INTRODUCTION TO CLINICAL MICROBIOLOGY ○ Discovered the microbial etiology of tuberculosis.

Using differential
staining techniques, careful microscopy and solid agar methods, he
Historical Development isolated the causative agent Mycobacterium tuberculosis, in pure
● Girolamo Fracastoro or Hieronymous Fracastorius - wrote a treatise on culture. Discovered also the causal agent of cholera.
the germ theory of disease entitled “de Contagione” ○ Developed the Koch’s Postulates (a set of criteria) which states that:
● Robert Hooke - made key observations on microscopic organisms the pathogen accounts for the clinical and pathological features of the
● Anton van Leeuwenhoek - credited with first identifying microorganisms, disease and must be found in every case in which the disease occurs;
or “little animals or animacules”, using his newly developed microscope in the pathogen is not found in other diseases as a non-pathogen; after
1677. being isolated from the body and repeatedly passed in pure culture, the
○ Accurate description of bacteria: He first accurately described the pathogen can induce the disease in animal models; and the same
different shapes of bacteria as cocci (spheres), bacilli (rods) and pathogen must be re-isolated from the experimental animal.
spirochetes (spiral filaments) and communicated them to the Royal ○ He was the first to use the Hanging Drop Method by studying bacterial
Society of London in 1683. motility
● Spontaneous generation (abiogenesis) - from the earliest times, people ● Paul Ehrlich – co-discoverer of antibodies, antigens, and chemotherapy
believed and supported spontaneous generation that living organisms for infectious diseases
could develop from nonliving matter. ● Richard Pfieffer – discovered bacterial endotoxin, the phenomenon of
● Ignaz Semmelweis (1818 – 1865) - advocated hand washing using dilute, bacteriolysis, and played a major role in the development of killed typhoid
chlorinated lime solution to help stop the spread of disease. vaccines
● John Snow (1813-1858) - published a pamphlet in which he speculated ● Emil von Behring – discoverer of serum therapy for diphtheria and
that cholera was a waterborne or foodborne, intestinal illness. tetanus
○ Considered as the “Founding Father of the field of Epidemiology”. ● Oswald Avery, Maclyn MacCarty, and Colin MacLeod – identified the
○ Recommended a number of sanitation maneuvers, such as washing “holy grail” of genetics in 1944 with their finding that the “transforming
the clothes and bed linens of cases, isolation of sick people from principle” or genetic material of Streptococcus pneumoniae was DNA, not
healthy ones, and boiling water supplies to help curtail the cases of protein as previously postulated.
cholera.
● Louis Pasteur (1822-1895) - convincingly demonstrated that Division of Clinical Microbiology
microorganisms were responsible for the fermentation of fluids thus ● Mycology - study of fungi, their relationship to each other and other
proving the germ theory of disease. organisms, and the unique biochemistry which sets them apart from other
○ Showed that heat sterilization, chemical sterilization, or filtration of air groups.
and water could maintain organic materials in sterile conditions ● Virology - scientific discipline concerned with the study of the biology of
indefinitely without microbial growth. Techniques of sterilization and viruses and viral disease, including the distribution, biochemistry,
“Pasteurization” of dairy products were soon introduced. physiology, molecular biology, ecology, evolution, and clinical aspects of
○ Established the Pasteur Institute which later became the International viruses.
Center For Microbiology, Immunology, And Medicine. ● Parasitology - scientific discipline concerned with the study of the biology
○ Coined the term “vaccine”. of parasite and parasitic disease, including the distribution, biochemistry,
○ Other works: discovery of attenuation and chicken cholera vaccine, physiology, molecular biology, ecology, evolution and clinical aspects of
developed live attenuated anthrax vaccine, developed rabies vaccine parasites, including the host response to these agents.
and noticed Pneumococci. ● Bacteriology - science and study of bacteria and their relation to medicine
● Charles Chamberland - Invented the autoclave and developed and to other areas such as agriculture and industry
Pasteurella vaccine
● Alexandra Yersin - Co-discoverer of plague bacillus Definition of Terms
● Emile Roux - Discovered diphtheria toxin and antitoxin ● Acid-fastness – ability of bacteria, such as the Mycobacterium spp., to
● Jules Bordet - Discovered whooping cough bacillus and complements retain dye when treated with mineral acid or an acid-alcohol solution
● Ilya Metchnikoff - Discovered the process of phagocytosis and provided ● Aerotolerant anaerobes – microorganism that grows best in the absence
the initial description of innate immunity. of oxygen but can tolerate low concentrations of oxygen
● Albert Calmette - Discovered cobra antivenin and developed ● Autotrophs – organisms that produce organic compounds from carbon
Bacillus-Calmette-Guerin, the first effective tuberculosis vaccine dioxide as a carbon source
● Joseph Lister (1827-1912) - “Father of Modern Surgery” ● Bacteria – also referred to as eubacteria; single celled microorganisms
○ Developed a system of antiseptic surgery – designed to prevent that lack a true cell nucleus and multiply by binary fission.
microorganisms from entering wounds. This approach was remarkably ● Bacterial interference – potential pathogens inhibited by the
successful and transformed surgery after Lister published his findings nonpathogenic resident microbiota.
in 1867. It also provided strong evidence for the role of microorganisms ● Biological agent – any microorganism or infectious substance, or any
in disease because phenol, which killed bacteria, also prevented naturally occurring, bioengineered, or synthesized component of any
wound infections. such microorganism or infectious substance, capable or causing death,
○ He established the guiding principles of antisepsis for good surgical disease, or other biological malfunction in a human, animal, plant, or
practice and was a milestone in the evolution of surgical practice from other living organism
the era of “laudable pus” to modern aseptic techniques. ● Capnophilic – used to describe microorganisms that require an increased
● Robert Koch (1843-1910) - “Father of Bacteriology” concentration of CO2
○ Identified anthrax bacilli in the blood of infected sheep and successfully ● Capsule –organelle in some prokaryotic cells, such as a bacterial cell
transmitted the infection into healthy experimental animals. Using located outside the cell wall of bacteria. It is usually made up of
careful photomicroscopy and detailed drawings, he accurately polysaccharides but could be composed of other materials
described the life cycle of anthrax and the process of endospore ● Differential media – media that allow grouping of microbes based on
formation. different characteristics demonstrated on the medium
○ He also pioneered a number of laboratory techniques such as the use ● Differential stains – stains such as the Gram stain that stain components
of oil immersion microscope to study bacteria; developed new staining of the smear differently so that each component can be recognized .
methods for bacterial identification; and invented procedures for the ● Eukaryotes – organism with complex cell; structures in which the genetic
isolation of pure bacterial cultures (Koch plate technique) on solid material is organized into a membrane-bound nucleus
media facilitated by the use of agar as the solidifying agent in flat ● Facultative anaerobes – microorganism that does not require oxygen for
“Petri” dishes, named after the inventor Richard Petri. growth but will use oxygen and grow better if it is present
● Fermentation – process in which a molecule is oxidized to produce ● Ionizing (gamma) radiation
energy without an exogenous electron acceptor. Organic molecules ■ Chemical methods
usually serve as electron donors and electron acceptors.
● Fimbriae – nonflagellar, sticky, proteinaceous, hairlike appendages that Sterilization: Physica methods
adhere some bacterial cells to each other and to environmental surfaces ● Incineration - most common method of treating infectious waste.
● Flagella – exterior protein filaments that rotate; used by microorganisms Hazardous material is literally burned to ashes at temperature of 870º to
for motility 980º C. Considered as the safest method to ensure that no infective
● Fusiform – spindle shaped or tapered at each end materials remain in samples or containers when disposed.
● Gram-negative bacteria – bacteria that do not retain the crystal violet ● Moist heat (Steam under pressure) - used to sterilize biohazardous trash
complex; stained red by the safranin counterstain and heat-stable objects; an autoclave is used or this purpose.
● Gram-positive-bacteria - bacteria that retain the crystal violet-iodine ○ Moist heat in the form of saturated steam under 1 atmosphere (15
complex and appear blue-black on Gram Stained smears psi) of pressure causes the irreversible denaturation of enzymes and
● Halophilic – “salt-loving”; an organism that grows best in media with an structural proteins.
increased concentration of NaCl. ○ The most common type of steam sterilizer in microbiology
● Heterotrophs – organism that requires organic substrates as a source of laboratories is the gravity displacement type.
carbon for growth and development ○ The two common temperatures are 121º C (250º F) and 132º C (270º
● Lysogeny – incorporation of the genetic material of a bacteriophage with F)
that of the host bacterium. ■ Items such as media, liquids, and instruments are usually
● Mesophiles – organism that grows best in moderate temperature, neither autoclaved for 15 minutes at 121º C.
hot nor cold (25 to 40 C). ■ Infectious medical waste is sterilized at 132º C for 30 to 60 minutes
● Microaerophilic – microorganisms that require environments containing to allow penetration of the steam throughout the waste and the
concentrations of oxygen lower than that present in the atmosphere displacement of air trapped inside the autoclave bag.
(about 20%). ● Dry heat - Requires longer exposure times (1.5 to 3 hours) and higher
● Minimal medium – laboratory growth medium whose contents are simple temperatures than moist heat (160º to 180º C)
and completely defined. ○ Used to sterilize items such as glassware, oil, petroleum, or powders
● Nutrient Media – culture media that are complex; made of extracts of ● Filtration - method of choice for antibiotic solutions, toxic chemicals,
meat or soybeans radioisotopes, vaccines, and carbohydrates, which are all heat sensitive.
● Obligate aerobes – microorganisms that require oxygen for growth ○ Filtration of liquid is accomplished by pulling the solution through a
● Obligate anaerobes – microorganisms that can live and reproduce only in cellulose acetate or cellulose nitrate membrane with vacuum.
a strict anaerobic environment (0% oxygen) ○ Filtration of air is accomplished using High-Efficiency-Particulate-Air
● Pathogenic bacteria – organisms capable of causing disease (HEPA) filters designed to remove organisms larger than 0.3 um from
● Pili – nonmotile, long, hollow protein tubes that connect two bacterial cells isolation rooms, operating rooms, and biological cabinets (BSCs)
and mediate DNA exchange ● Ionizing radiation - Ionizing radiation used in microwaves and radiograph
● Plasmids – extrachromosomal, circular pieces of DNA found in many machines are short wavelength and high-energy gamma rays.
strains of bacteria. ○ Used for sterilizing disposables such as plastic syringes, catheters, or
● Pleomorphic – demonstrating a variety of shapes and forms; for a Gram gloves before use
stain, neither distinctly coccoid nor rod-shaped; also used to describe the
Gram stain morphology of bacteria that exhibit a combination of cocci, Sterilization: Chemical Methods
bacilli, coccobacilli, and filamentous forms in a single stained smear. ● Most common sterilant is Ethylene oxide (EtO), which is used in gaseous
● Prokaryotes – microorganisms that lack a true nucleus and nuclear form for sterilizing heat-sensitive objects.
membrane ● Formaldehyde vapor and vapor-phase hydrogen peroxide (oxidizing
● Psychrophiles – bacteria that grow best at cold temperatures (optimal agent) have been used to sterilize HEPA filters in BSCs.
growth at 10 to 20 C) ● Glutaraldehyde, which is sporicidal (kills spores) in 3 to 10 hours, is used
● Selective media – media that support the growth of one type or one group for medical equipment such as bronchoscopes, because it does not
of microbes but not of another type. corrode lenses, metal, or rubber.
● Spores – unit of asexual reproduction that appears as a highly refractile ● Peracetic acid, effective in the presence of organic material, has also
body in the cell. been used for the surface sterilization of surgical instruments.
● Thermophiles – bacteria that grow best at high temperatures (optimal ● The use of peracetic acid or glutaraldehyde is called cold sterilization.
growth at 50 to 60 C)
● Transport medium – liquid or semisolid medium meant to preserve and Disinfection
maintain the integrity of the specimen for the period between specimen ● Disinfection - process whereby pathogenic organisms, but not necessarily
collection and laboratory processing of the sample. all microorganisms or spores, are destroyed.
● Virulence - degree of pathology caused by an organism; the ● Physical Methods of Disinfection
disease-evoking power of a microorganisms ○ Boiling at 100º C for 15 minutes, which kills vegetative bacteria.
○ Pasteurizing at 63º C for 30 minutes or 72º C for 15 seconds, which
kills food pathogens
SAFETY, SPECIMEN AND WASTE MANAGEMENT IN THE ○ Using nonionizing radiation such as ultraviolet (UV) light
MICROBIOLOGY LABORATORY ■ UV rays are long wavelength and low energy. They do not penetrate
well and organisms must have direct surface exposure, such as the
Sterilization working surface of a BSC, for this form of disinfection to work.
● Sterilization - process whereby all forms of microbial life, including ● Chemical Methods of Disinfection
bacterial spores are killed and can be accomplished by physical of ○ Chemical Disinfectants
chemical means ■ Phenolics
○ Methods of Sterilization ■ Alcohols
■ Physical methods ■ Aldehydes
● Incineration ■ Halogens (Iodine and Chlorine- Sodium hypochlorite)
● Moist Heat ■ Heavy metals and Quaternary ammonium compounds
● Dry Heat !!! Prepare a working solution of the compound EXACTLY according to the
● Filtration manufacturer’s package insert.
 ○ When chemicals are used to destroy all life they are called chemical mandated safety guidelines are followed. The plan identifies tasks that
sterilants, or biocides; however, these same chemicals used for shorter are hazardous to employees and promotes employee safety through the
periods are disinfectants. Disinfectants used on living tissue are called following:
antiseptics. ○ Employee education and orientation
○ Factors that influence the activity of disinfectants: ○ Appropriate disposal of hazardous waste
■ Type of organisms present ○ Standard Precautions
■ Temperature and pH of process ○ Engineering controls and safe work practices, as well as appropriate
■ Number of organism present (microbial load) waste disposal and use of biological safety cabinets.
■ Concentration of disinfectant ○ Personal Protective Equipment
■ Amount of organics (blood, mucus, pus) present ○ Postexposure plan involving the investigation of all accidents and a
■ Nature of surface to be disinfected (e.g., potential of corrosion; plan to prevent recurrences.
porous vs. nonporous surface) ● Disposal of Hazardous Waste
■ Length of contact time ○ All materials contaminated with potentially infectious agents must be
■ Type of water available (hard vs. soft) decontaminated before disposal. Infectious waste from microbiology
laboratories is usually autoclaved on-site or sent for incineration.
Chemical Safety ○ In 1986 the EPA published a guide to hazardous waste reduction to
● The United States Occupational Safety and Health Administration limit the amount of hazardous waste generated and released into the
(OSHA) published the Hazard Communication Standard, which provides environment. These regulations call for the following:
for certain institutional educational practices to ensure all laboratory ■ Substituting less hazardous chemicals when possible
personnel have a thorough working knowledge of the hazards of ■ Developing procedures that use less of a hazardous chemical
chemicals with which they work. This standard has also been called the ■ Segregating infectious waste from uncontaminated trash
“employee right-to-know” ■ Substituting miniaturized systems for identification and
○ It mandates that all hazardous chemicals in the workplace be identified antimicrobial susceptibility testing of potential pathogens to reduce
and clearly marked with a National Fire Protection Association (NFPA) the volume of chemical reagents and infectious wastes.
label stating the health risks, such as carcinogens, mutagens, or ○ Recently, several alternative waste-treatment machines were
teratogens, and the hazard class developed to reduce the amount of waste buried in landfills. These
○ Each laboratory should have a chemical hygiene plan that includes systems combine mechanical shredding or compacting of the waste
guidelines on proper labeling of chemical containers, manufacturer’s with either chemical (sodium hypochlorite, chlorine dioxide, peracetic
material safety data sheet (MSDS), and a written chemical safety acid), thermal (moist heat, dry heat), or ionizing radiation
training and retraining programs. (microwaves, radio waves) decontamination.
● The MSDSs include information on the nature of the chemical, the ○ Infectious waste should be placed into two leakproof, plastic bags for
precautions to take if the chemical is spilled, the disposal sturdiness; this is known as double-bagging in common laboratory
recommendations. The sections in the typical MSDS include: jargon.
○ Substance name; ○ Pipettes, swabs, and other glass objects should be placed into rigid
○ Name, address, and phone number of manufacturer cardboard containers before disposal.
○ Hazardous ingredient(s); ○ Broken glass is placed in thick boxes lined with plastic biohazard
○ Physical and chemical properties bags; when full, the box is incinerated or autoclaved.
○ Fire and explosion data; ○ Sharp objects, including scalpels and needles, are placed in Sharps
○ Toxicity containers, which are autoclaved or incinerated when full.
○ Health effects and first aid;
○ Stability and reactivity Standard Precautions
○ Shipping data; ● The essentials of Standard Precautions and safe laboratory work
○ Spill, leak, and disposal procedures practices are as follows:
○ Personal protective equipment; ○ Do not eat, drink, smoke, or apply cosmetics (including lip balm)
○ Handling and storage ○ Do not insert or remove contact lenses
● Fume hoods are provided in the laboratory to prevent inhalation of toxic ○ Do not bite nails or chew on pens
fumes ○ Do not mouth-pipette
○ Limit access to the laboratory to trained personnel only
Biosafety ○ Assume all patients are infectious for all blood-borne pathogens
● Individuals are exposed in various ways to laboratory acquired infections ○ Use appropriate barrier precautions to prevent skin and mucous
in microbiology laboratories. These involve the following: membrane exposure, including wearing gloves at all times and
○ Rubbing the eyes or nose with contaminated hands masks, goggles, gowns, or aprons if splash or droplet formation is a
○ Inhaling aerosols produced during centrifugation, vortexing, or spills risk.
of liquid cultures ○ Thoroughly wash hands and other skin surfaces after removing
○ Accidentally ingesting microorganisms by putting pens or fingers in gloves and immediately after any contamination.
the mouth ○ Take special care to prevent injuries with sharp objects, such as
○ Suffering percutaneous inoculation, that is, being punctured by a needles and scalpels.
needlestick. ● Precautions applies to blood and all body fluids, except sweat.
● In the clinical microbiology laboratory, shigellosis, salmonellosis, ● Food and drinks must be stored in refrigerators in areas separate from
tuberculosis, brucellosis, and hepatitis are the five most frequently the work area.
acquired laboratory infections. ● It is good practice to store sera collected periodically from all health care
● Viral agents transmitted through blood and body fluids cause most workers so that, in the event of an accidental, a seroconversion
infections in nonmicrobiology laboratory workers and in health care (acquisition of antibodies to an infectious agent) can be documented.
workers in general. These include Hepatitis B Virus, Hepatitis C Virus, ● All health care workers should follow Standard Precautions whether
Hepatitis D Virus, and HIV. working inside or outside the laboratory. When collecting specimens
outside the laboratory, individuals should follow these guidelines:
Exposure Control Plan ○ Wear gloves and a laboratory coat.
● It is the legal responsibility of the laboratory director and supervisor to ○ Deal carefully with needles and lancets.
ensure that an Exposure Control Plan has been implemented and that the ○ Discard sharps in an appropriate, puncture-resistant container.
 ○ Never recap needles by hand; if necessary, special safety devices are ○ Used in laboratory teaching exercises for undergraduate, secondary
available. educational training and teaching laboratories for students in
microbiology.
Engineering Controls ○ Includes Bacillus subtilis and Naegleria gruberi
● Laboratory Environment ○ Precautions working with BSL-1 agents include standard good
○ The biohazard symbol should be prominently displayed on laboratory laboratory technique.
doors and any equipment that contain infectious materials. ● Biosafety level 2 (BSL-2) Agents
○ The air-handling system of a microbiology laboratory should move air ○ Those that are most commonly being sought in clinical specimens
from lower to higher risk areas, never reverse. and used in diagnostic, teaching, and other laboratories.
○ Ideally, the microbiology laboratory should be under negative ○ Includes all the common agents of infectious disease, as well as HIV,
pressure, and air should not be recirculated after passing through HBV, Salmonella organisms, and several more unusual pathogens.
microbiology. ● Biosafety level 3 (BSL-3) Agents
○ The selected use of BSCs for procedures that generate infectious ○ Procedures have been recommended for the handling of material
aerosols is critical to laboratory safety. suspected of harboring organisms unlikely to be encountered in a
○ Subculturing blood cultures by puncturing the septum with a needle routine clinical laboratory and for such organisms as Mycobacterium
should be performed behind a barrier to protect the worker from tuberculosis, Coxiella burnetii, the mold stages of systemic fungi, and
droplets. some other organisms when grown in quantities greater than that
○ Procedures used to process specimens for culture, notably, mincing, found in patient specimens.
grinding, vortexing, and preparing direct smears for microscopic ○ Those working with BSL-3 agents should have baseline sera
examination should be performed in a BSC. specimens stored for comparison with acute sera that can be drawn
○ Access to microbiology laboratories should be limited to employees in the event of unexplained illness.
and other necessary personnel. ○ BSL-3 organisms are primarily transmitted by infectious aerosols
○ Care should be taken to prevent insects from infesting any laboratory ● Biosafety level 4 (BSL-4) Agents
area. ○ Are exotic agents that are considered high risk and cause
○ A pest control program should be in place to control rodents and life-threatening disease
insects. ○ Include Marburg virus or Congo-Crimean hemorrhagic fever
● Biologic Safety Cabinet ○ Personnel and all materials must be decontaminated before leaving
○ BSC is a device that encloses a workspace in such a way as to the facility and all procedures are performed under maximum
protect workers from aerosol exposure to infectious disease agents. containment.
○ Air that contains the infectious material is sterilized, either by heat, UV ○ Most facilities that deal with BSL-4 agents are public health or
light, most commonly by passage through a HEPA filter that removes research laboratories.
most particles larger than 0.3 um in diameter
○ Class I Cabinets Mailing Biohazardous Materials
■ Allow room (unsterilized) air to pass into the cabinet and around ● Packaging must meet the requirements of the International Air Transport
the area and material within, sterilizing only the air to be Association (IATA) and the International Civil Aviation Organization
exhausted. (IACO).
■ They have negative pressure, are ventilated to the outside, and ● The shipper is the individual (institution) ultimately responsible for safe
are usually operated with an open front. and appropriate packaging. Any fines or penalties are the shipper’s
○ Class II Cabinets responsibility.
■ Sterilize air that flows over the infectious material, as well as air to ● Infectious specimens or isolates should be wrapped with absorbent
be exhausted. material and placed inside a plastic biohazard bag, called a primary
■ The air flows in “sheets' ' which serve as barriers to particles from receptacle. The primary receptacle is then inserted into a secondary
outside the cabinet and direct the flow of contaminated air into the container, most often a watertight, hard plastic mailer. The secondary
filters; thus called vertical laminar flow BSCs. container is capped and placed inside an outer, tertiary container that
■ Have a variable sash opening though which the operator gains protects it from physical and water damage.
access to the work surface. ● The package must display the UN Packaging Specification Marking and
■ Depending on their inlet flow velocity and the percent of air that is must be labeled with a specific hazard label as an infectious substance.
HEPA filtered and recirculated, class II cabinets are further ● A packaging list and a Shippers Declaration for Dangerous Goods Form
differentiated into: must accompany the air bill or ground form.
● Type A (Class IIA) – is self-contained, and 70% of the air is ● Shippers should note that some carriers have additional requirements for
recirculated; mostly used in hospital clinical microbiology coolant materials, such as ice, dry ice, or liquid nitrogen
laboratory
● Type B (Class IIB) – exhaust air is discharged outside the MICROBIAL TAXONOMY, GENETICS, METABOLISM, AND STRUCTURE
building; is selected for radioisotopes, toxic chemicals, or
carcinogens will be used. Microbial Taxonomy
○ Class III Cabinets ● Bacterial taxonomy (or bacterial systematics) refers to the science of the
■ Offer the most protection to the worker because they are classification of bacteria.
completely closed and have negative pressure. ● It consists of three interrelated disciplines:
■ Air coming into and going out of the cabinet is sterilized, and the ○ Nomenclature - it is concerned with the assignment of names to
infectious material within is handled with rubber gloves that are taxonomic groups as per published rules. ; binomial system; naming
attached and sealed to the cabinet. ○ Identification - it represents the practical side of taxonomy, which is the
■ It is important to the proper operation of laminar flow cabinets that process of determining that a particular isolate belongs to a recognized
an open area for 3 feet from the cabinet be maintained during taxon.; genotypic and phenotypic characteristics; describing
operation of the air circulating system; this ensures that infectious ○ Classification - it refers to the arrangements of bacteria into groups or
material is directed through the HEPA filters. taxa (sing., taxon) on the basis of their mutual similarity or evolutionary
Classification of Biologic Agents based on Hazard relatedness.; classifying; hierarchical classification system
● Biosafety level 1 (BSL-1) Agents
○ Include those that have no known potential for infecting healthy
people and are well defined and characterized.
Microbial Classification ● Bacilli in chains (streptobacillus)
● In bacterial taxonomy, a bacterium is placed within a small but ● Bacilli in packets (palisade bacillus)
homogenous group in a rank or level (hierarchical classification system). ■ Cell size
● Classification- a method for organizing microorganisms into groups or ■ Colonial morphology: size, shape, texture, elevation, pigmentation,
taxa based on similar morphologic, physiologic, and genetic traits (Tille, and effect on growth medium
2014). ■ Arrangement of flagella: monotrichous, lophotrichous,
● Groups of this rank or level unite creating a group of higher rank or level. amphitrichous, peritrichous
● In bacterial taxonomy, the most commonly used ranks or levels in their ■ Cell motility mechanism: motile (flagellate), non-motile
ascending order are: (non-flagellate)
○ Species - the basic taxonomic group; contains a group of similar ■ Ultrastructural characteristics: surface structure of bacterial cells
qualities or characteristics (e.g., flagella, pili, fimbriae, slime layer, capsule)
○ Genera sing., genus - contains a group of similar species ■ Staining behavior:
○ Families- contains a group of similar genera ● Gram staining: Gram-positive/negative
○ Orders - contains a group of similar families ● Acid-fast staining: Acid-fast/non-acidfast/partially acid-fast
○ Classes - contains a group of similar orders ■ Endospore formation: non-sporing/sporing
○ Phyla: or Division - contains a group of similar classes ■ Spore morphology and location: endospores may be centrally
○ Kingdom - a group of similar phyla. The Kingdom Monera includes all located (central), close to one end (subterminal), or definitely at the
single celled prokaryotic organisms. end (terminal). The mother cell that carries the endospore is called
○ Domain - the highest rank. the sporangium. Sometimes, an endospore becomes so large that it
swells or creates a bulge on the sporangium (mother cell).
Table 1: Taxonomic ranks in ascending order ○ Physiological and Metabolic
■ Some physiological and metabolic characteristics are very useful in
Rank or Level Example classifying and identifying microorganisms because they are directly
Species Escherichia coli related to the nature and activity of microbial enzymes and transport
Genus Escherichia proteins.
Family Enterobacteriaceae ■ Some of the most important physiological and metabolic
Order Enterobacteriales characteristics used in microbial taxonomy are the following:
Class y-Proteobacteria ● Nutritional types: photolithotrophs, chemolithotrophs,
Phylum Proteobacteria photoorganotrophs, chemoorganotrophs
Kingdom Monera ● Cell wall components: peptidoglycan (murein), teichoic acid and
Domain Bacteria teichuronic acid, protein, polysaccharides, pseudomurein, etc.
● Carbon sources: carbon dioxide, sugar acids, sugar alcohols,
Identification - is the process by which a microorganism’s key features are polysaccharides, organic acids
delineated. Once those features have been established, the profile is ● Nitrogen sources: molecular nitrogen, ammonium salts, nitrate,
compared with those of other previously characterized microorganisms organic nitrogenous compounds
(Tille, 2014). ● Energy metabolism: cellular respiration, fermentation, inorganic
substrate oxidation, nitrate and sulfate oxidation
Characteristics Used in Bacterial Taxonomy ● Osmotic tolerance
● Classical or Phenotypic Characteristics ● Oxygen relationships: obligate aerobes, microaerophiles,
○ Macroscopic and microscopic morphology facultative anaerobes, aerotolerant anaerobes, obligate anaerobes
○ Staining characteristics ● Temperature relationships: mesophiles, facultative thermophiles,
○ Environmental and nutritional requirements obligate thermophiles, hyperthermophiles, psychrophiles
○ Resistance profiles ● Salt requirements and tolerance
○ Antigenic properties ● Secondary metabolites
○ Subcellular properties ○ Environmental or Ecological
● Molecular or Genotypic Characteristics - relate to an organism’s genetic ■ Ecological characteristics, i.e., the characteristics of the relationship
makeup, including the nature of the organism’s genes and constituent of microorganisms to their environment significantly contribute to
nucleic acids. microbial taxonomy
○ Deoxyribonucleic acid (DNA) base composition ratio ■ Some of the most important ecological characteristics used in
○ Nucleic acid (DNA and ribonucleic acid [RNA]) base sequence microbial taxonomy are – life cycle patterns, the nature of the
analysis, including hybridization assays symbiotic relationship, pathogenic nature, and variations in the
requirements for temperature, pH, oxygen, and osmotic
Classical or Phenotypic Characteristics concentrations
● Phenotypic characteristics - are based on features beyond the genetic ■ Some recent molecular approaches such as genomic DNA GC
level and include both readily observable characteristics and ratios, nucleic acid hybridization, nucleic acid sequencing,
characteristics that may require extensive analytic procedures to be ribotyping, and comparison of proteins have become increasingly
detected. important and are used routinely for determining the characteristics
○ Morphological of microorganisms to be used in microbial taxonomy.
The following are the various morphological features normally used to ● Genomic DNA GC ratios (G + C content): Genomic DNA GC ratio
classify and identify bacterial microorganisms: (G + C) is the first and possibly the simplest, molecular approach
■ Cell shape: cocci, bacilli, curved/spiral, spirochete to be used in microbial taxonomy. The GC ratio is defined as the
■ Cell arrangement: single, pairs, chains, clusters, packets percentage of guanine plus cytosine in an organism’s DNA
● Single (monococcus) ● Nucleic acid hybridization (Genomic hybridization): Nucleic acid
● Cocci in pairs (diplococcus) hybridization or genomic hybridization measures the degree of
● Cocci in chains (streptococcus) similarity between two genomes (nucleic acids) and is useful for
● Cocci in clusters (staphylococcus) differentiating two bacteria.
● Cocci in fours (tetracoccus; tetrad; quartet) ○ DNA-DNA hybridization is useful to study only closely related
● Coccis in cubes of eight (sarcina; octet) bacteria.
● Single bacillus (monobacillus)
● Bacilli in pairs (diplobacillus)
 ○ DNA-RNA hybridization helps in comparing distantly related ○ Bacterial nucleic acid structure and organization
bacteria. ○ Replication and expression of genetic information
● Nucleic acid sequencing: Nucleic acid (DNA and RNA) ○ Genetic exchange and diversity
sequencing is another molecular characteristic that helps directly ● Metabolism (cellular processes)
compare the genomic structures. Most attention has been given ○ Fueling
to the sequencing of 5S and 16S rRNAs isolated from the 50S ○ Biosynthesis
and 30S subunits of 70S prokaryotic ribosomes, respectively. ○ Polymerization and assembly
● Ribotyping: ○ Waste removal
○ Ribotyping is a technique that measures the unique pattern that ○ Motion and other responses to the environment
is generated when DNA from a bacterium (all other organisms ● Structure
also) is digested by a restriction enzyme and the fragments are ○ Bacterial morphology
separated and probed with an rRNA probe. ○ Bacterial cell morphology
○ The technique does not involve nucleic acid sequencing.
Ribotyping has proven useful for bacterial identification and has Bacterial Nucleic Acid Structure and Organization
found many applications in clinical diagnostics and for the ● The function of DNA and RNA
microbial analyses of food, water, and beverages. ○ DNA: Deoxyribonucleic acid contains the information for making
○ Ribotyping is a rapid and specific method for bacterial proteins, and for the development and function of organisms. In
identification; it is so specific that it has been given the eukaryotes, the cell's genetic material is contained within an organelle
nickname ‘molecular fingerprinting’ because a unique series of called the nucleus, where it is organized in long molecules called
bands appear for virtually any bacterium (any organism). chromosomes. A small amount of DNA can also be found in the
● .Comparison of proteins: The amino acid sequences of proteins mitochondria.
are direct reflections of mRNA sequences and therefore closely ○ RNA: Ribonucleic acid accurately copies the information
related to the structures of the genes coding for their synthesis. In (transcription) from DNA to produce proteins (translation). In
the light of this, the comparisons of proteins from different bacteria eukaryotes, it is contained in the nucleolus (mRNA), ribosomes
prove very useful taxonomically. (rRNA), and cytoplasm (tRNA).
● Classical characteristics are quite useful in the routine identification of ● Structure:
bacteria and also provide clues for phylogenic relationships amongst ○ DNA is a long polymer composed of nucleotide subunits bonded
them as well as with other organisms. together covalently by 3’-5’- phosphodiester bonds to make a
polynucleotide
Microbial nomenclature ○ Nucleotides are the basic units or “building blocks” of nucleic acids
● Nomenclature is the naming of microorganisms according to established (e.g., DNA and RNA).
rules and guidelines set forth in the International Code of Nomenclature ○ A DNA nucleotide molecule consists of a:
of Bacteria (ICNB) or the Bacteriological Code (BC). It provides the ■ nitrogen-containing base;
accepted labels by which organisms are universally recognized (Tille, ■ phosphate group;
2014). ■ sugar (deoxyribose)
● Binomial Nomenclature (Two-Name) System: ○ The three-dimensional structure of the DNA molecule is composed of
○ Every organism is assigned a scientific name or label composed of a two strands wound into a double-stranded helical structure.
genus and species designation ○ The DNA molecule consists of two complementary chains or strands
■ Greek or Latin derivation; of nucleotides.
■ Characteristics of the sample or organism ○ Each strand of DNA or polynucleotide chain is composed of four
■ Personal names of scientists or discoverers; types of nucleotide subunits, and these two strands are held together
■ Geographical locations (e.g., Hafnia [Copenhagen, Denmark], by hydrogen bonds between the base portions of the nucleotides.
Providencia [Providence, Rhode Island, USA]) ○ Adenine always pairs with thymine with two hydrogen bonds. Guanine
■ Name of institutions or events (e.g., Legionella from American pairs with cytosine with three hydrogen bonds
Legion Convention) ● The Polarity of the DNA Strands
● While the specific name is written in lowercase letters, the first letter of ○ The ends of a DNA molecule are called 3' (three prime) and 5' (five
the generic name is always written in UPPERCASE form. prime) ends, based on the numbering of carbon atoms in deoxyribose
● The two components of the scientific name are used SIMULTANEOUSLY sugars.
and written in italics or underlined in script or text. For example: ○ Each DNA strand has two ends.
○ Staphylococcus aureus ○ The 5' end of the DNA is the one with the terminal phosphate (PO4
○ Escherichia coli 3-) group on the 5' carbon of the deoxyribose
○ Clostridium tetani ○ The 3' end is the one with a terminal hydroxyl (OH) group on the
● For the generic epithet, all names derived from people, places, or events deoxyribose of the 3' carbon of the deoxyribose
must be in the female nominative case (adding the suffix –ia, –a, or – ○ The members of each base pair can fit together within the double
ella). For example: o Escherichia, Legionella, Salmonella, Listeria, helix because the two strands of the helix run antiparallel to each
Bartonella, Ehrlichia, etc. other—that is, they are oriented with opposite polarities
● Alternatively, the scientific name may be abbreviated by using the ● Characteristics of Prokaryotic (Bacterial) Nucleic Acid Structure and
uppercase form of the first letter of the generic epithet followed by a Organization
period (.) and the full specific name, which is NEVER abbreviated ○ Single (haploid) chromosome.
○ Staphylococcus aureus → S. aureus; Escherichia coli → E. coli; ○ Genetic material is not contained in membrane bound organelles
Clostridium tetani → C. tetani (e.g., nucleus).
● Frequently, an informal designation may be used to label a particular ○ Bacterial chromosome exists as a double stranded, closed, circular,
group of organisms. These designations are not capitalized or italicized. supercoiled (extensively folded and twisted) macromolecule
For example: ○ Bacterial plasmids exist as double-stranded, closed, circular,
○ Staphylococci, streptococci, enterococci, gonococci, mycobacteria, autonomously replicating non chromosomal elements.
actinomycete, spirochete, etc ■ Plasmids: may contain genes that enhance the survival of an
organism, either by killing other organisms or by defending the
Microbial Viability and Survival host cell by producing toxins, and providing genetic advantages
● Genetics
 such as antimicrobial resistance. Some plasmids facilitate the ○ The number of dividing cells equal the number of dying cells = no
process of replication in bacteria. overall population growth.
● 4 Stages of Bacterial DNA Replication ○ Under the less favorable conditions, competition for nutrients
1. Unwinding (relaxation) of supercoiled chromosome DNA increases and the cells become less metabolically active.
2. Separation of complementary strands of the parent DNA ○ Spore-forming bacteria produce endospores and pathogenic bacteria
3. Synthesis of new DNA strands by DNA polymerase III begin to generate virulence factors.
4. Termination of replication and segregation of the two identical ● Death phase
chromosomes to each daughter cell ○ As nutrients become less available and waste products increase, the
number of dying cells continues to rise.
Bacterial DNA Replication ○ In the death phase, the number of living cells decreases exponentially
● Initiation and population growth experiences a sharp decline.
○ The site of active replication is referred to as the replication fork; two ○ As dying cells lyse or break open, they spill their contents into the
bidirectional forks are involved in the replication process. environment, making these nutrients available to other bacteria. This
1. Uncoiling (relaxation) of supercoiled bacterial chromosome → helps spore-producing bacteria to survive long enough for spore
Topoisomerase II (DNA gyrase) production.
2. Breaking of hydrogen bonds between nucleobase pairs → Helicase ○ Spores are able to survive the harsh conditions of the death phase
3. As the DNA is unzipped, Y-shaped structures called replication forks
are formed Bacterial Structure and Function
4. A primer composed of 5-10 complementary RNA nucleotides are ● Requirements (Fueling reactions)
added to the template DNA strand → RNA primase (a type of RNA ○ Precursor Metabolites
polymerase) ■ Gasses: Carbon dioxide (CO2), Oxygen (O2), Ammonia (NH3)
● Elongation ■ Organic compounds, including amino acids
○ DNA polymerase I will remove the RNA primers and replaces it with ■ Water (H2O)
newly synthesized DNA ■ Nitrate (NO3 -)
○ Okazaki fragments are small DNA segments separated by the RNA ■ Phosphate (PO4 3-)
primers ■ Hydrogen sulfide (H2S)
○ DNA ligase catalyzes the formation of phosphodiester bonds between ■ Sulfate (SO4 2-)
the 3’-OH and 5’-phosphate end ■ Potassium (K+)
1. Addition of complementary DNA nucleotides (building blocks) to the 3’ ■ Magnesium (Mg2+)
end → DNA polymerase III ■ Calcium (Ca2+)
2. Holds DNA polymerase III in place as it continues to add DNA ■ Sodium (Na+)
nucleotides → Sliding clamp ■ Iron (Fe3+) - organic iron complexes
3. Prevents over-winding of DNA strand ahead of replication fork → ○ Nutrients, Other Growth Requirements
Topoisomerase II (DNA gyrase) ■ Glucose-6-phosphate
4. Removal of RNA primers from the lagging strand → DNA polymerase ■ Fructose-6-phosphate
I (with exonuclease activity) ■ Pentose-5-phosphate
5. Formation of phosphodiester bonds between nucleotides, including ■ Erythrose-4-phosphate
the Okazaki fragments → DNA ligase ■ 3-Phosphoglycerate
● Termination ■ Phosphoenolpyruvate
1. Circular chromosomes (DNA) are interlocked and must be separated ■ Pyruvate
from each other ■ Acetyl CoA
2. Bacterial topoisomerase IV introduces double-stranded breaks into ■ α—Ketoglutarate
DNA molecules, allowing them to separate from each other. It then ■ Succinyl CoA
reseals the circular chromosomes. ■ Oxaloacetate

Bacterial Growth
Bacteria are prokaryotic organisms that most commonly replicate by the
asexual process of binary fission. These microbes reproduce rapidly at an
exponential rate under favorable conditions.
● Lag Phase
○ This initial phase is characterized by cellular activity but not growth.
○ A small group of cells are placed in a nutrient-rich medium that allows
them to synthesize proteins and other molecules necessary for
replication.
○ These cells increase in size, but no cell division occurs in the phase
● Exponential or log phase
○ This is the time when the cells are dividing by binary fission and
doubling in numbers after each generation time.
○ The metabolic activity is high as DNA, RNA, cell wall components,
and other substances necessary for growth are generated for division. ● Bacteria are unicellular prokaryotic organisms. Bacterial cells have
○ It is in this growth phase that antibiotics and disinfectants are most simpler internal structure.
effective as these substances typically target bacteria cell walls or the ● It lacks all membrane bound cell organelles such as mitochondria,
protein synthesis processes of DNA transcription and RNA lysosome, Golgi, endoplasmic reticulum, chloroplast, peroxisome,
translation. glyoxysome, and true vacuole.
● Stationary or plateau phase ● Bacteria also lack true membrane bound nucleus and nucleolus.
○ The population growth experienced in the log phase begins to decline ● The bacterial nucleus is known as nucleoid.
as the available nutrients become depleted and waste products start ● Structures outside the cell wall:
to accumulate. ○ Capsule - The capsule is a 0.2-µm-thick viscous outer layer to the cell
wall.
 ■ The capsule is 98% water and 2% polysaccharide or glycoprotein/ ■ Lipid-A: it is phosphorylated glucosamine disaccharide. It is
polypeptide or both. antigenic
■ There are two types of capsules. ■ Polysaccharide: it consists of core-polysaccharide and
■ Macro-capsule: the thickness of 0.2µm or more, visible under a O-polysaccharide
light microscope ○ Cytoplasmic membrane
■ Microcapsule: thickness less than 0.2µm, visible under an electron ■ Cell membrane is the inner layer that lies inside the cell wall and
microscope encloses the cytoplasm.
■ The capsule is a very delicate structure. It can be removed by ■ It is also known as cytoplasmic membrane or plasma membrane. o
vigorous washing. It is about 80-nm-thick.
■ The capsule is the most important virulence factor of bacteria. ■ Cell membrane of bacteria is composed of phospholipid and
■ Functions: proteins
● It helps in attachments as well as it prevents the cell from ○ Mesosome - spherical or round-like structure found commonly in
desiccation and drying. Gram-positive bacteria
● Capsule resist phagocytosis by WBCs ■ Function: It is the site for respiration in bacterial cell.
○ Flagella - It is 15-20 nm hair-like structure that emerges from the cell ○ Ribosome - Bacterial ribosome is of 70s type.
wall ■ Ribosomes are rounded granules found freely floating in the
■ Flagella is not straight but is helical. cytoplasm
■ It is composed of flagellin protein (globular protein) and known as ■ Ribosomes are known as universal cell organelle because it is
H antigen. found in both bacterial cell and eukaryotic cell.
■ A single flagellum has three parts: a basal body, hook, and ■ Chemically the ribosomes are made up of nucleic acids
filament. (particularly RNA and proteins).
■ Function: it helps in the motility of the bacterial cells. ○ Cytoplasm - colorless, viscous fluid present inside the cell membrane
○ Pili or Fimbriae ■ All the cell organelles and inclusions are found floating in
■ Pili are hollow filamentous and nonhelical structures. cytoplasmic fluid.
■ They are numerous and shorter than flagella. ■ It contains proteins, lipids, minerals, nucleic acids, glycogen, water,
■ Pili is the characteristic feature of Gram-negative bacteria. etc.
■ Pili is composed of pilin protein. ■ Functions:
■ Bacteria containing pili: Shigella, Proteus, Neisseria gonorrhoeae, ● It helps to distribute water, oxygen as other substances
E. coli throughout the cell.
■ Functions: ● Literally, all the cellular content including nucleus, and other cell
● Attachment: pili helps the bacteria to attach to the host cell organelle are floating in the cytoplasm
surface. Most of the human pathogens of the respiratory tract ○ Spores (Endospores)
and the urinary tract are attached with the help of pili. ■ Spore is a metabolically dormant structure produced during
● Pili (fimbriae) possess an antigenic property unfavorable conditions by the process called sporulation.
■ Specialized function: ■ Sporulation occurs during late log phase or early stationary phase.
● Some pili are modified for a specialized function e.g., sex pilus ■ Under favorable conditions, spores germinate to give vegetative
(F-pili) help in transfer of DNA from donor to recipient cell during cells.
conjugation.
● F-pili also act as receptor for bacteriophage. HOST-MICROORGANISM INTERACTION
● Structures inside the cell wall
○ Cell wall - an important structure of a bacteria The Encounter Between Host and Microorganism
■ It gives shape to the organism. ● The Human Host’s Perspective
■ On the basis of cell wall composition, bacteria are classified into ○ Microbial Reservoirs and Transmission
two major group i.e., Gram-positive and Gram Negative ○ Human and Microbe Interactions
● Gram-positive cell wall - Cell wall composition of Gram Positive ○ Animals as Microbial Reservoirs
bacteria: ○ Insects as Vectors
○ Peptidoglycan - It consists of glycan backbone formed by ○ The Environment as a Microbial
repeated unit of NAG (N-acetyl glucosamine) and NAM ○ Reservoir
(N-acetyl muramic acid) and the glycan backbone is ● The Microorganism’s Perspective
cross-linked by peptide bonds. ○ All of the human host’s perspective are microbial survival (fulfillment
■ Peptidoglycan layer is present in cell wall of both of their metabolic and genetic needs)
Gram-positive as well as Gram-negative bacteria. ● Bacteria and microbe encounters
■ However, Gram-positive have thick layer of peptidoglycan. Humans are exposed to the organism through various means (modes of
○ Teichoic acid - water-soluble polymer of glycerol or ribitol transmission)
phosphate present in Gram-positive bacteria ○ Humans are directly exposed to the same environment where the
■ It constitutes about 50% of the dry weight of the cell wall. organism lives; or
■ It is the major surface antigen of Gram-positive bacteria ○ Humans are exposed to the organism through indirect means (e.g.,
○ Lipids vectors, vehicles)
● Gram-negative cell wall - Cell wall composition of Gram
Negative bacteria: Microbial Reservoirs and Transmission
○ Peptidoglycan ● Microbial Reservoirs: origin of the etiologic agent or location from which it
○ Outer membrane - additional layer present in Gram-negative disseminates (e.g.,water, food, insects, animals, other humans)
bacteria ○ Humans
■ composed of a lipid bilayer, proteins, and ○ Animals
lipopolysaccharide (LPS) ○ Food (from plant and
○ Lipids, proteins ○ animal sources)
○ Lipopolysaccharide (LPS) - composed of lipid-A and ○ Water
polysaccharide ○ Air
○ Soil
● Mode of Transmission: means by which etiologic agents are brought in ● Provides dry, acidic, and cool conditions that
contact with the human host (e.g., direct and indirect transmission) limit bacterial growth
○ Direct transmission: transmitted by direct contact between reservoir Hair follicles, sweat ● Produce acids, alcohols, and toxic lipids that
and host glands, sebaceous glands limit bacterial growth
■ Horizontal transmission Eyes/conjunctival ● Flushing action of tears removes
epithelium microorganisms
● Direct contact (e.g., skin-to-skin contact; kissing; unsafe sexual
● Tears contain lysozymes that destroys bacterial
practices; direct inoculation through needlestick or animal bite; cell wall
transfer of agent to open wounds) ● Mechanical blinking of the eyelid removes
● Blood or body fluid physical contact or exposure; Droplet spread microorganisms
(>5 microns) Skin-associated lymphoid Mediates specific and nonspecific protection
■ Vertical transmission tissues mechanisms against microorganisms that
● Perinatal (transmission of etiologic agent from mother to fetus) penetrate outer tissue layers
○ Indirect transmission: transmitted to host via vectors and vehicles
(inanimate objects); transmitted to host via intervening agents (there is Table 3-2 Protective Characteristics of Mucous Membranes
no direct human-to-human contact) Skin Structure Protective Activity
■ Airborne transmission (<5 microns): droplet nuclei suspended in Mucosal cells ● Rapid sloughing for bacterial removal
● Tight intercellular junctions prevent bacterial
air
penetration
■ Vehicles are non-living entities that are contaminated with the Goblet cells ● Mucus productions: Protective lubrication of
etiologic agent (e.g. water, food, air, blood products, medical cells; bacterial trapping; contain specific
devices) and as such is the mode of transmission for that agent antibodies with specific activity against bacteria
■ Vectors are living entities such as animals, blood-feeding ● Provisions of antibacterial substances to
arthropods (e.g., ticks, fleas, mosquitoes), or another human, that mucosal surface:
transmit the etiologic agent ○ Lysozyme (degrades bacterial cell wall)
○ Lactoferrin (competes for bacterial iron
supply)
Human and Microbe Interactions ○ Lactoperoxidase (production of substances
● Human play a substantial role as microbial reservoirs toxic to bacterial)
Examples Mode of Transmission Mucosa-associated ● Mediates specific responses against bacteria
Conjunctivitis of the newborn (also Direct mother-to-baby transmission with lymphoid tissue that penetrate outer layer
known as ophthalmia neonatorum) the mother's birth canal that is infected
with an STI (e.g., Neisseria
gonorrhoeae, Chlamydia trachomatis)
Group A streptococcal pharyngitis Direct person-to-person transmission
(Streptococcus pyogenes, “strep through saliva or nasal secretions from
throat”) an infected person
Gonococcal urethritis (Neisseria Direct person-to-person transmission
gonorrhoeae) through unsafe sexual practices
Syphilis (Treponema pallidum)
Pulmonary tuberculosis Indirect transmission mainly through
(Mycobacterium tuberculosis) airborne transmission of droplet nuclei
Cholera (Vibrio cholerae) Indirect transmission through ingestion
of fecal- contaminated water

Animals as Microbial Reservoirs (Zoonotic Infections)

● Animals also play a substantial role as microbial reservoirs.
Examples Mode of Transmission
Cat-scratch disease (Bartonella Direct (animal-to-human) transmission
henselae) through a cat bite or scratch; cat licks
an open wound
Rocky Mountain spotted fever Indirect (animal-to-human) transmission
(Rickettsia rickettsii) through a bite from an infected tick
(Dermacentor spp.)
Lyme disease (Borrelia burgdorferi) Indirect (animal-to-human) transmission
through a bite from an infected tick
(Ixodes spp.) ● Microbial Factors Contributing to Colonizations of Host Surfaces
Tularemia (Francisella tularensis) Indirect (animal-to-human) transmission ○ Survival against environmental conditions
through handling infected animals, ■ Localization in moist areas
ingestion of contaminated food or water, ■ Protection in ingested or inhaled debris
inhalation of infective aerosols, and ■ Expression of specific metabolic characteristics (e.g., salt
arthropod bites (ticks and insects)
tolerance)
Salmonella spp., Campylobacter Indirect (animal-to-human) transmission
spp., Enterohemorrhagic E. coli usually through ingestion of raw or ○ Achieving attachment and adherence to host cell surfaces
(EHEC) undercooked meat, poultry or eggs ■ Pili
■ Adherence proteins
Microorganism Colonization of Host Surfaces ■ Biofilms
● Internal and external body surfaces are our first lines of defense against ■ Various proteins adhesins
invading pathogens. ○ Other factors
■ Motility
Table 3-1 Protective Characteristics of the Skin and Skin Structures ■ Production of substances that compete with host for acquisition of
Skin Structure Protective Activity essential nutrients (e.e., siderophores for capture of iron)
Outer (dermal) layers ● Acts as a physical barrier to microbial ■ Ability to coexist with other colonizing microorganisms
penetration ● Normal flora: microorganisms that inhabit many surfaces of the human
● Sloughing of outer layers removes attached body (also referred to as colonizers, normal microbiota, and resident
bacteria microflora).
Microorganism Entry, Invasion, and Dissemination ○ Avoid Phagocyte-Mediated Killing
● First line of defense - non-specific or innate immune responses ■ Inhibiting phagosome-lysosome fusion
○ Physical barriers: skin and mucous membranes ■ Being resistant to destructive agents (e.g. lysozyme) released by
○ Secretions, mucus lysosomes
○ Reflexes ■ Actively and rapidly multiplying within a phagocyte
○ Normal flora ■ Releasing toxins and enzymes that damage or kill phagocytes
↓ ○ Avoid Effects of the Complement System
Disruption of the host’s surface barriers - (e.g., dryness of skin, trauma, ■ Using a capsule to hide surface molecules that would otherwise
needlestick injury, surgical and penetrating wounds, burns) activate the complement system, including the formation of a complex
- (e.g., loss of stomach acidity, smoking, toxic gases, trauma, implantation protein polysaccharide matrix referred to as biofilm
of medical devices, malignancies, alcoholism, childbirth, overuse of ■ Producing substances that inhibit the processes involved in
antibiotics) complement activation
↓ ■ Producing substances that destroy specific complement proteins
● Second line of defense - non-specific or innate immune responses ● Pathogens: microorganisms that cause infection or disease.
○ Phagocytosis by monocytes, neutrophils, and macrophages ● Virulence factors: the characteristics of organisms that enable them to
○ Inflammatory response cause disease, to evade host’s defenses, or to mediate damaging effects
○ Naturally occurring antimicrobial substances (e.g., complement on host cells.
proteins, cytokines, coagulation proteins) ● Pathogenicity: refers to the organism’s ability to cause disease. An
● Second line of defense: Phagocytosis organism of high pathogenicity is very likely to cause disease, whereas
○ Three possible outcomes an organism of low pathogenicity is much less likely to cause infection.
■ Destruction of bacteria ● Virulence: refers to the measure or degree of pathogenicity of an
■ Long-term survival of bacteria organism. The degree of severity decreases with diminishing virulence of
■ Destruction of phagocyte the microorganism.
● Second line of defense: Complement system
○ Classical pathway Microbial Strategies for Surviving the Host’s Immune System
○ Lectin pathway ● Rapid multiplication and invasion to complete host cell destruction or to
○ Alternative pathway increase its virulence
● Second line of defense: Inflammatory response ● Invasion and destruction of host cells involved in the immune response
○ Inflammatory response (inflammation): occurs when tissues are injured (production of M protein, collagenase, hyaluronidase, nuclease, etc.)
by bacteria, trauma, toxins, heat, or any other cause. The damaged ● Pathogen survives unrecognized in host cells and avoids detection by
cells release chemicals including histamine, bradykinin, and immune system
prostaglandins. These chemicals cause blood vessels to leak fluid into ● Pathogen covers its antigens with a capsule or biofilm so that an immune
the tissues, causing swelling. response is not activated
○ Blood vessel responses: ● Pathogen changes antigens so that immune system is constantly fighting
■ Changes in vascular flow and caliber a primary encounter
■ Increased vascular permeability ● Pathogen produces enzymes (proteases) that directly destroy or
■ Responses of lymphatic vessels inactivate antibodies
○ Leukocyte responses
■ Recruitment of leukocytes to the sites of injury Microbial Toxins
■ Recognition of microbes (and other stimuli) Toxins: these are biochemically active substances released by
■ Removal/destruction of offending agents (e.g., bacteria) microorganisms that have a particular effect on host cells. Microorganisms
○ Inflammation (swelling, redness, heat, pain) use toxins to establish infections and multiply within the host.
■ Attract cells and biochemical mediators of defense ● Endotoxins - its presence can be detected in-vitro
■ Facilitate removal of infectious agents by lymphatics system ○ General toxin common to almost all Gram- negative bacteria
■ Wall off and limit extension of invasion ○ Composed of lipopolysaccharide (LPS) portion of Gram-negative cell
■ Supplement and interact with immune system defenses envelope
↓ ○ Released when Gram-negative bacterial cell is destroyed
Interleukin-1 (IL-l): secreted by activated phagocytes (Mo, PMN) ○ Effects on host include:
Interleukin-2 (IL-2): secreted by activated TH cells ■ Disruption of clotting, causing clots to form throughout the body
↓ (i.e., DIC)
● Third line of defense - specific or acquired immune responses ■ Fever
■ Activation of complement and immune systems
■ Circulatory changes that lead to hypotension, shock, and death
● Exotoxins - General toxin common to almost all Gram- positive bacteria
○ Produced and released by living bacteria; do not require bacterial
death for its release
○ Specific toxins target specific host cells; type of toxin varies with
bacterial species
○ Some toxins kill host cells and some help spread bacteria in tissues
(e.g., enzymes)
○ Some toxins interfere with specific cellular activities (e.g., protein
synthesis, internal signaling, neuromuscular interruption, etc.)
● In-vitro test for bacterial endotoxins
○ Limulus Amebocyte Lysate (LAL) Test: an in-vitro bacterial endotoxin
test that is necessary to quantify this gram-negative bacteria within a
cell wall. Performed as a lot release test, the LAL assesses medical
● Microbial Strategies for Surviving Inflammation
devices coming in contact with cerebrospinal fluid or the
○ Avoid killing by phagocytes (polymorphonuclear leukocytes)
cardiovascular system. The bacterial endotoxin test uses the lysate
■ Producing a capsule, thereby inhibiting phagocytes ability to ingest
them
 from blood cells from horseshoe crabs (Limulus polyphemus) to 2. Accuracy in diagnosing the etiologic agent (microorganism causing
detect bacterial endotoxins. the infection)
■ Its blue blood is used in medicine to ensure that anything that gets 3. Whether the patient receives appropriate treatment for infection
injected or implanted into the human body is free of potential (depends on the accuracy of diagnosis)
bacterial contamination.
Preventing of Infectious Diseases
Outcome of Infectious Diseases ● Preventing Transmission
● Signs and Symptoms of Infection and Infectious Disease ○ Avoid direct contact with infected persons or body fluids; wear
○ General or localized aches and pains appropriate PPE
○ Headache ○ Block the spread of airborne microorganisms
○ Fever ○ Use sterile medical techniques
○ Fatigue ● Controlling Microbial Reservoirs
○ Swollen lymph nodes ○ Sanitation and disinfection
○ Rashes ○ Sewage treatment
○ Redness and swelling ○ Food preservation
○ Cough and sneezes ○ Water treatment
○ Congestion of nasal and sinus passages ○ Control of pests and insect vector populations
○ Sore throat ● Minimizing Risk Before or Shortly After Exposure
○ Nausea and vomiting ○ Immunization or vaccination
○ Diarrhea ○ Cleansing and use of antiseptics
● Acute infections: infectious processes that develop quickly. ○ Prophylactic use of antimicrobial agents
● Chronic infections: infectious processes that develop and progress slowly,
sometimes over a period of years.
● Clinical signs: are measurable indications or physical observations, such
as an increase in body temperature (fever) or the development of a rash
or swelling.
● Symptoms: are indicators as described by the patient, such as headache,
aches, fatigue, and nausea.

GRAM-POSITIVE COCCI: Staphylococcus, Micrococcus, and Similar

Organisms

Gram Staining: Review

● It separates bacteria into two large groups (Gram-positive and
Gram-negative) based on their different cell wall constituents.
● Three Basic Shapes of Bacteria:
○ Round
○ Rod
○ Curved

Gram-Positive Cocci
● Gram-positive cocci are those that stain positive with the Gram staining
method (purple color)
● The peptidoglycan layer is substantially thicker in Gram-positive bacteria
● Common isolates in the clinical microbiology laboratory
● Most Gram-positive cocci are members of the indigenous microbiota
(normal flora)
● Some species are pathogenic (disease-causing)
● The signs and symptoms reflect the stages of infection. In turn, the
● They are initially differentiated by the catalase test.
stages of infection generally reflect the stages in host- microorganism
○ The catalase test is used to differentiate staphylococci (+) from
interactions.
streptococci (–).
● Controlling or clearing an infectious process depends on the following
○ This test determines the presence of the enzyme catalase
factors:
○ Catalase is an enzyme that breaks down the harmful substance,
1. Severity of infection (determined by host and microbial interactions)
hydrogen peroxide, into water and oxygen.
 ○ If an organism can produce catalase, it will produce bubbles of Micrococcus spp. Normal flora: skin, Endogenous strain: uncertain
oxygen when 3% hydrogen peroxide is added to it. Kocuria spp. mucosa, and Rarely implicated in ifxn
○ Positive result: effervescence Kytococcus spp. oropharynx Immunocompromised hosts: brain
○ Catalase-positive: Staphylococcus spp. abscess, meningitis, pneumonia,
endocarditis
○ Catalase-negative: Streptococcus spp.
● They are initially differentiated by the coagulase test.
○ The catalase enzyme is a significant virulence factor (disease-causing
○ This test determines the presence of the enzyme staphylocoagulase
factor) for the Staphylococcus spp.
○ This enzyme is a virulence factor for Staphylococcus aureus
○ Staphylococcal catalase protects intra- phagocytic microbes by
destroying hydrogen peroxide produced by the phagocyte
○ The catalase test detects the presence of this enzymes
○ A positive catalase test is indicated by effervescence or formation of
bubbles
○ Staphylococcus aureus cultivated on Mannitol Salt Agar
○ The positive result is the formation of clot in plasma
○ Staphylococcus aureus cultivated on Sheep Blood Agar (showing
■ Organism: Staphylococcus aureus
beta-hemolytic pattern / clear zone around the colonies)
○ Negative coagulase test is the absence of clot in plasma
○ Important:
■ Organism: Staphylococcus epidermidis
■ The test organisms should not be taken from blood agar culture.
○ Staphylococci that are able to produce this enzyme are called
■ Human red blood cells contain catalase and their presence will
coagulase- positive staphylococci
give a false-positive test.
○ Slide - Bound or cell-bound coagulase (clumping factor)
■ Culture should be 18 to 24 hours old.
○ Tube -Free, extracellular coagulase (staphylocoagulase)
■ Hydrogen peroxide must be fresh as it is very unstable.
● Coagulase-positive staphylococci (CoPS):
○ Reaction Principle:
○ Staphylococcus aureus – most commonly isolated, most virulent and
clinically significant CoPS
○ Animal-associated species (less frequently isolated from humans)
○ Genera to be Considered ■ Staphylococcus intermedius
■ Staphylococcus aureus (coagulase-positive) ■ Staphylococcus delphini
■ Coagulase-negative staphylococci (most common ones) ■ Staphylococcus lutrae
● Staphylococcus epidermidis ■ Some strains of Staphylococcus hyicus
● Staphylococcus saprophyticus ● Coagulase-negative staphylococci (CoNS):
● Staphylococcus haemolyticus ○ Clinically significant and commonly recovered CoNS:
● Staphylococcus lugdunensis ■ Staphylococcus epidermidis – nosocomial infecti
■ Micrococcus spp. ■ Staphylococcus saprophyticus – urinary tract infection (UTI)
■ Rothia, Aerococcus, Alloiococcus spp. ● Occasional isolates and can be significant pathogens:
○ Staphylococcus haemolyticus – wound, septicemia (blood), UTI,
Staphylococcus spp. native valve infections
● Gram-positive cocci ○ Staphylococcus lugdunensis – catheter-related bacteremia and
● They produce the enzyme catalase endocarditis
● Exhibit spherical shapes (0.5 to 1.5 μm) that may appear singly, in pairs, ● Coagulase as a virulence factor
and in clusters ○ Coagulase-positive staphylococci showing bound coagulase
● The microscopic appearance is NOT specific or characteristic for (clumping factor) on the bacterial cell wall.
staphylococci ○ The fibrin capsule may protect the bacterium from phagocytosis and
● They resemble some members of the family isolate it from other defenses of the host. The fibrin coat or capsule
● Micrococcaceae, such as the genus Micrococcus can therefore make the bacteria more virulent.
● They are non-motile
● They are non-spore-forming Staphylococcus aureus
● They may be aerobic or facultative anaerobic, except for Staphylococcus ● Most clinically significant species of staphylococci
saccharolyticus (obligate anaerobe) ● Can be recovered from almost any specimen
● Colonies (after 18-24 hours of incubation at 37oC): medium-sized (4-8 ● An important cause of nosocomial (healthcare-acquired) and
mm) and appear cream-colored, white or rarely light gold, and community-acquired infections
buttery-looking ● Associated with suppurative skin and wound infections
● Some are ß-hemolytic ○ Abscess filled with pus and surrounded by necrotic tissue and
● They are one of the most commonly isolated organisms damaged leukocytes
○ Examples: folliculitis, furuncles (boils), carbuncles (multiple furuncles
Epidemiology with fever and chills), and bullous impetigo (a highly contagious
Organism Habitat (Reservoir) Mode of Transmission infection; larger pustules surrounded by small zone of erythema)
S. aureus Normal flora: anteriorEndogenous strain: sterile site by ● Staphylococcus aureus (Blood Agar Plate) - Notice the ß-hemolytic
nares, nasopharynx, traumatic introduction (e.g., surgical pattern (clear zone around the bacterial colony) on sheep blood agar
skin, perineal area, wound or microabrasions)
plate. Colonies are medium-sized and buttery-looking.
and colonizer of Direct contact: person to person;
mucosa fomite ● Staphylococcus aureus (Gram stain) - Notice the Gram-positive
Indirect contact: aerosolized spherical bacterial cells. They may appear singly, in pairs, or in clusters.
S. epidermidis Normal flora: skin and Endogenous strains: sterile site, by ● On microscopy of Gram-stained bacterial smears, S. aureus cells occur
mucous membranes implantation of medical devices (e.g. in grape-like clusters, may occur singly or in pairs. Colonies produce a
shunts, prosthetic devices) golden pigment, staphyloxanthin
Direct contact: person to person ● MSA contains the sugar mannitol and the pH indicator phenol red. If an
S. haemolyticus Normal flora: skin and Same as previously indicated for S. organism can ferment mannitol, an acidic byproduct that will cause the
S. lugdunensis mucous membranes epidermidis
phenol red in the agar to turn yellow.
(low numbers)
S. saprophyticus Normal flora: skin, Endogenous strain: sterile urinary ● Mannitol Salt Agar (MSA) is both selective and differential.
genitourinary tract, tract, notably in young sexually active Staphylococcus spp. can tolerate a high-salt (7.5% NaCl) environment
and mucosa females ● Disease-Causing Factors (or Virulence Factors)
○ Enzymes: catalase, staphylocoagulase, clumping factor, ○ Protein A - identified in the cell wall of Staphylococcus aureus
hyaluronidase, protease, lipase, nuclease, fibrinolysin ■ Its most significant role is its binding ability to the Fc portion
○ Enterotoxin B – implicated in staphylococcal food poisoning; stimulates (crystallizable fragment) of immunoglobulin G (IgG) antibodies.
cytokine release and inflammation ■ Binding IgG inhibits opsonization, which is an important step in
○ Toxic shock syndrome toxin-1 (TSST-1) – causes desquamation phagocytosis and negates the protective effect of IgG antibody
(peeling of the skin), high fever, rash, hypotension
○ Exfoliative toxin – implicated in staphylococcal scalded skin syndrome Table: Pathogenesis and Spectrum of Disease
(SSSS, Ritter’s disease); targets the protein desmoglein-1 (DG-1)
○ Cytolytic toxin – lyses RBCs (hemolysin) and WBCs (leukotoxin)
○ Protein A – can bind to the Fc portion of IgG antibody, blocking
opsonophagocytosis
○ Coagulase
■ Bound coagulase (or clumping factor) – bound to cell wall of the
bacterium
■ Has the ability to stimulate clot formation in plasma
■ Facilitates in the conversion of fibrinogen to fibrin in the
immediate vicinity of the bacterium as means of self-protection→
fibrin capsule → inhibits phagocytosis
■ Mainly produced by S. aureus; coagulase is also produced by
other coagulase-positive staphylococci and Yersinia pestis
○ Hyaluronidase - enzyme hydrolyzes hyaluronic acid that makes up
your connective tissues
■ Damage to connective tissues will permit the spread of bacteria
during infection
○ Lipase - enzyme is produced by both coagulase-positive and
coagulase-negative staphylococci
■ Lipase acts on the lipids present on the surface of the skin,
particularly the fats and oil secreted by the sebaceous glands
■ Acting together with protease and hyaluronidase, these enzymes
are capable of destroying tissue and may facilitate the spread of
infection to adjoining tissues
○ Enterotoxins - Cause symptoms such as vomiting and diarrhea
■ Staphylococcal enterotoxins are heat-stable exotoxins (stable at
100 C for 30 minutes) → reheating contaminated food does not
prevent disease or infection
■ Staphylococcal food poisoning is most commonly caused by
enterotoxins A, B, and D → BAD enterotoxins
■ Enterotoxins B and C (sometime G and I) are associated with
toxic shock syndrome
■ Enterotoxin B is associated with staphylococcal
pseudomembranous enterocolitis
○ Toxic Shock Syndrome Toxin-I (TSST-I) - Causes nearly all cases of
menstruating-associated toxic shock syndrome
■ Previously called enterotoxin F
■ A superantigen that stimulates T-cell proliferation and subsequent
Summary of the Key Tests for the Identification of the Most Clinically
production of cytokines that are responsible for the symptoms
Significant Staphylococcus Species Affecting Humans
■ At low concentrations, TSST-1 causes leakage by endothelial
cells
Test S. aureus S. epidermidis S. saprophyticus
■ It is cytotoxic to these cells at higher concentrations
Gram Staining Gram(+) Gram(+) Gram(+)
○ Exfoliative toxin - epidermolytic toxin
Colony pigment Positive Negative Negative
■ It causes the epidermal layer of the skin to slough off Staphylocoagulase Positive Negative Negative
■ Associated with scalded skin syndrome (SSS), which is Clumping factor Positive Negative Negative
sometimes referred to as Ritter disease; it is a bullous exfoliative Mannitol fermentation Positive Negative Negative
dermatitis primarily in newborns and previously healthy young Novobiocin Sensitive Sensitive Resistant
children
■ Also has been implicated in bullous impetigo GRAM-POSITIVE COCCI: STREPTOCOCCUS, ENTEROCOCCUS, AND
○ Cytolytic toxin SIMILAR ORGANISMS
■ An extracellular protein that affect red blood cells and white blood
cells General Characteristics
● Hemolysins – affects red blood cells; cause hemolysis! Streptococcus spp. and Enterococcus spp.
● Leukocidin – affects white blood cells ● Gram-positive (purple color)
■ S. aureus produces four (4) hemolysins: alpha-, beta-, gamma-, ● Spherical or ovoid shape, non-motile, non-spore-forming, and occurs in
and delta-hemolysin pairs (diplococci) or chains (streptococci)
■ Beta-hemolysin (sphingomyelinase C, “hot-cold” lysin) – acts on ● Aerotolerance: most streptococci are facultative anaerobes, and some
the sphingomyelin in the RBC plasma membrane are obligate (strict) anaerobes
■ Gamma-hemolysin is associated with the Panton-Valentine ● Most require enriched media (blood agar)
Leukocidin (PVL), which is lethal to neutrophils and contributes to ● Oxidase-negative (add oxidase reagent→ no blue color)
the invasiveness of the organism by suppressing phagocytosis; ● Catalase-negative (add H2O2 → no bubbles)
associated with severe skin infections, and necrotizing pneumonia
 ● Many of these organisms are part of the normal human flora (e.g., skin,
genital, respiratory and digestive tract) ● Indirect (Passive) Agglutination - Known soluble antigens are
● Become pathogenic → organisms gain entry to sterile sites coated on to other cells (e.g.,erythrocytes) or inert particles (e.g.,
latex, bentonite, charcoal, polystyrene), which act as passive
Epidemiology carriers of antigens.
● Streptococcus pyogenes, S. pneumoniae, S. agalactiae, viridans ● Reverse (Passive) Agglutination - Specific antibodies are coated
streptococci, Enterococcus faecalis, and Enterococcus faecium are on to other cells (e.g., erythrocytes) or inert particles (e.g., latex,
clinically important pathogens bentonite, charcoal, polystyrene), which act as passive carriers of
● Streptococcus pneumoniae is the leading cause of bacterial pneumonia antibodies.
and meningitis ○ Beta-Hemolytic, Group A Streptococci - Streptococcus pyogenes
● Streptococcus pyogenes, designated as the “flesh-eating bacteria,” is ■ Virulence Factors
associated with bacterial acute pharyngitis (strep throat) and skin ● Protein F – mediates epithelial cell binding (fibronectin
infections such as impetigo and erysipelas component)
● The main beta-hemolytic streptococci that cause pharyngitis are under ● M protein – inhibits phagocytosis (anti-phagocytic)
the Lancefield group A, C, and G ● Hemolysins (Streptolysin O and S) – cause lysis of cells
● Streptokinase – activates plasminogen to plasmin
Pathogenesis and Spectrum of Disease ● DNAse – helps bacteria escape neutrophil extracellular traps
● Beta Hemolytic Streptococci (NETs)
○ This is the most clinically significant streptococci belonging to the ● Hyaluronidase – helps bacteria penetrate into tissues
Lancefield group A ● Streptococcal pyrogenic exotoxins – mediate production of rash
○ Produces virulence factors such as hemolysins streptolysin O and (i.e. scarlet fever) or multisystem effects
streptolysin S → responsible for the beta-hemolytic pattern on blood ● Complement C5a peptidase – destroy complement system
agar plate (clear zone of hemolysis around the colony) chemotactic factors for phagocytes
○ Streptolysin O ■ Habitat (Reservoir)
■ Oxygen-labile (unstable) exotoxin ● This organism is not considered a normal flora
■ Broken down by oxygen ● Its presence in clinical samples is almost always considered
■ Immunogenic hemolysin clinically significant
■ Protein: MW of 61.5 kDa ● It inhabits the skin and upper respiratory tract of humans
■ Capable of lysing the cell membrane of blood cells e.g., ● It is carried on nasal, pharyngeal, and sometimes, anal mucosa
erythrocytes, leukocytes, and platelets ■ Mode of Transmission
■ Produce hemolysis only in the absence of oxygen ● Direct contact: person to person
■ It is inhibited by the presence of cholesterol in skin lipids ● Indirect contact: aerosolized droplets from coughs or sneezes
■ Body cannot produce protective antibodies associated with skin ■ Disease or infx
infections ● Acute pharyngitis
○ Streptolysin S ● Impetigo, cellulitis, erysipelas
■ Oxygen-stable exotoxin ● Necrotizing fasciitis and myositis
■ Non-immunogenic hemolysin ● Bacteremia with potential for infection in any of several organs
■ Polypeptide: MW 28 amino acids ● Pneumonia
■ Also capable of lysing the cell membrane of blood cells ● Scarlet fever - disease caused by Streptococcus pyogenes. The
■ Can produce hemolysis in the presence of room air signs and symptoms include a sore throat, fever, headaches,
○ Immunogen - any foreign substance capable of producing an immune swollen lymph nodes, and a characteristic rash. The rash is red
response. and feels like sandpaper and the tongue may be red and bumpy.
○ If a substance has the ability to induce an immune response, then it is ● Streptococcal toxic shock syndrome
considered immunogenic. ● Post_streptococcal Sequelae (After Streptococcal Infection):
○ Laboratory Determination of Anti-Streptolysin O (ASO) ○ Rheumatic fever and PSGN are inflammatory diseases that
■ Method: ASO Latex Agglutination can develop (10-14 days after streptococcal infection) when
■ Principle: Indirect (Passive Agglutination) strep throat or scarlet fever aren't properly treated. They are
■ ASO latex agglutination test method is based on an immunologic not caused by the bacteria itself, but by the body’s immune
reaction between streptococcal exotoxins (Streptolysin O) bound to reaction to the bacteria
biologically inert polystyrene latex particles and streptococcal ○ Rheumatic fever – cross-reactions of antibodies produced
antibodies in the test sample. Visible agglutination occurs when against streptococcal antigens and human heart tissue
increased antibody level, are present in the test specimen. ○ Post-streptococcal glomerulonephritis (PSGN) – deposition of
■ This is a qualitative test: it only detects the presence or absence of antibody- streptococcal antigen immune complexes in kidney
the analyte of interest. (glomerular basement membrane) results in damage to
■ Our analyte (unknown substance of interest) is Anti-Streptolysin O glomeruli
antibodies. ● Laboratory test for Post-Streptococcal Sequelae
■ Positive Result: Presence of visible agglutination or clumping of ○ Anti-Streptolysin O (ASO) titer
latex particles. It indicates the presence of antibodies to Streptolysin ■ Anti-streptolysin O is the antibody made against
O (ASO) streptolysin O, an immunogenic, oxygen-labile
■ Negative Result: Absence of visible agglutination or no clumping of streptococcal hemolytic exotoxin produced by most strains
latex particles. It indicates the absence of antibodies to Streptolysin of group A and many strains of groups C and G
O (ASO) streptococci.
■ Types of Agglutination Reactions ■ This test helps determine whether you have had a recent
● Direct agglutination - Cells (such as bacteria, fungus,and streptococcal infection with the bacteria group A,
erythrocytes) and insoluble particulate antigens can be directly beta-hemolytic streptococci.
agglutinated by their specific antibodies. ■ It also helps diagnose complications resulting from a
streptococcal infection such as rheumatic fever or
Passive Agglutination requires cells or inert particles as carriers glomerulonephritis, a form of kidney disease.
of antigens or antibodies.
○ Beta-Hemolytic, Group B Streptococci - Streptococcus agalactiae Streptococcus pneumoniae
■ Clinically significant streptococci belonging to the Lancefield ● Gram-positive
group B ● Spherical, encapsulated, lancet-shaped bacteria
■ Infections are usually associated with neonates and are acquired ● Usually occurring in pairs (diplococci)
before or during the birthing process. ● Non-motile and non-spore-forming
■ It is associated with septicemia, pneumonia, and meningitis in ● Facultative anaerobic
newborns. ● Produce alpha (under aerobic conditions) or beta-hemolytic (under
■ Habitat (Reservoir) anaerobic conditions) patterns on blood agar
● Normal flora of the female genital tract and lower ● Alpha-hemolytic streptococci must be differentiated from the viridans
gastrointestinal tract streptococci (also alpha-hemolytic)
● Occasional colonizer of the upper respiratory tract ● Virulence Factors:
● Some endogenous strains cause disease if they gain access to ○ C polysaccharide – unrelated to the Lancefield grouping; the
sterile sites polysaccharide capsule that inhibits phagocytosis (anti-phagocytic) is
■ Mode of Transmission primary virulence factor
● Direct contact: person to person from mother in-utero or during ○ Pneumolysin – has various effects on host cells: activates the
delivery classical complement pathway and mediates suppression of the
● Nosocomial transmission by unwashed hands of mother or oxidative burst in phagocytes
healthcare personnel ○ Secretory IgA protease – proteolytic enzymes that cleave specific
■ Virulence factor - Capsular material interferes with the phagocytic peptide bonds in the human immunoglobulin A1 (IgA1) hinge region
activity and complement system cascade activation sequence
■ Disease or infx: ○ Phosphorylcholine – binds receptors for platelet-activating factor in
● Infections in neonates often present as multisystem problems, endothelial cells, leukocytes, platelets, and tissue cells of the lungs
including sepsis, fever, meningitis, respiratory distress, and meninges providing for entry and spread of the organism
lethargy, and hypotension ● Habitat (Reservoir) - nasopharynx
● Infections in immunocompromised adults include bacteremia, ● Disease or infx - Leading cause of bacterial meningitis and pneumonia
pneumonia, endocarditis, arthritis, osteomyelitis, and skin and with or without bacteremia; also causes sinusitis and otitis media
soft tissue infections ○ S. pneumoniae is capable of spreading to the lungs, paranasal
○ Beta-Hemolytic Streptococci - Lancefield Group C, F, and G sinuses, and middle ear. In addition, this organism accesses the
Streptococci bloodstream and the meninges to cause acute, purulent, and often
■ Normal flora of the skin, nasopharynx, gastrointestinal tract, and life-threatening infections
genital tract; some endogenous strains may gain access to sterile ● Mode of Transmission - Direct contact: person to person with
sites contaminated respiratory secretions
■ Cause similar types of acute infections in adults as described for
S. pyogenes and S. agalactiae, but usually involve Viridans group streptococci
immunocompromised patients; do not cause post-infection ● Gram-positive
sequelae ● Catalase and coagulase-negative
■ A notable proportion of infections caused by Lancefield group G ● Non-motile and non-spore-forming
streptococci occur in patients with underlying malignancies ● Facultatively anaerobic
■ Lancefield group C organisms occasionally have been associated ● Produce alpha-hemolytic pattern (greenish) on blood agar – must be
with acute pharyngitis differentiated from S. pneumoniae (also alpha-hemolytic)
○ Beta-Hemolytic Streptococci - Lancefield Group C Streptococci ● Viridans streptococci can be distinguished from S. pneumoniae, by
■ Group C streptococci (GCS) are livestock pathogens and they resistance to optochin and lack of bile solubility
often cause zoonotic diseases in humans ● Normal flora:
■ They are Gram-positive, in mostly β-hemolytic and facultative ○ Oral cavity, gastrointestinal tract, female genital tract
anaerobes ○ Endogenous strains may gain access to sterile sites; most notably
■ Organisms under this group: results from dental manipulations
● S. dysgalactiae subs. equisimilis (also under group A and G) ● Virulence factors:
● S. equi subs. equi ○ Generally considered to be of low virulence
● S. equi subs. zooepidemicus ○ Production of extracellular complex polysaccharides (e.g., glucans
● S. constellatus subs. pharyngis and dextrans) enhance attachment to host cell surfaces, such as
○ Beta-Hemolytic Streptococci - Lancefield Group F Streptococci cardiac endothelial cells or tooth surfaces in the case of dental caries
■ Group F streptococci are part of the oropharyngeal, bowel, and ● Diseases or infx:
perineal flora ○ Slowly evolving (subacute) endocarditis, particularly in patients with
■ Group F β-hemolytic streptococci cause purulent disease and previously damaged heart valves
bacteremia in adults. Infections with these organisms are rare in ○ Bacteremia and infections of other sterile sites do occur in
previously healthy children. immunocompromised patients
■ Produce minute beta-hemolytic pattern on blood agar ○ Meningitis can develop in patients suffering trauma or defects that
■ Organism under this group - Streptococcus anginosus allow upper respiratory flora to gain access to the central nervous
○ Beta Hemolytic Streptococci - Lancefield Group G Streptococci system
■ Group G streptococcus (GGS) are commonly regarded as ○ Streptococcus mutans plays a key role in the development of dental
commensals usually found in association with the normal flora of caries (tooth cavities)
human skin, pharynx, and intestine.
■ Organisms and their Reservoirs: Enterococcus spp.
● S. dysgalactiae subspecies equisimilis – humans ● Formerly part of the group D streptococci
● S. milleri – humans ● Causes nosocomial and wound infections
● S. canis – dogs ● Normal flora (reservoir):
● S. intestinalis – pigs ○ Humans, animals, and birds
○ Gastrointestinal tract and female genitourinary tract
○ Endogenous strains may gain access to sterile sites
 ● Clinically significant organisms: ○ Catalase test: Streptococcus, Enterococcus, and similar organisms do
○ Enterococcus faecalis – linked to increased virulence not produce this enzyme, hence they test negative (catalase-negative)
○ Enterococcus faecium ○ Hemolytic patterns on 5% sheep blood agar
● Virulence factors: ● Other biochemical tests for presumptive identification:
○ Antibiotic resistance ○ PYR test: detection of PYRase enzyme
○ Enterococcal surface protein – allows the bacteria to aggregate and ○ Bile solubility test: test for clearing in the presence of bile salts
form bioflims ○ Optochin sensitivity test: test for inhibition in the presence of optochin
○ Aggregation substance – allows the microbe to bind to target cells and ○ CAMP test: test for enhanced (arrowhead) hemolysis
it facilitates the transfer of genetic material between cells. ○ Hippurate hydrolysis test: test for hippuricase enzyme
○ Superoxide, hydrogen peroxide, and hydroxyl radicals production
○ Gelatinase and hyaluronidase – hydrolyzes peptides Identification: Hemolytic Patterns on BA
○ Cytosolin – is a protein found in the cytosol and lyses erythrocytes.
● The enterococci are accountable for up to 20% of all cases of infective
endocarditis, with Enterococcus faecalis being the primary causative
isolate.
● Enterococcus faecium has multidrug resistance characteristics.
Incidence is on the rise in hospitals due to its resistance to vancomycin
and other antibiotics.

Nutritionally Variant Streptococci

● Abiotrophia spp. and Granulicatella spp.
○ Normal flora of the oral cavity
○ Abiotrophia spp. grow well in blood, but fail to grow when subcultured
on conventional agar media.
○ Grow as satellite colonies if the plates are streaked with S. aureus, or if
the growth medium is enriched with vitamin B6 (pyridoxine).
○ Associated with endocarditis Organism Hemolytic Pattern
○ Culture media of choice: S. pyogenes Beta-Hemolysis
S. agalactiae Beta-Hemolysis
■ The nutritionally variant streptococci will not grow on solid media
S. pneumoniae Alpha-Hemolysis(aerobic)
(e.g., BA or CA) unless you supplement these culture media with
Beta-Hemolysis (anaerobic)
pyridoxal (vitamin B6) or by cross-streaking the media with Viridans streptococci Alpha -Hemolysis
Staphylococcus aureus E. faecalis Gamma-Hemolytic
■ Liquid media (e.g., nutrient and enrichment broths) also support the E. faecium Gamma-Hemolytic
growth of these organisms ● Type of Hemolytic Patterns (on BA Plates)
■ Consider these organisms if there is no growth on solid media, but ○ Beta-Hemolysis - complete hemolysis (clear zone around colony)
there is growth on liquid media; and Gram-stain shows ○ Alpha-Hemolysis - partial hemolysis (green zone around colony)
Gram-positive cocci in chains ○ Gamma-Hemolysis - no hemolysis

Identification: PYR Test

Culture media of choice:
● Used for the presumptive identification or separation of group A
● Streptococcus spp. and Enterococcus spp.
ß-hemolytic streptococci (Streptococcus pyogenes) and Enterococcus
○ Organisms discussed will grow on standard laboratory media such as
spp. from other streptococci
nutrient and enrichment broths (e.g., thioglycollate, brain-heart
● It tests for the presence of the enzyme called
infusion, blood culture bottle), 5% sheep blood agar (BA) and
L-pyroglutamyl-aminopeptidase that breaks down the substrate
chocolate agar (CA)
L-pyrrolidonyl-ß-naphthylamide (PYR) to produce ß-naphthylamine,
● Solid media: BA and CA
which is detected by the reagent called N,N-methyl-
○ 5% Sheep Blood Agar (BA) - BA is an enriched and differential medium
aminocinnamaldehyde
○ Chocolate agar (CA) - CA is an enriched medium
● Positive result: red-colored product
■ composed of heated blood agar; no chocolate ingredient is added!

Organism Hemolytic Pattern

Incubation Conditions
S. pyogenes Positive
● Most organisms in this group are facultative anaerobes with some
E. faecalis Positive
preferring a CO2 -enriched environment.
E. faecium Positive
● Laboratories typically incubate BA and CA plates in 5% to 10% carbon S. pneumoniae Positive
dioxide (may be achieved using a candle jar) – this is the preferred S. agalactiae Negative
atmosphere for Streptococcus pneumoniae and other genera discussed Viridans streptococci Negative
in this lecture.
● Beta-hemolytic patterns are enhanced when BA plates are incubated in Identification: Bile Solubility Test
an anaerobic environment (may be achieved by stabbing the inoculating ● Purpose and Principle
loop into the agar several times. ○ Used to differentiate Streptococcus pneumoniae [aerobic incubation:
○ Oxygen-labile hemolysins (Streptolysin O) are produced in the alpha hemolysis] (positive) from alpha-hemolytic streptococci
absence of oxygen, producing subsurface hemolysis of the BA. (negative).
● CA plates incubated inside a candle jar ○ Bile or a solution of bile salt (sodium deoxycholate) rapidly lyses or
● Enhanced beta-hemolysis may be achieved by stabbing the inoculating dissolves pneumococcal colonies
loop into the agar several times. ○ Bile salts lower the surface tension between the bacterial cell
membrane and the medium (accelerates autolytic process of the
Approach for Identification organism)
● Initial laboratory tests for identification ○ Positive result: colony disintegrates or lysed by bile salt solution
○ Gram staining: organisms under this group are Gram-positive cocci in (clear)
pairs (S. pneumoniae) or chains (others) ○ Negative: intact colonies
Organism Hemolytic Pattern
S. pneumoniae Positive
Viridans streptococci Negative
Enterococcus spp. Negative

Identification: Optochin Sensitivity Test

● Purpose and Principle
○ Also called Taxo P sensitivity test
○ Used to determine the effect of optochin (ethylhydrocupreine
hydrochloride) on an organism.
○ Optochin lyses pneumococci (positive test), but alpha-hemolytic
streptococci are resistant (negative test)
○ Positive: presence of zone of inhibition around optochin disk (>14 mm)
○ Negative: absence of zone of inhibition around optochin disk (<14 mm)
○ The strain is only identified as S.pneumoniae (pneumococcus) if the
result for bile solubility is positive
○ Only S. agalactiae is positive

Identification: CAMP Test

● Purpose and Principle
○ CAMP stands for Christie-Atkins-Munch- Peterson
○ Used to presumptively identify group B β- hemolytic streptococci
(Streptococcus agalactiae) based on their formation of a substance
(CAMP factor) that enlarges or enhances the area of hemolysis formed
by the β-hemolysin elaborated from Staphylococcus aureus.
○ Positive: enhanced hemolysis is indicated by an arrowhead-shaped
zone of beta- hemolysis at the juncture of the two organisms.
○ Negative: no enhancement of hemolysis
○ S. agalactiae is positive and S. pyogenes is negative

Identification: Hippurate Hydrolysis Test

● Purpose and principle
○ Hippurate hydrolysis test is used to detect the ability of bacteria to
hydrolyze substrate hippurate into glycine and benzoic acid by action
of hippuricase enzyme present in bacteria.
○ It is used for the presumptive identification of S. agalactiae (positive)
○ Glycine is deaminated by the ninhydrin reagent, which is reduced in
the process.
○ Positive: deep purple color indicates hippurate has been hydrolyzed
○ Negative: slightly yellow-pink or colorless
○ S. agalactiae is positive and S. pyogenes is negative

Identification: Neufeld’s Quellung Reaction

● Quellung reactions are based on swelling of the capsules surrounding
individual organisms in the presence of specific antisera and may be
performed by the microscopic examination of cover-slipped slides with a
mixture of one loopful each of an overnight Todd-Hewitt broth culture,
specific antiserum, and methylene blue (Weisbroth and Freimer, 1969).
● Positive result: capsules look “swollen” and refractile