methods:
highly absorbent tampons, although some cases appeared in men, ▪ Infections associated with the use of implants, such as indwelling catheters ● Staphylococci ferment glucose, whereas micrococci fail to produce acid
children, and non-menstruating women. and prosthetic devices, are often caused by isolates shown to produce a under anaerobic conditions.
▪ The two categories of TSS are menstruating-associated and non- biofilm. Biofilm production is a key component in bacterial pathogenesis ● Catalase test: Presence of an enzyme that has an ability of the organism to
menstruating-associated. and is a complex interaction between host, indwelling device, and convert hydrogen peroxide (reagent) to water and oxygen. All
▪ Although non menstruating TSS has been associated with nearly any bacteria. staphylococci are catalase positive!
staphylococcal infection, many cases have been seen with postsurgical ⮚ Staphylococcus saprophyticus ● Positive reaction= presence of foam
infections and secondary to influenza virus infections.
 ● Coagulase test: Enzyme that has an ability to convert fibrinogen to fibrin. and pose a serious threat to healthinstitutions. Control of MRSA requires strict ● The streptococci and similar organisms can produce numerous exotoxins
Staphylococcus aureus are coagulase positive and the rest of the species adherence to infection control practices, including barrier protection, that damage intact red blood cells (RBCs).
are not. contact isolation, and handwashing compliance.
Hemolysis: Description:
● Positive reaction= presence of clot ● Vancomycin remains the treatment of choice for MRSA, but concerns with
Alpha (α) Partial lysis of RBCs around colony
● Bacitracin Susceptibility Test: Coagulase negative Staphylococci versus increasing resistance to glycopeptides call for the restrictive use of these
Greenish discoloration of area around colony.
Micrococci: drugs and selective reporting by the laboratory.
Beta (β) Complete lysis of RBCs around colony
● Bacitracin Resistant: Staphylococcus species ⮚ Vancomycin-Resistant Staphylococci:
Clear area around colony
● Bacitracin Susceptible: Micrococcus species ● Vancomycin is the drug of choice and sometimes the only drug available
Nonhemolytic No lysis of RBCs around colony
● Oxidase Test: The oxidase test detects the presence of a cytochrome for serious staphylococcal infections, and the development of vancomycin
No change in agar.
oxidase system that will catalyse the transport of electrons between resistance has been a serious concern for the medical community. In 1996,
Alpha-prime (α′) Small area of intact RBCs around colony
electron donors in the bacteria and a redox dye- tetramethyl-p-phenylene- the first vancomycin-intermediate Staphylococcus aureus (VISA) strains
or wide zone surrounded by a wider zone of complete
diamine. were recovered in Japan.
hemolysis
● Oxidase Positive: Micrococcus species (purple color)
GRAM POSITIVE COCCI: STREPTOCOCCI:
● Oxidase Negative: Staphylococcus species
o General Characteristics:
CLINICALLY SIGNIFICANT STREPTOCOCCI AND STREPTOCOCCUS-LIKE SPECIES:
● Streptococcus and Enterococcus spp. belong to the family
Streptococcaceae. Take note!
● Members of both genera are catalase-negative, gram-positive cocci that - Clinically isolated streptococci have historically been separated into β-
are usually arranged in pairs or chains. hemolytic streptococci or pyogenic (pus-forming) streptococci and species
● A negative catalase test result differentiates streptococci and enterococci that are non-β-hemolytic (nonpyogenic).
Biochemical Tests: Results:
from staphylococci. - Pyogenic streptococci isolated frequently from humans include
● Weak false-positive catalase reactions can be seen when growth is taken Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus
S. aureus S. chromogenes S. epidermidis
from media containing blood, owing to the peroxidase activity of dysgalactiae subsp. equisimilis, and Streptococcus anginosus group (some
Gram Stain Gram positive Gram positive Gram positive
hemoglobin. species are α-hemolytic or nonhemolytic).
Motility Negative Negative Positive
● Compared with other gram-positive cocci, the cells of enterococci and
Oxidase Negative Negative Negative
some streptococci appear more elongated than spherical. The
Catalase Positive Positive Positive ⮚ Streptococcus pyogenes
streptococcal cells are more likely to appear in chains when grown in broth
● Antigenic Structure and Virulence Factor:
Growth on high salt Positive Negative Negative cultures.
▪ S. pyogenes has a cell wall structure similar to that of other streptococci
containing media ● Most members of the genera Streptococcus and Enterococcus behave like
and gram-positive bacteria. The group antigen is unique, placing the
(Mannitol Salt Agar) facultative anaerobes. Because they grow in the presence of oxygen but
organism in Lancefield group A.
Acetoin Production Positive Negative Positive are unable to use oxygen for respiration, they should be considered
▪ M protein is attached to the peptidoglycan of the cell wall and extends
Mannitol fermentation Positive Variable Negative aerotolerant anaerobes.
to the cell surface.
Presence of DNase Positive Negative Negative ● Carbohydrates are metabolized fermentatively with lactic acid as the
Coagulase and Clumping Positive Negative Negative major end product; gas is not produced. Some species are capnophilic, Virulence Factor: Description:
factor requiring increased concentration of carbon dioxide (CO2), whereas the M protein M protein is attached to the peptidoglycan of the cell wall

growth of other species is stimulated by increased CO2, but CO2 is not and extends to the cell surface. The M protein is essential

required. for virulence. S. pyogenes colonizes the throat and skin on

⮚ Methicillin-Resistant Staphylococci
o Antigenic Structure: humans, making these sites the primary sources of
● The incidence of MRSA has been increasing for the past couple of
● Cell Wall Structure: Streptococci possess a typical gram-positive cell wall transmission.
decades.
consisting of peptidoglycan and teichoic acid. Streptolysin O It is responsible for hemolysis on SBA plates incubated
● The incidence of MRSA has been increasing for the past couple of
● Most streptococci, except for many of the viridans group, have a group or anaerobically.
decades. In addition, since the 1990s, incidence of community associated
common C carbohydrate (polysaccharide), which can be used to classify SLO lyses leukocytes, platelets, and other cells as well as
methicillin-resistant Staphylococcus aureus (CAMRSA) infections has
an isolate serologically. RBCs. SLO is highly immunogenic,and infected individuals
increased (>50% of S. aureus isolates in some areas of the United States),
● This classification scheme was developed in the 1930s by Rebecca readily form antibodies to the hemolysin. These antibodies
and these infections can befound in patients who lack traditional health
Lancefield. Lancefield was able to divide the streptococci into serologic can be measured in the antistreptolysinO (ASO) test to
care–associated risk factors, such as recent hospitalization, long-term care,
groups, designated by letters. Organisms in group A possess the same determine whether an individual has had a recent
dialysis, or indwelling devices.
antigenic C carbohydrate, organisms in group B have the same C infection with S. pyogenes.
● A third type of MRSA is termed health care–associated community-onset
carbohydrate, and so on. Streptolysin S Streptolysin S is oxygen stable, lyses leukocytes, and is
methicillin-resistant Staphylococcus aureus (HACO-MRSA
o Hemolysis: nonimmunogenic. The hemolys is seen around colonies
● MRSA infections—whether hospital-associated methicillinresistant
Staphylococcus aureus (HA-MRSA), HACO-MRSA,or CA-MRSA—are costly
 that have been incubated aerobically is due to
streptolysin S.
Hyaluronidase It is an enzyme that solubilizes the ground substance of
(spreading factor) mammalian connective tissues (hyaluronic acid).
It was postulated that the bacteria use this enzyme to
separate the tissue and spread the infection; however, no
evidence to support this hypothesis exists.
Streptococcal These toxins function as superantigens.
pyrogenic Streptococcal superantigens belong to a family of highly
exotoxins mitogenic proteins secreted individually or in
(erythrogenic toxins) combinations by many S. pyogenes strains
o Clinical Infections:
● Bacterial Pharyngitis
▪ The most common clinical manifestation of the Group A Streptococci
infection are pharyngitis and tonsillitis. Most cases of bacterial
pharyngitis are due to S. pyogenes.
▪ “Strep throat” is most often seen in children between 5 and 15 years of
age. After an incubation period of 1 to 4 days, an abrupt onset of illness
ensues, with sore throat, malaise, fever, and headache. Nausea,
vomiting, and abdominal pain are not unusual. The tonsils and pharynx
are inflamed. The cervical lymph nodes are swollen and tender.
▪ It is not unusual to isolate a nearly pure culture of S. pyogenes from the
throat of a child with fever and complaint of only a mild sore throat. The
symptoms subside within 3 to 5 days unless complications, such as
peritonsillar abscesses, occur.
● Pyodermal Infections
▪ Skin or pyodermal infections with Group A Streptococci (GAS) result in
impetigo, cellulitis, erysipelas, wound infection, or arthritis.
▪ Impetigo, a localized skin disease, begins as small vesicles that progress
to weeping lesions. The lesions crust over after several days. Impetigo is
usually seen in young children (2 to 5 years) and affects exposed areas
of the skin. Inoculation of the organism occurs through minor abrasions
or insect bites.
Pyodermal Description:
infections:
Impetigo A localized skin disease, begins as small vesicles that
progress to weeping lesions. The lesions crust over after
several days. Impetigo is usually seen in young children (2 to
5 years) and affects exposed areas of the skin. Inoculation
of the organism occurs through minor abrasions or insect
bites.
Erysipelas It is a rare infection of the skin and subcutaneous tissues
observed frequently in elderly patients. It is characterized
by an acute spreading skin lesion that is intensely
erythematous with a plainly demarcated but irregular
edge.
Cellulitis Can develop following deeper invasion by streptococci.
The infection can be serious or life-threatening with
bacteremia or sepsis. In patients with peripheral vascular ▪ Two serious complications, or sequelae, of GAS disease are rheumatic fever
disease or diabetes, cellulitis may lead to gangrene. and acute glomerulonephritis.
Scarlet Fever Infection with strains of S. pyogenes that produce 1. Rheumatic Fever:
streptococcal pyrogenic exotoxins can result in scarlet ● Rheumatic fever typically follows S. pyogenes pharyngitis. It is
fever. Strains of S. pyogenes infected with the temperate characterized by fever and inflammation of the heart, joints, blood
bacteriophage T12 produce streptococcal pyrogenic vessels, and subcutaneous tissues.
exotoxins. Scarlet fever, which appears within 1 to 2 days ● Attacks usually begin within 1 month after infection. The most serious
after bacterial infection, is characterized by a diffuse red result is chronic, progressive damage to the heart valves. Repeated
rash that appears on the upper chest and spreads to the infections can produce further valve damage.
trunk and extremities. The rash disappears over the next 5 to ● Rheumatic fever is rare in most developed countries, and it is no
7 days and is followed by desquamation longer a reportable disease in the United States. Acute rheumatic
fever and its chronic sequela, rheumatic heart disease, remain
problematic in developing countries and in some poor populations in
● Necrotizing Fasciitis:
industrialized countries.
▪ It is an invasive infection characterized by rapidly progressing inflammation
● The pathogenesis of rheumatic fever is poorly understood. Several
and necrosis of the skin, subcutaneous fat, and fascia. Although
theories have been proposed, including antigenic crossreactivity
uncommon, NF is a life-threatening infection.
between streptococcal antigens and heart tissue, direct toxicity
▪ Many different bacteria can cause destruction of the soft tissue in this
resulting from bacterial exotoxins, and actual invasion of the heart
manner, a clinical feature that has been described as “flesh-eating
tissues by the organism. Most evidence favors crossreactivity as
disease.” Depending on which organisms are cultured, NF may be
being responsible for the effects.
categorized as type 1, 2, or 3.
2. Acute Glumerulonephritis:
▪ Cases of NF were described in the eighteenth century, but the term was not
● It sometimes occurs after a cutaneous or pharyngeal infection. It is
conceived until 1952. In addition to flesh-eating bacteria syndrome, other
more common in children than in adults.
terms for NF have included suppurative fasciitis, hospital gangrene, and
● The pathogenesis appears to be immunologically mediated.
necrotizing erysipelas.
Circulating immune complexes are found in the serum of patients
▪ NF can occur as a result of trauma, such as burns and lacerations. In most
with acute glomerulonephritis, and it is postulated that these
cases, NF occurs in an individual with an underlying illness who has
antigen-antibody complexes deposit in the glomeruli.
sustained trauma to the skin. The break in the skin may become the portal
● Complement is subsequently fixed, and an inflammatory response
of entry for the bacteria. However, NF infections caused by Group
causes damage to the glomeruli, resulting in impairment of kidney
A streptococci occur in young, healthy individuals, and a break in the skin
function.
that served as portal of entry is not found in many cases.
● Treatment:
● Streptococcal Toxic Shock Syndrome:
Penicillin: drug of choice!
▪ Streptococcal TSS is a condition in which the entire organ system collapses,
Erythromicin: for patients allergic to Penicillin
leading to death. The exact portal of infection is unknown for most cases of
● Note: For patients who have a history of rheumatic fever,
streptococcal TSS, although minor injuries or surgeries have been
prophylactic doses of penicillin are given to prevent recurrent
implicated.
infections that might cause additional damage to the heart valves.
▪ The initial streptococcal infection is often severe (e.g., pharyngitis,
o Laboratory Diagnosis:
peritonitis, cellulitis, wound infections), and the symptoms that develop are
● An essential step in the diagnosis of streptococcal pharyngitis is proper
similar to symptoms of staphylococcal TSS. Patients are often bacteremic
sampling. The tongue should be depressed and the swab rubbed over the
and have NF.
posterior pharynx and each tonsillar area. If exudate is present, it should
▪ GAS associated with streptococcal TSS produce a
also be touched with the swab.
streptococcal pyrogen exotoxin, notably SpeA.
● Note: Examination of Gram stains of upper respiratory specimens or skin
▪ These toxins likely play a major role in the pathogenesis of this disease
swabs is of little value because these areas have considerable amounts of
functioning as superantigens leading to over simulation of the immune
gram-positive cocci as part of the normal bacterial biota.
response. Other virulence factors, such as SLO and various cell wall
● Colonies of S.pyogenes on sheep blood agar: small, transparent, and
antigens, can also contribute to toxic shock. Isolates with M1 and M3 are
smooth with a well-defined area of β-hemolysis
the most common strains associated with streptococcal TSS. Young
● Gram Stain: gram-positive cocci with some short chains
children, especially children with chickenpox (varicella), and elderly adults
● Suspect colonies can be Lancefield-typed using serologic methods, which
seem to be at greatest risk.
gives a definitive, rapid identification, or biochemical tests can be
● Poststreptococcal Sequelae:
performed. The correlation between presumptive identification using
 biochemical methods and the rapid definitive serologic method is high.
Most clinical laboratories choose to use serologic methods.
● Pyrrolidonyl α-naphthylamide (PYR) hydrolysis: PYR positive, hydrolyzes
PYR; whereas most other β-hemolytic groups are PYR negative.
● Bacitracin Susceptibility: Susceptible to Bacitracin; whereas most other β-
hemolytic groups are resistant
⮚ Streptococcus agalactiae:
● Antigenic Structure:
▪ All strains of S. agalactiae have the group B–specific antigen, an
acid-stable polysaccharide located in the cell wall.
▪ S. agalactiae is the only species that expresses group B antigen.
▪ Additionally, there are nine recognized capsular polysaccharide
serotypes. These type-specific antigens can be detected by
precipitin tests.
▪ Serotypes Ia, Ib, and II contain a terminal residue of sialic acid, which
is weakly immunogenic and can inhibit activation of the alternative
complement pathway.
Virulence Factor: Description:
homologous polysaccharide in the Prevents phagocytosis but is ineffective
capsule after opsonization. Sialic acid appears to
be the most significant component of
the capsule and a critical virulence
determinant.
Hemolysin No evidence exists that any of these
CAMP factor products plays a role in the virulence of
Neuraminidase this organism.
DNase hyaluronidase and protease
o CLINICAL INFECTIONS:
● Group B Streptococci were known for many years as the cause of mastitis
in cattle. It was not until Lancefield defined streptococcal classification in
the 1940s that their role in human disease was recognized. S. agalactiae
was identified in the 1970s as a significant cause of invasive disease in the
newborn, and it remains so today.
● Early-onset disease accounts for about 80% of the clinical cases in
newborns and is caused by vertical transmission of the organism from the
mother. Colonization of the vagina and rectal area with GBS is found in 10%
to 30% of pregnant women.
● Most infections of infants occur in the first 3 days after birth, usually within 24
hours. This infection is commonly associated with obstetric complications,
prolonged rupture of membranes, and premature birth.
● Early-onset infection often manifests as pneumonia and sepsis, and late-
onset infection manifests as meningitis and sepsis. The mortality rate is high,
and death usually occurs if treatment is not started quickly. The most
important determining factor seems to be the presence of GBS in the
VIRIDANS STREPTOCOCCI:
vagina of the mother.
● It is recommended that all pregnant women be screened for GBS at 35 to
37 weeks’ gestation.
GROUPS C AND G STREPTOCOCCI:
⮚ Streptococcus pneumoniae
● Also known as pneumococcus.
● Antigenic Structure:
▪ The cell wall of S. pneumoniae contains an antigen, referred to as C
substance, which is similar to the C carbohydrate of the various
Lancefield groups.
● Virulence Factors
▪ The characteristic of S. pneumoniae that is clearly associated with
virulence is the capsular polysaccharide. Strains that have lost the ability
to produce a capsule are nonpathogenic. In addition, opsonization of
the capsule renders the organism avirulent. Several toxins are produced,
including a hemolysin, an immunoglobulin A protease, neuraminidase,
and hyaluronidase. None of these have been shown to have a role in
disease production.
o Clinical Infections:
● S. pneumoniae is a common isolate in the clinical microbiology laboratory.
It is an important human pathogen that causes pneumonia, sinusitis, otitis
media, bacteremia, and meningitis.
● S. pneumoniae is the most frequently encountered isolate in children
younger than 3 years old with recurrent otitis media.
● S. pneumoniae, the number one cause of bacterial pneumonia, is
especially prevalent in elderly persons and in patients with underlying
disease. Of the more than 90 capsular serotypes, about a dozen account
for most pneumococcal pneumonia cases.
⮚ Pneumococcal Pneumonia:
● For an individual to contract pneumococcal pneumonia, the organism
must be present in the nasopharynx, and the individual must be deficient in
the specific circulating antibody against the capsular type of the colonizing
strain of S. pneumoniae.
● Pneumococcal pneumonia is characterized by sudden onset with chills,
dyspnea, and cough. The infection begins with aspiration of respiratory
secretions, which often contain pneumococci.
● The infecting organisms in the alveoli stimulate an outpouring of fluid, which
serves to facilitate the spread of the organism to adjacent alveoli. The
process stops when the fluid reaches fibrous septa that separate the major
lung lobes. This accounts for the “lobar” distribution of the infection—hence
the name.
o Laboratory Diagnosis:
● Gram Stain: appear as gram positive cocci in pairs (diplococci). The ends
of the cells are slightly pointed, giving them an oval or lancet shape.
● Colonies on Sheep Blood Agar: a large zone of α-hemolysis
● Bile Solubility Test: determines the lysis of S. pneumoniae in the presence of
bile salts. Bile SOLUBLE!
● Optochin Susceptibility: It correlates with optochin susceptibility; that is, S.
pneumoniae isolates are optochin SUSCEPTIBLE!
ENTEROCOCCUS:
- Viridans streptococci are constituents of the normal microbiota of the upper
respiratory tract, the female genital tract, and the gastrointestinal tract.
- The term viridans means “green,” referring to the α-hemolysis many species
exhibit.
- However, β-hemolytic and nonhemolytic species are also classified as viridans
streptococci. Viridans streptococci are fastidious, with some strains requiring
CO2 for growth.
- More than 30 species are now recognized. The current classification assigns
streptococci species in the viridans group to one of five groups:
1. S. mitis group (including S. mitis, S. pneumoniae, S. sanguis, S. oralis);
2. S. mutans group (including S. mutans and S. sobrinus);
3. S. salivarius group (including S. salivarius and S. vestibularis);
4. S. bovis group (including S. equinus, S. gallolyticus, S. infantarius, and S.
alactolyticus),
5. S. anginosus group (including S. anginosus, S. constellatus, and S.
intermedius).
o Clinical Infections:
● Viridans streptococci are oropharyngeal commensals that are regarded as
opportunistic pathogens. Although their virulence is low, they cause disease
if host defenses are compromised.
● These pathogens are the most common cause of subacute bacterial
endocarditis, a condition associated with a transient bacteremia. Viridans
streptococcal bacteremia is more common in children than in adults and is
usually more prevalent in patients with hematologic malignancies.
● Fatal cases have been characterized by fulminant cardiovascular collapse
or meningitis. Generally, the course of endocarditis is very slow; symptoms
may be present for weeks or months. Individuals whose heart valves have
been damaged by rheumatic fever are especially susceptible to
endocarditis from viridans streptococci.
● Besides bloodstream infections, oral infections such as gingivitis and dental
caries (cavities) are caused by viridans streptococci.
o Virulence Factors:
● Virulence factors that characterize the pathogenicity of viridians
streptococci have not been well established.
● A polysaccharide capsule and cytolysin have been identified in some
members of the anginosus group. Besides these, extracellular dextran and
cell surface–associated proteins (adhesins) have been implicated
inadherence and colonization of these organisms in endocarditis.
o Laboratory Diagnosis:
● It is extremely difficult to identify isolates of the viridans group to the species
level;
● All members are PYR-negative and leucine aminopeptidase (LAP)-positive.
● Viridans streptococci show typical Streptococcus characteristics on Gram
stain.
● On Sheep Blood Agar: Colonies are small and are surrounded by a zone of
α-hemolysis; some isolates are β-hemolytic or nonhemolytic.
o General Characteristics:
● Enterococci were previously classified as group D streptococci. This group
consists of gram-positive cocci that are natural inhabitants of the intestinal
tracts of humans and animals.
 ● The commonly identified species in clinical specimens are E. faecalis and E.
faecium. Other species such as E. durans, E. avium, E. casseliflavus, E.
gallinarum, and E. raffinosus are observed occasionally.
● All species produce the cell wall–associated group D antigen in the
Lancefield classification system
● Most enterococci are nonhemolytic or α-hemolytic, although some strains
show β-hemolysis.
● Enterococci sometimes exhibit a pseudocatalase reaction—weak bubbling
in the catalase test.
o Clinical Infections:
● Enterococci are frequent causes of nosocomial infections. Of these, urinary
tract infections (UTIs) are the most common. followed by bacteremia. UTI is
often associated with urinary catheterization or other urologic
manipulations. Prolonged hospitalization is a risk factor for acquiring
enterococcal bacteremia.
● Bacteremia is often observed in patients receiving hemodialysis,
immunocompromised patients with a serious underlying disease, or patients
who have undergone a prior surgical procedure. Endocarditis from
enterococci is seen mainly in elderly patients with prosthetic valves or
valvular heart disease.
● Enterococci account for about 5% to 10% of infections in patients with
bacterial endocarditis.
o Virulence factors:
● The virulence factors that contribute to the pathogenicity of enterococci
are incompletely understood. The enterococci have a survival advantage
over other organisms in that they can grow in extreme conditions and are
resistant to multiple antimicrobial agents.
● Extracellular surface adhesin proteins, extracellular serine protease, and
gelatinase of E. faecalis are thought to play a role in the colonization of the
species and adherence to heart valves and renal epithelial cells. E. faecalis
also produces a twosubunittoxin, termed cytolysin. This toxin shows similarity
to bacteriocins produced by gram-positive bacteria and is expressed by a
quorum-sensing mechanism
o Laboratory Diagnosis:
● Standard procedures for collection and transport of blood, urine, or wound
specimens should be followed. The specimens should be cultured as soon
as possible with minimum delay.
● Trypticase soy or brain-heart infusion agar supplemented with 5% sheep
blood is routinely used to culture enterococci. Enterococci grow well at 35°
C in the presence of CO2 but do not require a high level of CO2 for growth.
If the clinical specimen is obtained from a contaminated site or is likely to
contain gram-negative organisms, elective media containing bile esculin
azide, colistin– nalidixic acid, phenylethyl alcohol, chromogenic substrates,
orcephalexin-aztreonam-arabinose agar should be used for isolation of
enterococci.
● Enterococcus spp. are identified based on their:
1. ability to produce acid in carbohydrate broth,
2. ability to hydrolyze arginine,
3. tolerance of 0.04% tellurite,
4. utilization of pyruvate,
5. ability to produce acid from methyl-α-D-glucopyranoside,
6. growth around 100-μg efrotomycin acid disk
7. motility
● E. faecalis is easily identified by its ability to grow in the presence of tellurite
o General Laboratory Diagnosis of Streptococci:
● Microscopy: Gram positive cocci in chains!
● Catalase: Streptococci vs Staphylococci
Streptococci are negative
● Hemolysis Test on Sheep Blood Agar:
▪ Beta-hemolytic (complete hemolysis/clearing)= S. pyogenes, S.
agalactiae, Enterococcus sp.
▪ Alpha-hemolytic (partial hemolysis) = S. pneumoniae, Enterococcus sp.
▪ Non-hemolytic= Enterococcus sp.
● Optochin Disk Susceptibility:
▪ Susceptible= S.pneumoniae (capsulated)
▪ Resistant = Viridans Streptococci (non-capsulated)
TOPIC 5: GRAM NEGATIVE COCCI: NEISSERIACEAE
General Characteristics of Neisseriaceae:
- Most Neisseria spp. are aerobic, nonmotile, non–spore-forming, gram-negative
diplococci.
- All species are cytochrome oxidase- and catalase-positive except for N.
elongata and N. bacilliformis, which are catalase-negative.
- Many Neisseria spp. are capnophilic, requiring carbon dioxide (CO2) for
growth, and have optimal growth in a humid atmosphere.
- The natural habitat of Neisseria spp. is the mucous membranes of the
respiratory and urogenital tracts.
- Neisseria gonorrhoeae (often called gonococci) and Neisseria meningitidis
(meningococci) are the primary human pathogens of the genus.
⮚ Neisseria gonorrheae
• Humans are the only natural host for N. gonorrhoeae, the agent of
gonorrhea. Gonorrhea is an acute pyogenic infection of non-ciliated
columnar and transitional epithelium; infection can be established at
any site where these cells are found.
• Gonococcal infections are primarily acquired by sexual contact and
occur primarily in the urethra, endocervix, anal canal, pharynx, and
conjunctiva.
Pathogenesis: The organism adheres to the genitourinary epithelium via
pili, then invades the epithelial layer and provokes a local acute
inflammatory response. Variation in the proteins of the pili means that
infection does not provide protection against re-infection, therefore
infections with another strain of different antigenic structure are
possible.
History: The first use of the term gonorrhea, meaning a “flow of seed,”
was in the second century when the urethral discharge was mistaken for
semen. For centuries thereafter, the diseases syphilis and gonorrhea
were confused because the two were often present together in the
same individual.
Epidemiology: N. gonorrhoeae infections are most commonly
transmitted by sexual contact.
The primary reservoir is the asymptomatic carrier. In the United States,
gonorrhea is a national reportable disease; all culture-confirmed cases
must be reported to state health laboratories.
Gonorrhea is second to Chlamydia trachomatis in the number of
confirmed sexually transmitted bacterial infections.
Epidemiology: Sexually active teens and young adults have the highest
rates of infection. Most cases in women are seen between ages 15 and
19 years; most cases in men occur between ages 20 and 24 years.
o Clinical Infections
● Gonorrhea has a short incubation period of approximately 2 to 7 days. In
men, acute urethritis, usually resulting in purulent discharge and dysuria
(painful urination), is the most common manifestation.
● Complications in male patients include ascending infections such as
prostatitis and epididymitis.
● The endocervix is the most common site of infection in women. Symptoms
of infection, when present, include dysuria, cervical discharge, and lower
abdominal pain.
● However, 50% of cases in women may be asymptomatic leading to
complications such as pelvic inflammatory disease, which may cause
sterility, ectopic pregnancy, or perihepatitis (Fitz-Hugh–Curtis syndrome).
● Other conditions associated with N. gonorrhoeae include anorectal and
oropharyngeal infection.
● Gonorrhoea can be prevented by avoiding sexual contact with individuals
at high risk and using effective barrier contraception.
● Contacts of infected individuals should be traced and treated. At present
vaccine development is precluded by the antigenic variation that occurs
within the pili.
● Newborns can acquire ophthalmia neonatorum, a gonococcal eye
infection, during vaginal delivery through an infected birth canal. This
condition, which can result in blindness if not treated immediately, is rare in
the United States because application of antimicrobial eyedrops, generally
erythromycin, to every infant at birth is legally required.
● Treatment: Erythromycin ophthalmic ointment should be instilled into both
eyes of neonates as soon as possible after delivery, regardless of whether
they are delivered vaginally or by cesarean delivery.
o Laboratory Diagnosis:
A. Specimen Collection and Transport
• Specimen of choice: genital sources or from other sites, such as the
rectum, pharynx, and joint fluid.
• Urethral discharge in male patients and endocervical discharge in
female patients.
• In men, purulent discharge can be collected directly onto a swab for
culture. When no apparent discharge is present, the swab is inserted up
to 2 cm into the anterior urethra and slowly rotated to collect material.
• Swabs for rectal culture should be inserted 4 to 5 cm into the anal
canal.
 • Calcium alginate and cotton swabs are inhibitory to N. gonorrhoeae, so
Dacron or rayon swabs are preferred.
• N. gonorrhoeae is extremely susceptible to drying and temperature
changes, direct plating of the specimen to gonococcal selective media
gives optimal results.
• When direct plating is not possible, inoculated swabs should be placed
in a transport system such as Amies medium with charcoal, transported
to the laboratory promptly, and plated within 6 hours.
B. Microscopic examination: The demonstration of gram-negative intracellular
diplococci from the urethral discharge of a symptomatic male correlates at
a rate of 89% with culture and is evidence of gonococcal infection. The
gonococci are in pairs with adjacent sides flattened, giving them a kidney
shape. Because women have vaginal commensal microbiota that
resembles gonococci, direct Gram stain correlates in only 50% to 70% of
cases with culture.
C. Culture: N. gonorrhoeae typically does not grow on SBA; the medium of
choice for cultivation is Chocolate agar: small, gray to tan, translucent, and
raised after 24 to 48 hours of incubation.The gonococci can produce
autolytic enzymes that may make the isolate nonviable on prolonged
incubation. A fresh subculture should be used for identification tests.
Modified Thayer-Martin (MTM II) Agar is used for the isolation of pathogenic
Neisseria from specimens containing mixed flora of bacteria and fungi.
D. Oxidase Disk Test: In the filter paper method, oxidase reagent (1% dimethyl-
p-phenylenediamine dihydrochloride or tetramethyl-p-phenylenediamine
dihydrochloride) is placed on filter paper, and a colony from the plate is
rubbed onto the reagent with an applicator stick or a nonnichrome
loop. (+) = deep purple to black
E. Carbohydrate Utilization Test: The traditional method for the identification
of Neisseria spp. was carbohydrate utilization in cystine trypticase agar
(CTA), containing 1% of the individual carbohydrate and phenol red as a
pH indicator. If the organism uses the particular carbohydrate, acid,
characterized by a yellow color, is produced in 24 to 72 hours. N.
gonorrhoeae is positive for glucose only!!
Nonculture Methods: Alternatives to culture for N. gonorrhoeae are available. These
methods detect gonococcal antigen or nucleic acid directly in urine or cervical or
urethral exudates. Nucleic acid detection methods include both nonamplified and
amplified probe technologies. Nucleic acid amplification test (NAAT) on urethral or
vaginal swabs, or urine is the optimal diagnostic technique!
o Antimicrobial Resistance:
- Almost all strains of N. gonorrhoeae in the United States were previously
susceptible to penicillin. In 1976, the first plasmid-mediated penicillinase-
producing Neisseria gonorrhoeae (PPNG) strains were isolated, largely
imported from Southeast Asia or Africa. By 1980, more than half of the
reported PPNG cases were of domestic origin.
- In addition to penicillin resistance, N. gonorrhoeae exhibits plasmid-
mediated resistance and chromosomally mediated resistance to
tetracycline, spectinomycin, and fluoroquinolones (e.g., ciprofloxacin).
- Susceptibility testing for N. gonorrhoeae is not routinely performed.
o Treatment:
● Drug of Choice: Cephalosporins: ceftriaxone and cefixime.
● Because of the widespread increase in fluoroquinolone resistance
throughout the United States, the CDC issued a recommendation in 2007
that fluoroquinolones no longer be used as therapy for gonorrhea and
associated gonococcal infections.
⮚ Neisseria meningitidis
o General Characteristics:
- Similar to N. gonorrhoeae, N. meningitidis is also found only in
humans.
- However, N. meningitidis can be found as a commensal as well as
an invasive pathogen.
- It is an important etiologic agent of endemic and epidemic
meningitis and meningococcemia and rarely pneumonia, purulent
arthritis, or endophthalmitis.
o Epidemiology: N. meningitidis can be found on the mucosal surfaces of
the nasopharynx and oropharynx in 30% of the population.
Mode of transmission: close contact with respiratory droplet
secretions from a carrier to a new host and the highest incidence of
meningococcal infection being found in infants and adolescents.
o Clinical Infections: The incubation period ranges from 1 to 10 days. The
bacteria adhere to the nasopharyngeal mucosa leading to
colonization. When N. meningitidis enters the bloodstream, two main
diseases can occur: fulminant meningococcemia and may progress
to meningitis. Meningococcemia, or sepsis, may occur with or without
meningitis and carries a 25% mortality rate, even if treated.
o Manifestations:
- Purpura -hemorrhaging of blood into the skin and mucous
membranes producing bruises
- Petechial skin rash -pinpoint red spot caused by hemorrhage.
- Others include tachycardia, and hypotension can develop during
bacteremia, and thrombosis is common. In some cases, the disease
becomes fulminant and spreads rapidly, causing disseminated
intravascular coagulation, septic shock, or hemorrhage in the
adrenal glands (also known as the Waterhouse Friderichsen
syndrome).
- Death may occur in 12 to 48 hours from onset.
- Meningitis is characterized by an abrupt onset of frontal headache,
stiff neck (nuchal rigidity), confusion, and photophobia. The fatality
rate is 10% to 15%; an additional 10% to 20% have sequelae such as
neurologic complications or seizures.
o Pathogenesis:
● An antiphagocytic polysaccharide capsule that allows survival in the
bloodstream.
● Lipo-oligosaccharide activates complement and stimulates
cytokine release, which leads to shock and disseminated intravas-
cular coagulation (DIC).
● The organism hijacks the ß2-adrenoreceptor to cross the brain
vascular endothelium.
● Meningococci cross mucosal epithelium by
endocytosis. Meningococcal meningitis is characterized by fever,
neck stiffness and reduced consciousness. The petechial rash, a sign
of septicaemia, may be present without other signs of meningitis.
Septic or reactive arthritis may develop.
o Laboratory Diagnosis:
A. Specimen Collection and Transport: Specimen of choice: cerebrospinal
fluid (CSF), blood, nasopharyngeal swabs and aspirates, joint fluids.
B. Direct Microscopic Examination: They appear as intracellular and
extracellular gram-negative diplococci.
C. Direct Microscopic Examination: They appear as intracellular and
extracellular gram-negative diplococci. On Sheep Blood agar: grey
and medium sized and appear round, smooth, moist, glistening, and
convex, with a clearly defined edge. Encapsulated strains are
mucoid. On Chocolate agar: large, colorless-to-grey, opaque colonies
N. GONORRHOEA IS KIDNEY SHAPED N. MENINGITIDIS IS SEMICIRCULAR
WITH APPOSING ENDS CONCAVE. DIPLOCOCCUS WITH FLAT APPOSING
ENDS.
D. Biochemical tests: Carbohydrate Utilization Test: Neisseria meningitidis
ferments glucose and maltose!. ONPG test: A rapid o-nitrophenyl-β-
Dgalactopyranoside (ONPG) test, which detects lactose utilization, is
usually positive in 30 minutes for N. lactamica.
The diagnosis is usually made clinically and confirmed by culture of
blood, aspirate from the rash and CSF. Rapid antigen detection or NAAT
on CSF and blood are sensitive and reliable.
Infection is life-threatening and rapidly progressive; treatment should
not await laboratory confirmation or hospitalization
o Laboratory-Acquired Disease:
- In 2000, two cases of fatal laboratory-acquired meningococcal disease
were reported to the CDC. Both clinical microbiologists had examined
plates, performed Gram stains, subcultured, or performed slide
agglutination serogrouping on patient isolates on the open bench. Isolates
recovered from the laboratory scientists were identical to patient
organisms.
- Because exposure to N. meningitidis aerosols increases risk of infection, the
CDC recommends use of a biosafety level 2 cabinet for manipulation of
suspected isolates of N. meningitidis from sterile sites.
o Treatment: Drug of choice – for meningitis: penicillin; For meningococcemia is
best treated with third-generation cephalosporins. Routine susceptibility testing
is not recommended.
o Vaccine: The quadrivalent vaccine Menactra is a polysaccharide-protein
conjugated vaccine with antigens to serogroups A, C, Y, and W-135. This
conjugate vaccine is licensed for people 2 to 55 years old. This vaccine does
not protect against meningitis caused by serogroup B because group B
polysaccharide is a very poor immunogen in humans. Practices recommends
routine vaccination be administered to individuals at age 11 to 12 years with a
booster dose at age 16 years.
● A protein-conjugated serogroup C vaccine is more than 90% efficient and
has vastly reduced the incidence where it has been introduced.
 ● An effective vaccine against serogroup B is not available, although a
vaccine based on membrane proteins specific for epidemic strains has
shown promise.
● Close contacts of patients with meningococcal meningitis should be given
‘prophylaxis’ with rifampicin or ciprofloxacin.
⮚ Moraxella catarrhalis
o General Characteristics:
- M. catarrhalis is in the family Moraxellaceae, which contains three
genera: Moraxella, Acinetobacter, and Psychrobacter. Isolated only
from humans, M. catarrhalis is a commensal of the upper respiratory
tract.
- Severe infections are seen in immunocompromised hosts;
hospital outbreaks of M. catarrhalis respiratory infections have
occurred.
- It usually produces ß-lactamase.
o Clinical Infections:
- M. catarrhalis is an opportunistic pathogen and is recognized as a
cause of upper respiratory tract infection in otherwise healthy children
and the elderly.
- It also can cause lower respiratory infections, especially in adults with
chronic obstructive pulmonary disease. Predisposing factors include
advanced age, immunodeficiency, neutropenia, and chronic
debilitating diseases.
- M. catarrhalis has been reported as the third most common cause of
acute otitis media and sinusitis in children.
- Severe infections are seen in immunocompromised hosts;
hospital outbreaks of M. catarrhalis respiratory infections have
occurred.
⮚ Commensal Neisseria Species
o General Characteristics: Other Neisseria spp. exist as normal inhabitants of
the upper respiratory tract. Referred to as commensals, saprophytes, or
nonpathogens, these species are occasionally isolated from the genital
tract. The commensal Neisseria spp. rarely cause disease, but they have
sporadically been implicated in meningitis, endocarditis, prosthetic valve
infections, bacteremia, pneumonia, empyema, bacteriuria, osteomyelitis,
and ocular infections. In the clinical laboratory, when isolated from
respiratory specimens, the commensal Neisseria spp. are usually identified
only by Gram stain and gross colony morphology and are called Neisseria
spp. or “usual oral biota.” Further identification by biochemical tests is not
done. Common laboratory tests and observations do not always
adequately differentiate all the commensal species from one another or
from the pathogens. Additional tests used to help further differentiate these
organisms include growth on nutrient agar at 35°C, growth on SBA or CHOC
agar at 22°C, and the reduction of nitrate and nitrite.
● Neisseria cinerea: It has received considerable attention in recent years
because of its misidentification as N. gonorrhoeae in some commercial
identification systems.
● Neisseria flavescens: N. flavescens (flavescens means “yellow”) is a yellow-
pigmented Neisseria species that is asaccharolytic. It can be differentiated
from N. cinerea by its ability to grow on SBA or CHOC agar at 22°C and its
yellow colonies.
● Neisseria lactamica: N. lactamica is commonly found in the nasopharynx
of infants and children and, similar to Neisseria polysaccharea, is commonly
encountered in meningococcal carrier surveys.
- It is rarely isolated from adults. It is the only Neisseria species that uses
lactose—hence its species designation lactamica.
- N. lactamica can be misidentified as N. meningitidis because of its
similar colony morphology (N. lactamica is slightly smaller)
- ONPG test: A rapid o-nitrophenyl-β-Dgalactopyranoside (ONPG)
test, which detects lactose utilization, is usually positive in 30 minutes for
N. lactamica.
● Neisseria mucosa: Colonies of N. mucosa are large, often adherent to the
agar, and very mucoid, giving the species its name. N. mucosa is usually
isolated from the nasopharynx of children or young adults. It has also been
isolated from the airways of dolphins. This organism has been documented
to cause pneumonia in children.
● Neisseria sicca: The colonies of N. sicca are usually dry, wrinkled, adherent,
and breadcrumb-like. The word sicca in Latin means “dry.” N. sicca and N.
subflava biovar perflava are usually the two most common Neisseria spp.
found in the respiratory tract of adults.
● Neisseria subflava: The species name for N. subflava means “less yellow”.
The organism is considered to be part of the upper respiratory microbiota. It
consists of three biovars that differ from one another by their patterns of
carbohydrates
● Neisseria weaveri: N. weaveri is normal oral microbiota in dogs and can be
found in humans in infections following dog bites. It is catalase-positive and
does not produce acid from any of the carbohydrates traditionally used to
identify Neisseria spp.
● Neisseria elongata: Neisseria elongata, N. weaveri, and N. bacilliformis are
unique among the members of the genus Neisseria in that they are rod-
shaped.