BACTERIAL MORPHOLOGY, STRUCTURE AND CLASSIFICATION

Taxonomy

 Is an area of biologic science comprising three distinct, but highly interrelated, disciplines that
include classification, nomenclature and identification
 Is a formal system for organizing, classifying and naming living things
 CARL VON LINNE- a Swedish botanist, laid down the basic rules for taxonomic categories

I. CLASSIFICATION
 Is the organization of microorganisms that share similar morphologic, physiologic
and genetic traits into specific groups or taxa
 Is the arrangement of organisms into groups, preferably in a format that shows
evolutionary relationships
CLASSIFICATION SYSTEM IS HIERARCHIC AND CONSISTS OF THE FF. TAXA DESIGNATIONS
1. DOMAIN- Bacteria and Archaea (unicellular prokaryotic organisms)
2. KINGDOM- composed of similar divisions; similarities of DNA and RNA
3. DIVISION- composed of similar classes
4. CLASS- composed of similar orders
5. ORDER- composed of similar families
6. FAMILY- composed of similar genera
7. GENUS- composed of similar species
8. SPECIES- is the basic group; collection of bacterial strains with common physiologic and
genetic features
9. SUBSPECIES- species are subdivided based on phenotypic differences
a. Serotype- based on serologic differences
b. Biotype- based on biochemical differences

Epithet- proper word for the name of the species

II. NOMENCLATURE
 Is the naming of microorganisms according to established rules and guidelines
 Provides the accepted labels by which organisms are universally recognized
 Binomial system is used- two-name system of nomenclature, composed of the genus
and species
 Genus level should always have its first letter capitalized, while the species
designation should never be capitalized- both the genus and species should be
italicized in print but underlined when written in script
III. IDENTIFICATION
1. Genotypic characteristics
 Relates to the organism’s genetic makeup
 Involves detection of a gene or a part thereof, or an RNA product of a specific
organism
 Serves as a confirmatory method for the presence of the organism
 2. Phenotypic characteristics
 Based on features beyond the genetic level
 Includes readily observable characteristics and those characteristics that may
require extensive analytic procedures to be detected

Prokaryotes

 “Pro” meaning before and “karyon” meaning nucleus, nut or kernel

 These are organisms that do not contain a true nucleus
 They also do not contain organelles such as mitochondria, endoplasmic reticulum and golgi
apparatus, and all functions take place in the cytoplasm or cytoplasmic membrane
Bacteria
 Example of prokaryotic cells
 Unicellular organisms that lack a true nucleus, a nuclear membrane and membrane- bound
organelles

BACTERIAL CELL STRUCTURE

1. BACTERIAL NUCLEUS
 Not surrounded by a nuclear membrane
 Generally called a nucleoid or nuclear body; contains bacterial chromosome
2. BACTERIAL GENOME
 Refers to all genes that comprise the organism
 Can be chromosomal, non- chromosomal and transposable
 Bacterial Chromosome
 Composed of set of genes
 Found in the nucleoid of the bacteria
 Plasmids
 Capable of self- replication but not essential for bacterial growth
 Usually codes for antibiotic resistance, virulence factors and toxin
production
 Transposable Elements
 Pieces of DNA that move from one genetic element to another; from
plasmid to chromosome or vice versa
3. CELL ENVELOPE
 Outermost structure of the bacterial cell
 Composed of layers (cell wall, cell membrane and capsule) that surround the bacterium
 Some organisms have cell wall, some do not; some have capsule, some do not
 All bacteria contain cell membrane
4. CELL WALL
 Referred to as the peptidoglycan or murein layer
 Rigid structure that maintains the shape of the cell
 Composed of disaccharide- pentapeptide subunits; also made up of teichoic acid or
lipoteichoic acid
  Prevents bacterial cells from rupturing when the osmotic pressure inside the cell is
greater than outside the cell
 Point of anchorage for flagella
 Determines staining characteristics of a species

A. Gram- positive cell wall

 Composed of very thick protective peptidoglycan layer
 Contains teichoic acid
 Prime target of antimicrobial agents like penicillin, which acts by preventing the
synthesis of peptidoglycan
B. Gram- negative cell wall
 Composed of outer membrane and inner (thin peptidoglycan) membrane
 Has porins which contributes to the permeability of the cell wall
 Does not contain teichoic acid
C. Acid- fast cell wall
 Has gram- positive cell wall structure
 Also contains a waxy layer of glycolipids and fatty acids
D. Absence of cell wall
 Prokaryotes that lack a cell wall contain sterols in their cell membrane
 Examples: Ureaplasma and Mycoplasma
5. PLASMA MEMBRANE
 Deepest layer of the cell envelope; internal matrix of the cell
 Phospholipid bilayer with embedded proteins that surrounds the cytoplasm
 Acts as an osmotic barrier
 Site of respiration and photosynthesis
6. CYTOSOL
 Fluid portion of the cell; contains no organelles
 Contains ribosomes and various types of nutritional storage granules
7. PILI OR FIMBRIAE
 Hair- like structures made up of the protein pilin
 Exists in two classes:
 Ordinary (common) pili- involved in adherence and may serve as virulence factor
 Sex pili- for bacterial conjugation
8. FLAGELLA
 Organ for locomotion or motility
 Arrangement of flagella:
 Atrichous- without flagellum
 Monotrichous- single flagellum on one end
 Amphitrichous- single flagellum on both ends
 Lopotrichous- tuff/ group of flagella on one end or both ends
 Peritrichous- spread over the whole surface
9. ENDOSPORES
 Contains calcium dipicolinate (dipicolinic acid); found in Bacillus and Clostridium
  Metabolically inactive and resting bacterial cells that are highly resistant to heat and
chemicals

HISTORY OF MICROBIOLOGY

MICROBIOLOGY is the study of organisms that individually are too small to be seen by the naked
eye. In the beginning of this study, great minds have contributed to the discovery and evolution of
microbiology, and its relationship to medicine and other areas of biology.

DISCOVERY OF MICROORGANISMS

 Roman philosopher Lucretius(98-55 B.C.) and Girolamo Fracastoro(1478-1553)

 Suggested that diseases were caused by “invisible living creatures”
 Francesco Stelluti(1625-1723)
 Made the earliest microscopic observations on bees and weevils using a microscope
probably supplied by Galileo
 Anton Van Leeuwenhoek(1632-1723)
 First “true microbiologist”
 First person to observe and describe microorganisms accurately
 “Father of Protozoology and Bacteriology”
 Used his self-made single lens microscope with 50-300x magnification to study
protozoans and bacteria

SPONTANEOUS GENERATION- life arose from non-living matter

 Francesco Redi
 In 1668, he demonstrated that maggots do not arise spontaneously from decaying
meat
 John Needham
 He observed that boiled mutton broth eventually became cloudy with microorganisms
after pouring it into a flask and sealed tightly
 Lazzaro Spallanzani
 He improved the experiments of Needham by heating the broth placed in a sealed jar
 He observed that no growth took place as long as the flasks remained sealed
 He proposed that air carried microorganisms to the culture medium and that might be
the reason for the growth of organisms present already in the medium
 Laurent Lavoisier
 He showed the importance of oxygen to life

BIOGENESIS- living cells can arise only from preexisting living cells

 Rudolf Virchow
 He challenged spontaneous generation with the concept of “biogenesis”
 Theodore Schwann
  He observed that no growth occurred in a flask containing nutrient solution after
allowing air to pass through a red-hot tube
 Louis Pasteur
 He resolved the issue of spontaneous generation
 He stated that microorganisms are indeed present in the air and can contaminate
seemingly sterile solution, however the air itself does not create microbes
 He showed that microorganisms can also be present in non-living matter
 He stated that microbial life can be destroyed by heat(basis of the aseptic technique- a
technique to prevent contamination by unwanted microorganisms)
 He provided evidence that microorganisms cannot originate from mystical forces
present in nonliving materials
 John Tyndall
 He showed that dust carry germs which contaminates sterile broth
 “Tyndallization”- form of sterilization for three consecutive days

ANTISEPTIC SYSTEM
 Ignaz Semmelweis
 He demonstrated that routine handwashing can prevent the spread of disease
 Joseph Lister
 He developed the antiseptic system of surgery
 “Father of Modern Antisepsis”

GERM THEORY OF DISEASE- based on the concept that microorganism might cause disease

 Robert Koch
 He established the first proof that bacteria indeed cause diseases
 He discovered Bacillus anthracis- causative agent of anthrax
 He discovered Mycobacterium tuberculosis
 He was the first to cultivate bacteria on boiled potatoes, gelatin, meat extracts and
protein
 He developed culture media for observing growth of bacteria isolated from human body

Collaborators of Koch
 Fannie Hesse- she suggested the use of agar as a solidifying agent
 Julius Richard Petri- he developed the petri dish
 Martinus Beijerinck and Sergie Winogradsky- they developed the enrichment- culture
technique; introduced the use of selective media

CELL PHYSIOLOGY, METABOLISM AND BACTERIAL GENETICS

 MICROBIAL NUTRITION AND GROWTH

PHASES OF BACTERIAL GROWTH
1. LAG PHASE
  Period wherein there is no cell division; no abrupt increase in cell number
 Phase of adjusting to a new environment
2. LOG/ EXPONENTIAL PHASE
 Period wherein microorganisms are actively growing and dividing; bacterial numbers
increase logarithmically
 Cellular production is most active during this period
 Microorganisms are sensitive to radiation and antimicrobial agents
3. STATIONARY/ PLATEAU PHASE
 Period wherein there is a balance between cell division and dying organism- number of
viable microorganisms remains constant
 Metabolic activities of surviving cells slow down and nutrients are becoming limited
4. DEATH/ DECLINE PHASE
 Period wherein there is cessation of bacterial growth- the number of cell death exceeds
the number of living microorganisms
 There is loss of nutrients and increased amount of toxic waste

 PHYSIOLOGIC REQUIREMENTS OF BACTERIA

1. ACCORDING TO OXYGEN REQUIREMENT
A. OBLIGATE AEROBE- have absolute requirement for oxygen
B. OBLIGATE ANAEROBE- unable to grow and multiply in the presence of oxygen
C. FACULTATIVE AEROBE- fundamentally an anaerobe but can grow in the presence of
atmospheric oxygen
D. FACULTATIVE ANAEROBE- fundamentally an aerobe but can grow in the absence of
atmospheric oxygen
E. CAPNOPHILE- require increased carbon dioxide
F. AEROTOLERANT- does not grow well, but survives in the presence of atmospheric oxygen
G. MICROAEROPHILE- requires reduced oxygen
2. ACCORDING TO NUTRITIONAL REQUIREMENT
A. AS TO CARBON SOURCE
a. Autotrophs- use carbon dioxide as sole source of carbon
b. Heterotrophs- use reduced, preformed organic molecules from other bacteria
B. AS TO ENERGY SOURCE
a. Phototrophs- organisms that use light
b. Chemotrophs- organisms that use energy produced by the oxidation of organic or
inorganic compounds
C. AS TO ELECTRON SOURCE
a. Lithotrophs- reduce inorganic molecules
b. Organotrophs- require inorganic substances for growth and multiplication
3. ACCORDING TO TEMPERATURE REQUIREMENT
35°C- 37°C- optimum temperature for most bacteria
A. PSYCHROPHILE/CRYOPHILE- grows well at 0°C to a maximum of 20°C
B. MESOPHILE- grows between 20°C to 45°C
C. THERMOPHILE- grows between 50°C to 125°C
4. pH
A. ACIDOPHILE- pH 0-5.5
B. NEUTROPHILE- pH 5.5-8.0
C. ALKALOPHILE- pH 8.5-11.5
5. HIGH SALT CONCENTRATION
A. Halophiles- require increased concentration of sodium chloride

 BACTERIAL METABOLISM
METABOLISM-
 Sum of all chemical processes that take place in a living organism, resulting in growth,
energy generation, waste disposal and other functions in relation to distribution of cell
nutrients
 Divided into two major parts:
 Anabolism- constructive phase
 Catabolism- destructive phase
 Bacterial metabolism- consists of biochemical reactions to use to breakdown organic
compounds as well as those they use to synthesize new bacterial parts from the carbon
skeleton
ENERGY PRODUCTION
 Accomplished by the breakdown of chemical substrates through the degradative process of
catabolism coupled with oxidation-reduction reactions
2 GENERAL PROCESSES USED BY MICROORGANISMS TO PRODUCE ENERGY FROM GLUCOSE:
1. RESPIRATION
 An efficient ATP- generating process in which molecules are oxidized and the final
electron acceptor is an inorganic molecule
 In this process, glucose is completely broken down resulting to high energy production
 Aerobic process carried out by obligate aerobes and facultative anaerobes
 GLYCOLYSIS (EMBDEN-MEYERHOF-PARNAS PATHWAY)
 KREBS CYCLE
2. FERMENTATION
 Anaerobic process carried out by obligate anaerobes and facultative anaerobes
 Classified according to their end products:
 Alcoholic fermentation- ferments sugar to ethanol
 Homolactic fermentation- pyruvate is reduced to lactate
 Heterolactic fermentation- produce substances other than lactate such as
alcohol, carbon dioxide, formic acid and acetic acid
 Mixed acid fermentation- production of ethanol and acids such as lactic, acetic,
succinic and formic; detected by the Methyl-red test
 Butanediol fermentation- pyruvate is converted to acetoin which is detected by
Voges- Proskauer test
 Butyric acid fermentation- pyruvate is converted to butyric acid along with acetic
acid, carbon dioxide and hydrogen
  BACTERIAL GENETICS
3 MAJOR ASPECTS:
1. STRUCTURE AND ORGANIZATION OF GENETIC MATERIAL
 Bacteria contain a single, unpaired chromosome
 Bacterial chromosome contains all genes essential for viability and exists as a double
stranded, closed circular, naked macromolecule
2. REPLICATION AND EXPRESSION OF GENETIC INFORMATION
 Duplication of chromosomal DNA for insertion into a daughter cell
3. MECHANISMS BY WHICH GENETIC INFORMATION IS CHANGED AND EXCHANGED AMONG
BACTERIA
A. Mutation
B. Recombination

MECHANISMS OF GENE TRANSFER:

1. TRANSFORMATION- involves recipient cell uptake of free DNA released into the environment
when another bacterial cell dies and undergo lysis
2. TRANSDUCTION- transfer of bacterial genes by a bacteriophage from one cell to another; DNA
from two bacteria may come together in one cell
3. CONJUGATION- transfer of genetic material from a donor bacterial strain to a recipient strain;
occurs between to living cells

MICROBIAL CONTROL

Microbial control involves physical and chemical agents that destroy or inhibit microorganisms and
potential pathogens, and prevent their transmission.

STERILIZATION

 It refers to the removal or destruction of all forms of life, including bacterial spores

PHYSICAL METHODS

I. APPLICATION OF HEAT
 heat is the most commonly used method for the removal of microorganisms
A. Moist Heat Procedure
 destroys microorganism by coagulation of enzymes and structural proteins and
degradation of nucleic acids
1. Boiling
- Destroys vegetative bacteria (non-sporulating)
- Temperature and time of exposure: 100°C for 10-15 minutes
2. Autoclave
 - It is a chamber which is filled with hot steam under pressure
- Is the fastest and simplest method of sterilization- all organisms (except prions)
and spores are killed within 15 minutes
- It is used to sterilize biohazardous trash and heat-stable objects
- Principle: steam under pressure
- Biological indicator: B. stearothermophilus
a. 121°C, 15psi for 15 minutes- media, liquids, utensils, glass pipettes and
instruments for assay
b. 132°C, 15psi for 30-60 minutes- for decontaminating medical waste
3. Fractional/Intermittent Sterilization (Tyndallization)
- Temperature and time of exposure: 100°C for 30 minutes (3 consecutive days)
4. Inspissation
- It is used to sterilize protein-rich medium such as Lowenstein Jensen medium
- Principle: thickening of media through evaporation
- Temperature and time of exposure: 70°-80°C for 2 hours (3 consecutive days)
5. Pasteurization
- It is used to sterilize milk, dairy products and alcoholic beverages
- It eliminates foodborne pathogens and organisms responsible for food spoilage
- This method cannot eliminate bacterial endospores
a. Low Temperature Holding (LTH)/ Batch Method
 Treatment at this temperature reduces spoilage of food without
affecting its taste
 It destroys milk-borne pathogens
 Temperature and time of exposure: 63°C for 30 minutes
b. High Temperature Short Time (HTST)/Flash Pasteurization
 Temperature and time of exposure: 72°C for 15 seconds (quick
heating then immediate cooling)
c. Ultra High Temperature (UHT)
 Temperature and time of exposure: 140°C for 3 seconds (cooled very
quickly in a vacuum chamber)

B. Dry Heat Procedure

 It kills microorganism by denaturation of proteins; sterilization without water
 It is utilized for the sterilization of glassware, oil products and powder
 It is not applicable for heat- sensitive materials
 Biological indicator: B. subtilis
1. Flaming
- Direct heating
2. Oven
- It is used for glassware, oil, petroleum of powders
- Temperature and time of exposure: 160°C to 170°C for 1.5 to 2 hours
3. Incineration
 - Is the most common method of treating infectious waste and infected laboratory
animal
- Principle: burning materials into ashes (300°C to 400°C)
- Temperature of hazardous material (870°C to 980°C)
4. Cremation
- Used to control the spread of communicable disease

TERMINOLOGIES:

1. THERMAL DEATH POINT (TDP)- it refers to the lowest temperature at which a suspension of
bacteria is killed in 10 minutes
2. THERMAL DEATH TIME (TDT)- it refers to the shortest period of time needed to kill a
suspension of bacteria at a prescribed temperature and under specific conditions

II. FILTRATION
 It is the method of choice for sterilization of antibiotic solutions, toxins, chemicals,
radioisotopes, vaccines and carbohydrates (heat-sensitive solution)
2 TYPES OF FILTERS:
A. DEPTH FILTERS- consist of fibrous or granular material
B. MEMBRANE FILTERS- porous membranes; used to sterilize pharmaceuticals,
ophthalmic solutions, culture media, antibiotics and oil products
1. Liquid filtration
- Uses cellulose acetate/ cellulose nitrate membrane with a vacuum
2. Air filtration
- Uses high- efficiency particulate air (HEPA) filters
- HEPA filters remove organisms larger than 0.3µm from isolation rooms, operating
rooms and biological safety cabinets
3. Filtration of bacteria, yeasts, and molds
- Uses 0.45µm pores of membrane filters
4. Critical- sterilizing
- Uses 0.22µm membrane filters for parenteral solutions
III. LOW/COLD TEMPERATURE
 It is considered bacteriostatic, because it reduces the rate of metabolism
 Important in food microbiology
IV. DESSICATION AND LYOPHILIZATION
 Destroys bacteria by disruption of metabolism; involves removing water from
microbes (bacteriostatic)
 Lyophilization is the most effective method for long term preservation of microbial
cultures
V. OSMOTIC PRESSURE
 Use of high concentrations of salts and sugars in food create a hypertonic
environment
VI. RADIATION
  When it passes through the cells, it creates free hydrogen and hydroxyl radicals and
some peroxidases which in turn cause different intracellular damage
1. Ionizing radiation
 Use: plastic syringes, sutures, catheters, gloves
2. Non-ionizing radiation
 Use: exposed surfaces, air, operating room
 Example: ultraviolet rays

CHEMICAL METHODS

DISINFECTION

 Refers to removal, inhibition or killing microorganisms including potential pathogens by using

chemical agents usually on inanimate objects; it does not remove all bacterial spores

TERMINOLOGIES

1. ANTISEPTIC- applied topically to skin; inhibits sepsis formation

2. DISINFECTANT- usually applied to inanimate objects
3. BACTERICIDAL- precipitates bacterial protein; it kills all bacteria in the specimen
4. BACTERIOSTATIC- inhibits the growth of organism

COMMON CHEMICAL AGENTS USED IN MICROBIAL CONTROL

1. Acid and Alkali solutions

o Hydrolyzes and coagulates proteins
2. Phenol and Phenolic compounds
o First widely used antiseptic and disinfectant
o Destroys plasma membranes and denatures cell proteins
o Effective even in the presence of organics
3. Alcohol
o Causes denaturation of proteins and dissolution of lipid membrane
o Used both as an antiseptic and disinfectant
o Should be allowed to evaporate from the surface to which they were applied to achieve
maximum effectivity
4. Halogen
o Destroys microorganisms by oxidation mechanism
a. Iodophor
- Combination of iodine and neutral polymer (detergent)
b. Chlorine
- Used in the form of hypochlorite
- Should be made fresh daily
5. Salts of heavy metals
o Destroys microorganisms by inactivation and precipitation of cell proteins
6. Quaternary ammonium compounds
 a. Detergent- widely used as surface- active agents
b. Phenolics- use: hospital floors and surroundings; found in germicidal soap
7. Aldehydes
o Used to sterilize medical instruments
a. Formaldehyde- usefulness is limited- has irritability factor and carcinogenic
b. Glutaraldehyde
8. Gas sterilant
a. Ethylene oxide
o Most commonly used gas for sterilization
o Use: plastic petri dishes, sutures
b. Periacetic acid

MORPHOLOGY AND STAINING

MORPHOLOGY

A. SIZE
- Measured in microns or micrometer
- Size ranges between 0.2-5.0µm
- Smallest pathogenic bacillus- Haemophilus
- Largest pathogenic bacillus- Bacillus anthracis
B. SHAPE
- Can be usually determined with appropriate staining and light microscope
a. Coccus- round
b. Bacillus- rod-like
c. Curved
d. Coccobacilli
e. Spirillum- rigid helical rod
f. Spirochete- flexuous helical rod
g. Fusiform- bacilli with tapered, pointed ends
C. ARRANGEMENT
a. Staphylo- grape-like clusters
b. Strepto- chains
c. Diplo- pairs
d. Tetrads- groups of four
e. Sarcinae- packs of eight
f. Pleomorphic- bacterial species that varies in size and shape in pure culture
g. Palisades- bacteria that align themselves side by side each other
STAINING PROCEDURE

STAINING TECHNIQUES

1. SIMPLE STAINING
- A single stain is used
2. DIFFERENTIAL STAINING
- It divides bacteria into separate groups (ex. Gram staining, AFB staining)
Steps in Differential Staining

a. Application of primary stain

b. Application of mordant
c. Application of decolorizing agent
d. Application of secondary stain/counterstain
GRAM STAINING- most commonly used differential stain in the clinical microbiology laboratory

STEPS GRAM POSITIVE GRAM NEGATIVE

CRYSTAL VIOLET Purple-blue Purple-blue
GRAM’S IODINE Purple-blue Purple-blue
ACETONE-ALCOHOL Purple-blue Colorless
SAFRANIN Purple-blue Pink-red
ACID-FAST STAINING- stains the mycolic acid in the cell wall of bacteria; organisms with mycolic acid
in cell wall are difficult to gram stain

STEPS ACID-FAST NON ACID-FAST

CARBOL FUCHSIN Red Red
ACID-ALCOHOL Red Colorless
METHYLENE BLUE Red blue

SPECIAL STAINS- stains that are used for the demonstration of a specific part of the cell or specific
microorganisms

Examples:

A. CAPSULE- Hiss stain, Tyler, Welch’s

B. FLAGELLA- Leifson, Gray’s
C. METACHROMATIC GRANULES- Albert, Ljubensky, Neisser
D. ENDOSPORES- Schaeffer-Fulton, Dorner’s
E. GLYCOGEN VACUOLES- Periodic Acid Schiff
F. MITOCHONDRIA- Janus green B
G. FUNGI- Lactophenol blue
H. SPIROCHETES- Fontana-Tribondeau
CULTURE AND CULTURE MEDIA

CULTURES- are growth of microorganisms on a culture medium

CULTURE MEDIUM- is a liquid, semi-solid or solid medium utilized to observe growth patterns or
microorganism as well as for transport and storage

CLASSIFICATION OF CULTURE MEDIUM

A. ACCORDING TO CONSISTENCY
1. Liquid medium
 it contains 0% agar
 allows growth of aerobes, anaerobes and facultative anaerobes
 2. Semi-solid medium
 It contains 0.5 to 1% agar
3. Solid medium
 It contains 2-3% agar
B. ACCORDING TO COMPOSITION
1. Synthetic or defined medium
 It is a medium where all the components are known to the user
 It is used for research purposes
2. Non-synthetic or complex medium
 It is composed of some unknown substances (peptone, meat and yeast extracts)
 It is very useful for isolation of bacteria
3. Tissue culture medium
 It is used for obligate intracellular bacteria
C. ACCORDING TO HOW THE MEDIUM IS DISPENSED/DISTRIBUTED
1. Plated medium
2. Tube medium- liquid, slant, butt and slant, and butt only
D. ACCORDING TO USE
1. Simple media/ general purpose media/ supportive media
 Are routinely used in the laboratory and without added supplement
 Are media that support the growth of most non-fastidious bacteria
 Composition: meat and soybean extracts
2. Enrichment media
 It is used to propagate the growth of certain group of bacteria from a mixture of
organism
 It contains specific nutrients; liquid-type media
 It is incubated for a certain period and then must be subcultured to isolate the desired
organism
 It can also be used as supplement to agar plates to detect aerobes, anaerobes and
microaerophiles
3. Enriched media/ nonselective media
 These are media with added supplements such as blood, vitamins and yeast extract,
necessary for the growth of fastidious organisms
 These are solid- type media
4. Differential media
 These are media that allow visualization of metabolic differences between groups of
bacteria
5. Selective media
 These are media incorporated with antibiotics, dyes or chemicals to inhibit the growth
of other organisms while promoting the growth of the desired organism
6. Special media
 Is used to isolate bacteria with specific growth requirements
INOCULATION TECHNIQUES

1. Streaking
 the most commonly performed inoculation technique
 a. Radial streak
b. Overlap streak
c. Multiple streak
d. Multiple interrupted
e. Interrupted streak
 Specimens can be inoculated onto agar plates by using a general- purpose isolation
streak to yield a semi quantitative estimate of growth- plates are usually struck out in 4
quadrants
 It may be necessary to flame the loop in between quadrants, depending on the number
and type of bacteria present in the specimen
 When more than one medium is used, the loop is flamed in between media to prevent
carryover of possible contaminants from one plate to another
 Streaking for isolation- the microorganisms present in the specimen are successively
diluted out as each quadrant is streaked until finally each morphotype is present as a
single colony
2. Placement of fluid specimens or swabs into culture broth
 Broth media can be inoculated by immersing the inoculating loop with specimen into
the tube or by placing a few drops of the liquid specimen
 Swab can also be placed into the broth
3. Stabbing the medium
 This technique is applied to semi-solid medium like SIM
 It is also used to inoculate streptococci into BAP
4. Stab and streak technique
 It is used to inoculate organism into the slant and butt tube medium such as TSI and
LIA
5. Pour plate technique
 Is a technique where the organism is added to a molten agar, mixed and then allows
the medium to solidify
Notes to remember:

 The inoculating loop is sterilized and allowed to cool thoroughly before use
 Specimens on swab are applied by rolling the swab onto a small area at the edge of the
plate, and then streak
 Bedside or direct inoculation of specimen to culture media is optimal for isolation of the
pathogen- specimens collected at the bedside are more susceptible to contamination
BIOCHEMICAL TESTS

 MOTILITY
 Can be determined by microscopic examination or by observing growth in a semisolid
medium (hanging drop preparation and flagellar stain)
 Semisolid medium or motility test media (SIM or MIO)
 Characteristic motilities of some organisms:
 Darting motility- Campylobacter
 Tumbling motility- Listeria monocytogenes
 Gliding motility- Capnocytophaga
  Swarming motility- Proteus

 MICRODASE OR MODIFIED OXIDASE TEST

 Positive result: development of blue to purple-blue color
 (+) Micrococcus (-) Staphylococcus

 OXIDATION- FERMENTATION TEST

 Media: Hugh and Leifson’s OF Medium

OPEN TUBE CLOSED TUBE

(non-sealed; aerobic) (overlain with sterile mineral oil; anaerobic)
OXIDATIVE POSITIVE NEGATIVE
FERMENTATIVE POSITIVE POSITIVE
ASACCHAROLYTIC NEGATIVE NEGATIVE
 CATALASE TEST
 Reagent: 3% Hydrogen Peroxide
 Positive result: gas production, bubbles or effervescence
 (+) Staphylococcus and Micrococcus (-) Streptococcus

 SUPEROXOL TEST
 Reagent: 30% Hydrogen Peroxide
 Positive result: gas production, bubbles or effervescence
 (+) Neisseria
 COAGULASE TEST
 2 types of coagulase:
 Bound coagulase- known as clumping factor; detected by slide coagulase test
 Free coagulase- detected using tube coagulase test
 Reagent: EDTA- rabbit plasma
 Positive result: macroscopic clumping or clot formation
 (+) Staphylococcus aureus (-) other Staphylococci
 NOVOBIOCIN SUSCEPTIBILITY TEST
 Reagent: 5ug of novobiocin and sheep blood agar
 Positive result: zone of inhibiton greater than 16mm
 Susceptible: Staphylococcus epidermidis
 Resistant: Staphylococcus saprophyticus

 DNA HYDROLYSIS TEST(DNASE TEST)

 Reagent: DNAse agar (toluidine blue or methyl green as indicator)
 Positive result: methyl green is converted from green to colorless
 (+) Serratia, Moraxella, Staphylococcus aureus

 PYR TEST
 Reagent: PYR disk
 Positive result: bright red
 (+) Streptococcus pyogenes, Enterococcus faecalis
 BACITRACIN SUSCEPTIBILITY TEST
 Reagent: 0.04U bacitracin (Taxo A)
 Positive result: zone of inhibition
 Susceptible: S. pyogenes, Micrococcus
 Resistant: S. agalactiae, S. aureus

 HIPPURATE HYDROLYSIS TEST

 Reagent: to detect glycine: ninhydrin to detect benzoic acid: ferric chloride
 Positive result: deep purple color
 (+) S. agalactiae, Enterococcus (-) S. pyogenes

 CAMP TEST
 Reagent: sheep blood agar plate, S. aureus isolate
 Positive result: enhanced hemolysis, arrowhead zone of beta hemolysis
 (+) S. agalactiae (-) S. pyogenes
 NEUFELD-QUELLUNG TEST
 Positive result: swelling of capsules of organism
 (+) S. pneumonia (-) Viridans Streptococci

 OPTOCHIN TEST
 Reagent: 6ug or 10ug of Taxo P (ethyl hydrocupreine hydrochloride)
 Positive result: zone of inhibition
 (+) S. pneumonia (-) Viridans Streptococci

 BILE ESCULIN AGAR

 Positive result: brown-black precipitate
 (+) Group D Enterococcus and Non- Enterococcus (-) Viridans Streptococci
 SALT TOLERANCE TEST OR 6.5% SALT TURBIDITY TEST
 Reagent: heart infusion broth with 6.5% NaCl and bromcresol purple
 Positive result: visible turbidity or change in color of indicator from purple to yellow
 (+) Enterococcus (-) Non enterococcus

 LEUCINE AMINOPEPTIDASE TEST

 Reagent: LAP disk
 Positive result: red color
 (+) Enterococcus faecalis (-) Leuconostoc

 BUTYRATE DISK
 Reagent: butyrate disk
 Positive result: blue-colored indigo compound formation
 (+) Moraxella catarrhalis (-) Neisseria gonorrhoeae
 CETRIMIDE TEST
 Reagent: cetrimide agar slant
 Positive result: growth
 (+) Pseudomonas aeruginosa (-) Escherichia coli

 ACETAMIDE UTILIZATION TEST

 Reagent: acetamide medium
 Positive result: change of the color of medium from green to royal blue
 (+) Pseudomonas aeruginosa (-) Stenotrophomonas maltophilia

 ACETATE UTILIZATION TEST

 Reagent: acetate slant/medium
 Positive result: change of the color of the medium from green to blue
 (+) Escherichia coli (-) Shigella flexneri
 CITRATE UTILIZATION TEST
 Reagent: Simmons citrate agar
 Positive result: growth and change in color of the medium from green to blue
 (+) Klebsiella pneumonia (-) Escherichia coli

 INDOLE TEST
 Reagent: tryptophan containing media; Kovac’s/ Ehrlich reagent (p-
dimethylaminobenzaldehyde)
 Positive result: red ring (pink to wine-colored ring)
 (+) Escherichia coli (-) Klebsiella

 MUG TEST
 Reagent: MUG disk (4-methylumbelliferyl-beta-D-glucuronide)
 Positive result: electric blue fluorescence
 (+) Escherichia coli (-) Pseudomonas aeruginosa
 H2S PRODUCTION TEST
 Reagent: biochemical medium (TSI, LIA); requires an organic source of sulfur and a
source of metal
 Positive result: black

 ONPG TEST
 Reagent: O-nitrophenyl-beta-D-galactopyranoside
 Positive result: yellow
 (+) rapid and late lactose/slow lactose fermenters (-) non-lactose fermenter

 UREA HYDROLYSIS(UREASE) TEST

 Reagent: urea agar/slant with phenol red as pH indicator
 Positive result: color change from light orange to magenta
 (+) rapid urease producers (positive in 4 hours)- Proteus, Providencia, Morganella
 (+) slow urease producers (positive in 24 hours)- Klebsiella, Enterobacter, Yersinia,
Citrobacter, Serratia (-) solution remains buff to yellow
  GELATIN HYDROLYSIS TEST
 Positive result: partial or total liquefaction of the inoculated tube
 (+) Proteus vulgaris (-) Enterobacter aerogenes

 PHENYLALANINE DEAMINASE TEST

 Reagent: phenylalanine slant and 10% ferric chloride
 Decarboxylase is an enzyme that removes the carboxyl group from the amino acids
 Deaminase is an enzyme that removes the amino group from amino acids
 Positive result: green color
 (+) Proteus, Providencia, Morganella (-) Escherichia coli

PLATING MEDIA FOR ROUTINE BACTERIOLOGY

MEDIUM PRIMARY PURPOSE
ALKALINE PEPTONE WATER For Vibrio spp
BORDET-GENGOU AGAR For Bordetella pertussis
BISMUTH SULFITE AGAR For isolation of Salmonella spp
BRAIN HEART INFUSION For cultivation of non-fastidious and moderately
fastidious microorganisms

BUFFERED CHARCOAL-YEAST EXTRACT AGAR For Legionella spp

CETRIMIDE AGAR Selects Pseudomonas aeruginosa in specimens

with mixed flora

CAMPY-BLOOD AGAR For Campylobacter spp

CEFSULODIN-IRGASAN NOVOBIOCIN AGAR For Yersinia spp

CHOCOLATE AGAR For fastidious organisms, such as Haemophilus

and pathogenic Neisseria spp

COLUMBIA COLISTIN-NALIDIXIC AGAR For gram-positive cocci

CYCLOSERINE-CEFOXITIN FRUCTOSE AGAR For Clostridium difficile

CYSTINE-TELLURITE BLOOD AGAR For Corynebacterium diphteriae

EOSIN METHYLENE BLUE AGAR For isolation and differentiation of lactose-

fermenting and non-lactose fermenting enteric
bacilli

FLETCHER SEMI-SOLID MEDIUM For Leptospira

FOOT PADS OF MICE AND ARMADILLO For Mycobacterium leprae

HEKTOEN ENTERIC AGAR Differentiation of Salmonella and Shigella

LIM BROTH For Streptococcus agalactiae

LOWENSTEIN-JENSEN MEDIUM For Mycobacterium spp
MACCONKEY AGAR Isolation and differentiation of lactose-
fermenting and non-lactose fermenting enteric
bacilli

MANNITOL SALT AGAR Selective isolation of Staphylococci

PETRAGNANI MEDIUM For Mycobacteria

REGAN LOWE Preferred medium for Bordetella pertussis

SALMONELLA-SHIGELLA AGAR Differentiation of Salmonella and Shigella

SKIRROW AGAR For campylobacter spp

TINSDALE AGAR For Corynebacterium diphteriae
THIOSULFATE CITRATE-BILE SALT SUCROSE For Vibrio spp
AGAR
TODD-HEWITT BROTH For Streptococcus agalactiae in female genital
 specimens

PATHOGENESIS OF INFECTION

Pathogenesis- source or cause of an illness or abnormal condition

INFECTION

 Involves the growth and multiplication of microorganisms that result in damage to the host
 Invasion of the body by pathogenic microorganism that reproduce and multiply, causing
disease by local cellular injury, secretion of a toxin, or antigen-antibody reaction in the host
TYPES OF IN INFECTION AS TO CAUSE:

1. AUTOGENOUS INFECTION
- Caused by a microorganism from the microbiota of the individual
2. IATROGENIC INFECTION
- An infection that occurs as the result of medical treatment or medical procedures
3. OPPORTUNISTIC INFECTION
- An infection in immunocompromised hosts that do not cause disease in individuals with a
normal immne system
- May be due to overuse of antibiotics, immunosuppressive drugs and chemotherapeutic
agents
4. NOSOCOMIAL INFECTION
- Also known as hospital acquired infection
TYPES OF INFECTION ACCORDING TO DISTRIBUTION IN THE HOST

1. LOCAL INFECTION
- Signs and symptoms are confined to one area
- Examples: wound, boil, abscess, acne
2. FOCAL INFECTION
- Starts as a local infection and spread to other parts of the body
3. SYSTEMIC (GENERALIZED INFECTION)
- Microbes are spread throughout the body by blood or lymph
a. Bacteremia- presence of bacteria in the blood
b. Septicemia- active multiplication of bacteria in the blood
c. Pyemia- condition wherein pus-producing organism repeatedly invade the bloodstream
and localized at different parts of the body
d. Toxemia- prevent of toxins in the blood; when bacteria are killed in one area but
produce a toxin, which spreads and absorbed by the body cells
EXTENT OF INFECTION

1. PRIMARY INFECTION- initial infection causing the illness

2. SECONDARY INFECTION- caused by opportunistic pathogen after primary infection has
weakened host immune system
 3. LATENT INFECTION (SILENT PHASE)- is clinically silent inside the body without any noticeable
illness in the host before suddenly causing severe and acute infection
4. MIXED INFECTION- caused by 2 or more organisms
5. ACUTE INFECTION- type of infection that develops and progress slowly
6. CHRONIC INFECTION- an infection with milder but longer-lasting symtoms
ROUTES OF INFECTION

1. DIRECT TRANSMISSION
a. Congenital contact
b. Sexual contact
c. Hand-to-hand transmission
d. Infectious respiratory secretions of droplets
2. INDIRECT TRANSMISSION
a. Fomites
b. Water
c. Arthropod vectors
DISEASE

 Is a specific illness or disorder characterized by a recognizable set of signs and symptoms

attributable to hereditary, infection, diet or environment
 It results when the infection produces notable changes in human physiology that are often
associated with damages to one or more of the body’s organ system
CLASSIFICATION OF INFECTIOUS DISEASES

1. COMMUNICABLE DISEASE
 Spread from one host to another, directly or indirectly
2. CONTAGIOUS DISEASE
 It is spread easily from one person to another
3. NON-COMMUNICABLE DISEASE
 It is not spread from one host to another
 It is caused by microbes that live outside the body or by opportunistic pathogens that
live inside the body
CLASSIFICATION OF DISEASE AS TO OCCURRENCE

1. SPORADIC DISEASE
 It occurs occasionally
2. ENDEMIC DISEASE
 Constantly present in a particular location or population
3. EPIDEMIC DISEASE
 Many people acquire the disease in a particular location or population
4. PANDEMIC DISEASE
 An epidemic that spans the world
EFFECTS OF INFECTIOUS DISEASE

1. SIGNS
  These are objective changes that can be measured
2. SYMPTOMS
 These are subjective feelings not obvious to a person
3. SYNDROME
 Is a group of signs and symptoms that are associated with a disease
PHASES OF INFECTIOUS DISEASE

1. INCUBATION PERIOD
- Time between the exposure to a pathogenic organism and the onset of symptoms
2. PRODROMAL PERIOD
- Appearance of signs and symptoms
3. CLINICAL/ILLNESS PERIOD
- Peak characteristic signs and symptoms of an infection or a disease
4. DECLINE PERIOD
- Period wherein the signs and symptoms begin to subside as the host condition improves
5. CONVALESCENT PERIOD/PERIOD OF RECOVERY
- Full recovery of the surviving host
PATHOGENS- are microorganisms that cause infection and/or disease

PATHOGENICITY- pertains to the ability of a pathogenic agent to produce a disease in a susceptible

individual

2 GENERAL CLASSES OF PATHOGENIC MICROORGANISMS

1. TRUE PATHOGENS
- They are able to invade the tissues of healthy individuals through some inherent ability of
their own
2. OPPORTUNISTIC PATHOGENS
- These are organisms that normally do not cause disease in their natural habitat in a
healthy person- they may cause disease if the host is weakened or if they enter a different
part of the body
- These are organisms that only cause infection when one or more of the host’s defense
mechanisms are disrupted, damaged, changed or malfunctioning
HOST-MICROBE RELATIONSHIP

1. SYMBIOSIS- is the association of 2 organisms living together

2. MUTUALISM- is a symbiotic relationship where both the host and organism benefit from one
another
3. COMMENSALISM- is a relationship where the organism benefits, but there is no beneficial or
harmful effect to the host
4. PARASITISM- is a relationship where the organism benefits at the expense of the host
VIRULENCE

 Power of microorganisms to produce disease

 Degree of pathogenicity
  Measured by the number of microorganisms necessary to cause infection in the host
VIRULENT

 It pertains to a very pathogenic microorganism or rapidly progressive condition

 Those organisms than can establish infection with a relatively low infective dose are more
virulent that those that require high numbers for infection
FACTORS AFFECTING MICROBIAL VIRULENCE

1. TOXIC FACTORS- toxins are poisonous substances produces by pathogenic microorganisms

2. ENZYMATIC FACTORS- these are produced by bacteria
3. CELLULAR STRUCTURE- capsule resists phagocytosis
HOST RESISTANT FACTORS

1. PHYSICAL BARRIERS
- The skin serves as the physical and chemical barrier to microorganisms
- Continuous shedding dislodge bacteria that are attached to the outer layers
2. CLEANSING MECHANISM
- Nasal hairs keep out airborne particles that may contain microorganisms
- Cough-sneeze reflex contributes to the removal of potentially infective agents
3. ANTIMICROBIAL SUBSTANCES
- Lysozymes destroy bacterial cell walls; bile salts destroy bacterial membranes
4. INDIGENOUS/NORMAL MICROBIAL FLORA
- These are microorganisms that are commonly found on or in body sites of healthy
persons
5. PHAGOCYTOSIS
- Is the process by which certain cells engulf and dispose- off microorganisms and cell
debris
6. INFLAMMATION
- It plays an important role as a reinforcement mechanism against microbial survival and
proliferation in tissues and organs
7. IMMUNE RESPONSE
- It provides the human host with the ability to mount a specific protective response to
the presence of a microorganism
- Immune system has a “memory” so that if a microorganism is encountered second or
third time, an immune-mediated defensive response is immediately available
INFECTIOUS AGENT FACTORS

1. ADHERENCE
- The infectious agent must attach to host cells before infection occurs
- The main adhesins in the bacteria are the pili and surface polysaccharides
2. PROLIFERATION
- In order to establish itself and cause disease, a pathogen must be able to replicate
following attachment to host cells
3. TISSUE DAMAGE
- A disease or an infection is noticeable only if tissue damage occurs
 4. PRODUCTION OF TOXINS
DIFFERENCES BETWEEN EXOTOXIN AND ENDOTOXINS
EXOTOXINS ENDOTOXINS
PARENT ORGANISM Gram-positive bacteria Gram-negative bacteria
CHEMICAL NATURE Simple proteins Lipopolysaccharide(LPS)-lipid a
LOCATION Outside the living cell Part of outer membrane
HEAT STABILITY Labile- inactivated at 60 ºc Stable- 121ºc
TOXICITY High Low
REPRESENTATIVE Gas Typhoid fever,uti,meningococcal
DISEASES gangrene,tetanus,botulism,diptheria, meningitis
scarlet fever
FEVER Usually do not produce fever Produce fever by release of il-1
GENETICS Carried by extrachromosomal genes Synthesized directly by chromosomal
such as plasmids genes
IMMUNE RESPONSE Highly antigenic Weakly antigenic
EFFECTS ON HOST Destroys particular part of the host’s Disruption of clotting(DIC),fever
cell hypotension,shock,death

5. INVASION
- The process of penetrating and growing in tissues
6. DISSEMINATION
- The spread of organisms
ROUTES OF TRANSMISSION

1. AIRBORNE TRANSMISSION
- Respiratory spread is common
- Some infectious agents may be transmitted by dust particles
2. TRANSMISSION BY FOOD AND WATER
- Infections occur via the fecal-oral route
3. CLOSE CONTACT
- Refers to passage of organism by salivary, skin and genital contact
4. CUTS AND BITES
- Bites are infection by the normal flora of the mouth
5. ARTHROPODS
- Infectious agents multiply in the arthropod which then feeds off a human host and
transmits the microorganism
6. ZOONOSES
- Depends on the contact with animals or animal by-product

SPECIMEN COLLECTION, TRANSPORT AND PROCESSING

Specimen collection, handling and transportation are critical considerations, because any result the
laboratory generates is limited by the quality of the specimen and its condition on arrival in the
laboratory
GENERAL GUIDELINES FOR SPECIMEN COLLECTION

1. Specimen should be collected during the acute phase of an illness

2. Specimen should be collected at the correct anatomic site and should be representative of the
infection
3. Specimen should be collected before the initiation of antibiotic therapy or chemotherapeutic
agents
4. The quantity of the specimen should be sufficient enough for diagnostic testing
5. Specimen should be collected under sterile, aseptic condition to avoid contamination with
normal flora and organisms from adjacent tissues
6. Specimen must be labeled accurately with patient information, date and the specific anatomic
site
7. If specimens cannot be processed as soon as they are received they must be stored
8. Place specimen in containers designed to maintain viability of organism and avoid hazard that
result from leakage

SPECIMEN CONTAINERS AND OTHER MATERIALS FOR COLLECTION

 Specimen for microbiology cultures should be collected in sterile containers

 Swabs for specimens from URT, external ear, eye and genital tract
 Swabs are not recommended for routine collection because they are easily
contaminated and can become dried out
 If swabs are used, Calcium alginate, Dacron or Rayon swabs are preferred because
cotton swabs may release toxic fatty acids; Calcium alginate is not recommended for
viral specimen collection since it inactivates viruses

SPECIMEN TRANSPORT

 Ideally, specimens must be processed immediately after collection

 Primary goal in the transport of specimens to the laboratory is to maintain the specimen as
near to its original state as possible with minimal deterioration and to prevent risk to the
specimen handler
1. Specimen should be transported to the laboratory within 2 hours
2. All specimens should be transported within sealable, leak-proof, plastic bags; specimen
bags should be marked with a biohazard label

SPECIMEN PRESERVATION

 If transport of the specimen to the laboratory is delayed, or if it will not be processed

immediately, the specimen can be maintained with the use of preservatives, holding media
and even culture media

SPECIMEN REJECTION

 All rejected specimen require a phone call to the person in charge of collecting the specimen
  Specimens that are impossible to recollect or that would require the patient to undergo
another invasive procedure may need to be processed regardless of the situation
 If the physician insists on processing an inadequate specimen, it should be noted on the
requisition form

REASONS FOR SPECIMEN REJECTION

1. The information on the label does not match the information on the requisition slip
2. The specimen is transported at the improper temperature
3. The specimen has not been transported in the proper medium
4. The quantity of specimen is insufficient for testing
5. The transport of specimen exceeds 2 hours post collection and the specimen has not been
preserved properly
6. The specimen is preserved in a fixative
7. The specimen has been received for anaerobic culture from a site known to have anaerobes as
part of the normal flora
8. The specimen has already dried up when transported to the laboratory
9. The specimen is leaking
10.More than one specimen from the same source (except blood) and from the same patient was
submitted on the same day
11.Expectorated sputum in which the gram stain reveals <25 WBCs and >10 epithelial cells

SPECIMEN PRIORITIZATION

 When specimen is received with multiple requests but the amount of specimen is insufficient,
the clinical should prioritize the testing
 When multiple specimens arrive at the same time, priority should be given to those that are
most critical- CSF,tissue,blood and sterile fluids

LEVELS OF SPECIMEN PRIORITIZATION

LEVEL DESCRIPTION SPECIMENS
1 CRITICAL/INVASIVE AMNIOTIC FLUID,BLOOD,CSF,PERICARDIAL FLUID
2 UNPRESERVED BONE,FECES,SPUTUM,TISSUE,BODY FLUIDS
3 QUANTITATION REQUIRED CATHETER TIP,URINE TISSUE
4 PRESERVED URINE,FECES,SWABS IN HOLDING MEDIA

 Level 1 specimens represent potentially life-threatening illness and are from invasive source
 Level 2 specimens are unprotected and may quickly degrade or have overgrowth of
contaminating flora

SPECIMENS USED FOR BACTERIOLOGICAL STUDY

1. BLOOD
- Blood is normally sterile; used to evaluate bacteremia, septicemia and fever of unknown
origin
A. BACTEREMIA- presence of bacteria in the blood
 B. SEPTICEMIA- presence of toxins in the blood
- Blood culture bottles contain anticoagulant- 0.025%-0.50% sodium polyanethol sulfonate
- Disinfect skin prior to collection by applying 70% ethyl alcohol, then 2% iodine tincture or
Iodophor. Dry for 1 minute, then another alcohol to remove iodine
- Blood culture contaminants may be mistaken as pathogenic isolate
- Considered pathogenic when found in more than 1 culture bottle
2. CEREBROSPINAL FLUID
- Collected through lumbar puncture or spinal tap
- Used to evaluate for meningitis
- Smears of CSF are stained with gram stain and India ink
3. GASTROINTESTINAL TRACT SPECIMEN
- Stool, gastric secretions and rectal swab
- Stool is the specimen of choice for gastrointestinal pathogens
- Specimen should not be taken from the toilet bowl
- Specimen should not be contaminated with urine or water
4. URINARY TRACT SPECIMEN
- Ideally, first morning urine is used since it is more concentrated
- Suprapubic aspirate is collected via needle aspiration above the symphysis pubis through
the abdominal wall into the full bladder
5. RESPIRATORY TRACT SPECIMEN
A. Throat swab- swab in the posterior pharynx, tonsils and inflamed areas
B. Nasal swab- moistened swab in the nares/nose
C. Nasopharyngeal swab- flexible swab inserted through the nose and into posterior
nasopharynx and then rotated for 5 seconds
D. Sputum- ideally collected in the morning; 3 morning sputum specimens for acid fast smears
E. Bronchial washings and bronchoalveolar lavage
6. GENITOURINARY SPECIMEN
- Used to determine the cause of vaginitis, urethritis and cervicitis, as well as childbirth
infections
- Often used to determine STIs
7. WOUNDS AND ABSCESSES
- Proper aseptic technique must be applied; care must be taken during specimen collection
to prevent contamination with normal flora

GRAM POSITIVE COCCI

STAPHYLOCOCCI

 The genus name is derived from the Greek word staphle, meaning bunches of grapes
 They are gram-positive cocci that belong to the family Staphylococcaceae
 They are catalase-producing bacteria and facultatively anaerobic except S. saccharolyticus
 They are non-motile, non-sporeforming and glucose fermenter
 They are normal inhabitant of the skin, mucous membrane and intestine
  Microscopy: spherical cells that appear in clusters, some singly
 Culture: BAP- colonies on agar plate appear creamy, white or light gold

Micrococcus vs. Staphylococcus

Micrococcus Staphylococcus
Growth characteristics
Oxygen requirements Obligate aerobe Facultative anaerobe
Aerobic growth Positive Positive
Anaerobic growth Negative Positive
Anaerobic susceptibility
Bacitracin Susceptible Resistant
Furazolidone Resistant Susceptible
Lysostaphin Resistant Susceptible
Modified Oxidase (Microdase) Test Positive Negative
Carbohydrate utilization (OF medium) Oxidative Fermentative
Open Tube: Positive Positive
Closed tube: Negative Positive
Growth on Furoxone-Tween 80 oil red O positive Negative
agar

Micrococcus spp

 Gram positive cocci

 In tetrads or cuboidal packets or sheets
 Nitrate negative
 Grows on 5% NaCl but not on 7.5% NaCl
 White or yellow pigments
 Non-hemolytic
 Oxidizer

Staphylococcus aureus

 Most virulent species

 Coagulase positive
 Golden yellow colonies in Loeffler’s serum slant
 Can be isolated from cultures by use of medium with 7.5% salt concentration (MSA)
 DNAse positive

Pathogenic determinants of Staphylococcus aureus:

 Coagulase- converts fibrinogen to fibrin; may coat neutrophils with fibrin formed to protect
organisms from phagocytosis
 Staphylokinase- also known as fibrinolysis; dissolves fibrin clots and may enable infection to
spread once clot is dissolved
  Lipase- hydrolyze lipids in plasma and skin; enables Staphylococci to colonize certain body
areas; associated with initiation of skin infections such as boils, carbuncles and furuncles
 Hyaluronidase- cause hydrolysis of hyaluronic acid, which is present in connective tissue,
thereby resulting in the spread of infection; also known as the spreading factor, T factor or
the Duran-Reynal factor
 Deoxyribonuclease- causes degradation of DNA
 Exfoliatins- also known as epidermolytic toxin; hydrolyze tissue through cleavage of stratum
granulosum; associated with staphylococcal scalded skin syndrome (also known as Ritter
disease)
 Leukocidins (Panton-Valentine)- lysis of neutrophils and macrophages; inhibit phagocytosis
 Hemolysins- group of alpha, beta, gamma and delta hemolysins that lyse erythrocytes
 Enterotoxins- enterotoxin F is also known as the Toxic shock syndrome toxin (TSST-1). TSST-
1 is associated with sudden onset of fever, diarrhea, vomiting, hypotension, shock, as well as
rashes and skin desquamation. Most cases are in menstruating women, particularly using
tampons
 Beta lactamase- hydrolysis and inactivation of penicillin antibiotics through breakdown of
beta-lactam ring in penicillin molecule
 Protein A- cellular component identified in the cell wall of S. aureus; has the ability to bind the
Fc portion of IgG. Binding IgG in this manner neutralizes IgG and can block phagocytosis

Clinical significance of Staphylococcus aureus

 Cutaneous infections (boils, carbuncles, furuncles, folliculitis, bullous impetigo and purulent
abscesses)
 Food poisoning (most common cause of bacterial food poisoning in the US)
 Toxic shock syndrome
 Scalded skin syndrome
 Toxic epidermal necrolysis
 Staphylococcal pneumonia
 Staphylococcal osteomyelitis

Coagulase-negative Staphylococci

S. epidermidis S. saprophyticus
Novobiocin Susceptible Resistant

Staphylococcus epidermidis

 Is a normal flora of the skin

 Is a contaminant of medical instruments- catheters (indwelling and IV), CSF shunts and
prosthetic heart valve implants (implanted medical devices)
 It secretes poly-gamma-DL-glutamic acid(slime layer) which provides adherence to devices
 It has been known to cause various hospital-acquired infections
  Culture: BAP- small to medium-sized, non-hemolytic, nonpigmented, white opaque, pin-head
colonies
 Biochemical tests: -MSA; coagulase-negative staphylococci (CoNS)

Staphylococcus saprophyticus

 It is associated with community-acquired UTI in young, sexually active females

 It is the second most common cause of UTI in young women
 It adheres more effectively to the epithelial cells lining the urogenital tract than other CoNS
 It is rarely found on other mucous membranes or skin surfaces
 Culture: BAP- white opaque, pin-head slightly larger colonies; 50% of the strains produced
yellow pigment; nonhemolytic

Staphylococcus lugdunensis

 Is a coagulase-negative staphylococci by tube method

 It can be confused with S. aureus if the slide coagulase method is performed
 It is more aggressive than the other CoNS in its ability to be infective- associated with
catheter- related bacteremia and endocarditis
 It can cause both community-associated and hospital acquired infections
 It can be more virulent and can clinically mimic S. aureus infections

STREPTOCOCCI
 Belong to the family Streptococcaceae
 They are commonly found as part of normal human flora, however, when these organisms
gain access to normally sterile sites, they can cause life threatening infection
 Are facultative anaerobes; some species require increased CO 2 for growth (capnophiles)
 Some species grow in the presence of oxygen but are unable to use oxygen for respiration,
thus they may be considered aerotolerant anaerobes
 Their growth is enhanced by blood, serum or glucose incorporated in agar plate
 All streptococci except the viridans group and S. pneumonia are included in the Lancefield
classification
 Microscopy: gram-positive spherical cells, arranged in chains or pairs
 Culture: BAP- grayish, pinpoint, translucent to slightly opaque colonies some with mucoid
colonies
 Notorious pathogens: S. pyogenes and S. pneumonia

Classification of Streptococci:

A. Academic/ Bergey’s Classification- based on temperature requirement

1. Pyogenic group
- Neither grow on 10ºC or 45ºC, only at 37ºC
- Species: S. pyogenes
2. Viridans group
 - It will grow both at 45ºC and 37ºC
- It is not part of the lancefield group
3. Lactic group
- It will grow at 10ºC and 37ºC
- Often found in dairy products
4. Enterococcus group
- It will grow at 10ºC, 45ºC and 37ºC
- Normal flora of human intestine
- Species: E. faecalis
B. Smith and Brown Classification- based on hemolytic patterns
1. Alpha- hemolytic streptococci
- they have partial/incomplete hemolysis of red blood cells around colony
- Culture: green hemolysis/ discoloration around colony
- Species: S. pneumoniae
2. Beta- hemolytic streptococci
- They exhibit complete lysis of red blood cells around colony
- Culture: clear area/zone around colony
- Species: S. pyogenes, S. agalactiae
3. Gamma- hemolytic streptococci
- They exhibit no lysis of red blood cells around colony
- Culture: red cells immediately surrounding the colony are unaffected
- Species: Enterococci
C. Lancefield Classification (Antigen Serogrouping)
- It is based on the extraction of C carbohydrate from the streptococcal cell wall
- Rebecca Lancefield found out that the C carbohydrate can be extracted from the
streptococcal cell wall by placing the organisms in dilute acid and heating for 10 minutes

Group A Streptococci
 It is not considered part of the normal flora; pathogenic to man
 It is acquired through contaminated droplets by cough or sneeze
 It is resistant to drying and can be recovered from swabs after several hours of collection
 Species: Streptococcus pyogenes- “fever producing bacteria”; flesh eating bacteria
 Culture: small, translucent and smooth; well-defined β-hemolysis
 Principal virulence factor: M-protein (attached to the peptidoglycan; type-specific; anti-
phagocytic; for adherence to mucosal cells
 Other virulence factors:
a. Protein F- mediates epithelial cell attachment
b. Lipoteichoic acid- bacterial adherence to the respiratory epithelium
c. Hyaluronic acid capsule- weakly immunogenic; prevents opsonized phagocytosis; masks its
antigens
d. Hemolysins- Streptolysin O and Streptolysin S
e. Toxins
f. Enzymes
Pathogenic determinants:

 Hemolysins-

Streptolysin O Streptolysin S
Hemolysis type Sub-surface hemolysis Surface hemolysis
Characteristics Oxygen-labile Oxygen-stable
Antigenicity Immunogenic; can stimulate Non-immunogenic; cannot
production of antibodies stimulate production of
antibodies

 Erythrogenic toxins/pyrogenic toxins A,B,C- causes fever, alteration of blood-brain-barrier;

may be associated with organ damage and skin rashes of scarlet fever
 Streptokinase- fibrinolysin that lyses blood clots, prevents fibrin barrier and allows spread of
infection
 Streptodornase- group of four enzymes with nuclease activity degrade host DNA and/or RNA
 M protein- antiphagocytic; interferes with complement activity
 Hyaluronic acid- found in mucoid strains, present in capsules; anti-phagocytic

Clinical significance of Streptococcus pyogenes

 Acute pharyngitis (Strep throat)

 Scarlet fever- scarlet in form rash that spreads in the upper chest and trunk, neck, arms and
legs; rash formation is due to production of erythrogenic toxin; tongue becomes yellow-white
with red papillae and is described as strawberry tongue
 Impetigo, cellulitis, wound infection and erysipelas
 Complications: post-streptococcal acute glomerulonephritis and acute rheumatic fever

Group B Streptococci

 It is part of the normal flora of the female genital tract and lower GIT
 It is nosocomially transmitted by unwashed hands of mother or health care personnel to the
newborn or infant
 It causes infection of fetus during passage through the colonized birth canal and premature
rupture of mother’s membranes
 It is recommended that all pregnant women be screened for group B streptococci at 35-37ºC
weeks of gestation
 Species: Streptococcus agalactiae
 Virulence factor: capsule (sialic acid- significant component of the capsule)
 Avirulent factors: hemolysin, CAMP factor, neuraminidase, deoxyribonuclease, hyaluronidase
and protease
 Culture: grayish white, mucoid colonies with small zone of β-hemolysis

Laboratory tests for Group B Streptococci

1. CAMP test
 2. Hippurate Hydrolysis test
 S. agalactiae is resistant to both bacitracin and SXT

Group C and G Streptococci

 These organisms are recovered from URT, vagina and skin of humans
 They possess M protein just like the group A streptococci
 Group C streptococci are the main source of streptokinase; an animal pathogen

Viridans Streptococci
 Are also known as alpha-prime streptococci that lack the lancefield group antigens and do not
fall on the criteria for S. pneumonia; some isolates can be β-hemolytic and nonhemolytic
 Are normal microbiota or the URT, female genital tract and GIT
 Are opportunistic pathogens of low virulence; fastidious bacteria
 Are the most common cause of sub-acute bacterial endocarditis
 Virulence factors: capsule, cytolysin, extracellular dextran and adhesins

Laboratory tests for S. bovis group

1. Growth in bile esculin medium

2. 6.5% NaCl test
3. PYR test
4. Penicillin test

Enterococci

 They belong to the family Streptococcaceae

 Are formerly known as Group D Streptococci
 Are natural inhabitants of the intestinal tracts of humans and animals
 Are not highly pathogenic but they are frequent causes of nosocomial infections
 They can grow in extreme conditions- alkaline pH, grow at 45ºC and salt solutions
 Species: E. faecalis (most common isolate), E. faecium, E. avium, E. durans
 Virulence factors: extracellular serine protease, gelatinase and cytolysin

Enterococci are identified based on their ability to:

1. Produce acid in carbohydrate broth

2. Produce acid from methyl-α-glucopyranoside
3. Hydrolyze arginine
4. Tolerate 0.04% tellurite
5. Utilize pyruvate
6. Growth around 100µg efromycin acid disk

GROUP D STREPTOCOCCI Enterococcus Non-enterococcus

Bile esculin test Positive Positive
6.5% salt turbidity test Positive Negative
PYRase test Positive Negative
Growth at 45ºC Positive Positive
Growth at 10ºC Positive Negative
penicillin Resistant Susceptible

GROUP D STREPTOCOCCI Enterococcus faecalis Enterococcus faecium

Pyruvate broth test Positive Negative

Streptococcus pneumonia

 Also known as Diplococcus/Pneumococcus

 It is considered part of the normal flora of the URT of preschool children
 It is a common isolate both as a pathogen and as a member of the normal respiratory tract
 It is the causative agent of lobar pneumonia
 It is the most common cause of bacterial meningitis in adults
 Principal virulence factor: capsular polysaccharide
 Microscopy: gram-positive cocci in pairs, oval or lancet shape/bullet shape/flame shaped
diplococcic
 The capsule of S. pneumonia is antigenic and can be identified with appropriate antiserum; it
is composed of hyaluronic acid

Clinical significance of Streptococcus pneumonia

 Lobar pneumonia associated with rusty or anchovy red sputum

 Meningitis

Streptococcus pneumoniae Viridans Streptococci

Mouse inoculation Positive Negative
Inulin fermentation Positive Negative
Bile solubility Positive Negative
Optochin susceptibility Positive Negative
Neufeld Quellung test Positive Negative

GRAM NEGATIVE COCCI

NEISSERIA

 They are obligate aerobic; non-motile and non-hemolytic

 They are capnophilic and have an optimal growth in a moist temperature
 They are carbohydrate fermenters- primarily glucose and maltose
 Growth is best observed on enriched medium or media containing blood, serum, cholesterol or
oleic acid
  They are sensitive to drying and extremes of temperature- direct inoculation of specimen “at
the bedside” is required for these bacteria
 Microscopy: gram- negative diplococci with coffee or kidney bean shape
 Biochemical tests: (+) oxidase; (+) catalase
 Major pathogens: N. gonorrhoeae and N. meningitides

Neisseria gonorrhoeae

 Is never considered part of the human flora

 Transmitted by sexual contact (person to person); infected mother to newborn during birth
 It requires iron for growth
 Glucose fermenter
 Principal virulence factor: common pili
 Colonial types: T1 and T2 (virulent), T3-T5 (avirulent)
 Clinical infections:
o Gonorrhea
 Symptoms: purulent discharge, lower abdominal pain and dysuria (men); dysuria
and vaginal bleeding (women)
o Purulent urethritis
o Pharyngitis
o Conjunctivitis (Ophthalmia neonatorum)
 Gonococcal eye infection, during vaginal delivery through an infected birth canal
o Purulent arthritis
 Specimen collection and handling
o Specimen: pus and secretions from urethra, cervix, prostate, rectal mucosa, throat and
joint fluid
o Dacron or rayon swabs- for collection of specimen
 Notes to remember:
o Swabs should be placed in a transport medium and plated within 6 hours
o Cotton swabs should be avoided due to the presence of toxic fatty acids in the cotton
fibers

THAYER MARTIN VANCOMYCIN COLISTIN NYSTATIN

AGAR
MODIFIED THAYER VANCOMYCIN COLISTIN NYSTATIN TRIMETHOPRIM
MARTIN AGAR LACTATE
MARTIN LEWIS VANCOMYCIN COLISTIN ANISOMYCIN TRIMETHOPRIM
MEDIUM LACTATE
NEW YORK CITY VANCOMYCIN COLISTIN AMPHOTERICIN B TRIMETHOPRIM
MEDIUM LACTATE
Neisseria meningitidis
 Causative agent of epidemic meningococcal meningitis/ meningococcemia
 It may be found as a commensal inhabitant of the upper respiratory tract of the carriers
 Clinical infections:
o Meningococcemia
 Refers to the presence of N. meningitides in the blood and can occur as an acute
or chronic form
 It occurs with or without meningitis
 Notes to remember:
o The LOS- endotoxin complex activates the clotting cascade, depositing fibrin in small
vessels, producing hemorrhage in the adrenals (Waterhouse-Friderichsen syndrome),
altering peripheral vascular resistance, and leading to shock and death
o Individuals with a deficiency in complement C5-C8 are at risk of meningococcemia
 Specimen collection and handling
o Specimen: CSF, blood, nasopharyngeal swabs, petechial skin lesions
o Nasopharyngeal swabs should be plated immediately to the JEMBEC system, or
submitted on swabs placed in charcoal transport media
 Non- pathogenic Neisseria species
1. N. cinerea
 The colony morphology is similar to T3 colonies of N. gonorrhoeae on CAP
2. N. flavescens
 Is a yellow- pigmented Neisseria species; assacharolytic
3. N. lactamica
 Commonly found in the nasopharynx of infants and children
4. N. mucosa
 On culture, it is a large, often adherent to agar, and very mucoid colonies
 5. N. sicca
 On culture, it has dry, wrinkled, adherent and breadcrumbs-like colonies
6. N. elongate
 Is a “rod-shaped” Gram-negative cocci
7. N. weaver
 Is a “rod-shaped” Gram-negative cocci

Moraxella catarrhalis (Branhamella catarrhalis)

 This species resembles Neisseria by exhibiting “gram negative coccal” morphology

 The most commonly isolated member of the genus Moraxella
 Part of the normal flora of upper respiratory tract
 Microscopy: small gram- negative cocci that tend to grow in pair end-to-end; with adjacent
sides flattened
 Culture: smooth, opaque, gray to white colonies with “hockey puck” appearance- colonies
remain intact when pushed across the plate with a loop
 CHO utilization test- does not utilize any sugar; asaccharolytic in CHO degradation test
 DNAse test positive
 Butyrate Esterase test- positive (blue color)

DIFFERENTIAL TEST FOR PATHOGENIC Neisseria and M. catarrhalis

N. gonorrhoeae N. meningitidis M. catarrhalis

Superoxol + - -
DNAse - - +
Nitrate reduction - - +

ACID PRODUCTION

SUGAR N. gonorrhoeae N. meningitidis N. lactamica M. catarrhalis N. subflava

GLUCOSE + + + - +
MALTOSE - + + - +
SUCROSE - - + - -
LACTOSE - - - - -
 ENTEROBACTERIACEAE

 Are gram- negative, non- sporeforming, facultatively anaerobic bacilli

 Most are present in the intestinal tract as commensal flora except Salmonella, Shigella and
Yersinia
 All members are motile with peritrichous flagella except Klebsiella, Shigella and Yersinia
 All members ferment glucose and reduce nitrate to nitrite
 Some organisms may grow at low temperatures -1ºC to 5ºC (Serratia and Yersinia)
 Microscopy: gram- negative straight rods or coccobacilli with rounded ends
 2 GROUPS:
o Opportunistic pathogens
 They are part of the intestinal microbiota of both humans and animals
 They generally do not initiate disease in healthy, uncompromised human hosts
 They produce significant virulent factors
 Examples: Citrobacter, Enterobacter, Klebsiella, Proteus, Serratia
o Overt/true pathogens
 They are not present as commensal flora of the GIT
 Only inhabit the bowel at the time of infection and are required by ingestion of
contaminated food or water
 Examples: Salmonella typhi, Shigella, Yersinia pestis
 Antigen determinants that can be used for serological identification
o Somatic “O” antigen- heat stable; located in the cell wall; for E. coli and Shigella
serotyping
o Flagellar “H” antigen- heat labile; found in the flagellum; for Salmonella serotyping
o Capsular “K” antigen- heat labile polysaccharide; covers the O antigen; found as K 1
antigen of E. coli and Vi antigen of S. enterica subsp enterica serotype typhi

Escherichia coli
 The most significant species in the genus Escherichia
 It is part of the normal bowel flora of humans and may also inhabit female genital tract
 It is a primary marker of fecal contamination in water purification
 It is the leading cause of nosocomial infection- urinary tract infection
  E. coli “O” groups have shown cross reactivity with the “O” antigens of Shigella
 The K1 antigen is identical to the capsular antigen of N. meningitides group B
 Culture: MAC- flat, dry, with pink colonies
BAP- some strains are B- hemolytic, most non- hemolytic
EMB- greenish metallic sheen
 Virulence factors: endotoxin, common pili, K1 antigen, intimin
 iMViC reaction- ++--
 TSI reaction: A/A, (+)gas, (-) H2S

E. coli strains Infection Virulence factors Characteristics

Enteropathogenic E. Infantile diarrhea (+) H antigen and
coli (EPEC) (stool without blood) intimin
Enterotoxigenic E. coli Traveler’s diarrhea/ Heat-stable and heat- Persons with
(ETEC) Montezuma’s revenge labile enterotoxins achlorhydria are at
Vibrio-like infection high risk
Enteroinvasive E. coli Dysentery-like/ invasin Atrichous
(EIEC) Shigella like infection

Watery diarrhea (with

WBCs)
Enterohemorrhagic E. Hemorrhagic colitis cytotoxins
coli (EHEC) Hemolytic Uremic
Syndrome (HUS)
E. coli O157:H7- MUG
test negative and Bloody diarrhea
sorbitol non-fermenter (without WBCs)

Klebsiella

 It is usually found in the GIT of humans and animals

Klebsiella pneumonia

 Is formerly known as Bacillus capsulatus

 Is the most common isolated species of Klebsiella
  It is the causative agent of community acquired pneumonia (currant jelly-like sputum)
 Culture: MAC- pink, mucoid colonies
 Virulence factor: polysaccharide capsule
 TSI reaction: A/A, (+)gas, (-) H2S
 IMViC reaction- --++

BIOCHEMICAL TEST K. pneumoniae K. oxytoca

Indole - +
Methyl red - +
Vogues-Proskauer + +
Citrate + +
Urease + +
Lysine decarboxylase + +

Enterobacter

 it resembles Klebsiella when growing on Mac Conkey agar

 Culture: pink colonies and maybe with mucoid colonies- Mac Conkey agar

Biochemical tests:

 Ornithine decarboxylase test: positive

 Lysine decarboxylase test: positive (except: E. cloacae, E. gergoviae)
 Sorbitol fermentation: positive (E. aerogenes, E. cloacae)
 Urease test: positive (E. cloacae)
 IMViC reaction: --++ (E. aerogenes, E. cloacae)
 TSI reaction: A/A, (+)gas, (-) H2S (E. aerogenes, E. cloacae)

Enterobacter gergoviae

 Is found in respiratory samples and is rarely isolated from blood cultures

Enterobacter cancerogenus

 It has been isolated with osteomyelitis following traumatic wounds

BIOCHEMICAL TEST E. aerogenes E. cloacae

Lysine decarboxylase + -
Ornithine decarboxylasee + +
Urease - +

Cronobacter sakazakii

 It is a pathogen in neonates causing meningitis and bacteremia, often coming from powdered
infant formula

Pantoea agglomerans

 It causes nosocomial outbreak of septicemia due to contaminated IV fluids

Serratia

 Is an opportunistic pathogen associated with nosocomial outbreaks

 NLF; some strains are slow/ late lactose fermenters; resistant to a wide range of antibiotics

Serratia marcescens

 Is the most clinically significant species

 It causes the bacteremic outbreaks in nurseries, cardiac surgery and burn units
 It is a contaminant in antiseptic solutions used for joint injections causing an epidemic of
septic arthritis
 Biochemical test: urease producer; (+)gelatinase
 TSI reaction: K/A, (+) gas, (-) H2S

Serratia plymuthica

 It causes osteomyelitis following a motorcycle accident

Other Serratia species:

 S. marcescens, S. rubidaea and S. plymuthica- have pink to red pigment (prodigiosin) at 25ºC
 S. odorifera- musty-pungent odor or “potato-like” odor
 S. liquefaciens- pink to red pigment; (-) sorbitol fermentation

Proteus

 It is isolated from urine, wound and ear infections

  It can infect the proximal kidney tubules and can cause AGN, particularly in patients with
urinary tract defects and catheterization
 It is a rapid urease producer- urease splits urea in urine, raises urine pH and encourages renal
stone formation
 Culture: “swarming phenomenon” and “burnt chocolate” or “burnt-gun powder” odor

BIOCHEMICAL TEST P. mirabilis P. vulgaris

Indole - +
LIA R/A R/A
PAD + +
IMViC -+vv ++-v
TSI K/A, (+) gas, (+) H2S K/A, (+/-) gas, (+) H2S

Providencia

 Is one of the causes of nosocomial outbreaks involving burn units

 Urease test: positive
 PAD test: positive
 IMViC reaction: ++-+
 TSI reaction: K/A, (-) gas, (-) H2S

Providencia rettgeri

 Is a pathogen of the urinary tract

 It also causes diarrheal disease among travelers

Providencia stuartii

 It has been isolated from nosocomial outbreaks in burn units and in urine cultures
 It is mostly resistant to antimicrobial agents

Providencia alcalifaciens

 Is most commonly found in the feces of children with diarrhea

Morganella
  Same biochemical reaction with P. vulgaris except citrate negative
 Species: M. morganii
 PAD test: positive

Edwardsiella

 It has been isolated from cold-blooded and warm-blooded animals

 Species: E. tarda (human pathogen)
 Urease test: negative

Citrobacter

 It produces colonies on Mac Conkey agar that resemble E. coli and biochemically resembling
Salmonella
 It can cause false agglutination test with Salmonella
 All species grow on Simmon citrate agar; slow urease producers
 IMVic reation: -+-+ (C. freundii)
++-+ (C. koseri)

Citrobacter freundii

 It can be isolated in diarrheal stool cultures

 It has been associated with endocarditis in IV drug users
 The colony morphology on primary plated media can be mistaken for that of Salmonella when
isolated from stool cultures, since majority of the strains produce H 2S and a few fail to ferment
lactose

Citrobacter koseri

 It causes nursery outbreaks of neonatal meningitis and brain abscess

BIOCHEMICAL TEST C. freundii C. koseri

Indole - +
H2S production + -
TSI + -
IMViC A/A or K/A, (+) gas, (+) H2S K/A, (+) gas, (-) H2S
ONPG + -
Salmonella

 The most serious pathogenic enterobacteria for humans, causing enteric fever (typhoid fever)
and acute gastroenteritis (food poisoning)
 It inhabits the GI tract of animals
 Humans acquire this organism by ingestion of contaminated animal food products or
improperly cooked poultry, milk, eggs, and dairy products
 It may also be transmitted by human carriers
 Culture: MAC: clear, colorless colonies
Media with H2S indicators: colonies with black center (HE, BSA, XLD)
SSA: colorless colonies with black centers

Biochemical Characteristics

 All species are motile except S. serotype Pullorum and S. serotype Gallinarum
 All produce gas exceot S. serotype Gallinarum and S. serotype Typhi
 All produce H2S except S. serotype Paratyphi A

Notes to remember:

 Many Salmonella serotypes are typically found in cold- blooded animals as well as in rodents
and birds, which serve as their natural hosts

Salmonella bongori

 Is a rarely isolated species that is named after the town of Bongor in Chad, Africa
 It is isolated from lizard and other cold- blooded animals

3 General Categories of Salmonella infection

1. Gastroenteritis
 Is one of the most common forms of “food poisoning”
 The Salmonella strains associated with this infection are those found in animals, mostly
S. enterica subsp. Enterica
 For the peanue-butter outbreak, S. serotype Typhimurium is the causative agent
 The used of contaminated cooking utensils and cutting boards can spread the bacteria
to other food
 Inadequate refrigeration also allows the growth and multiplication of the organisms
  Sources of infection: poultry, eggs, and egg products, milk, and handling of pets
2. Enteric Fever
 Is also known as typhoid fever, and it is caused by S. serotype Typhi
 It is a febrile disease that results from the ingestion of contaminated food originating
from infected individuals or carriers
 Direct transmission through fomites is also possible
 Sources of infection: human carriers, contaminated food and water
 Causes of outbreaks: improper disposal of sewage, poor sanitation and lack of modern
water system
 The characteristics “rose spots” appear during the 2nd week of fever
 The gallbladder is the site of long-term carriage of S. serotype Typhi
 Complications: necrosis in the gallbladder (necrotizing cholecystitis) and Peyer’s patches
3. Bacteremia
 It occurs with and without extraintestinal foci of infection caused by non typhoidal
Salmonella
 It is characterized by prolonged fever and intermittent bacteremia

Notes to remember:

 “rose spots” (blanching rose-colored papules) appear around the periumbilical region and it is
a sign of infection
 Individuals who recover from the infection may harbor the organisms in the gallbladder, which
becomes the site of chronic carriage
 Carriers of Salmonella excrete the organisms in their feces continuously or intermittently
 The carrier state may be terminated by antimicrobial therapy if gallbladder infection is not
evident
 Cholecystectomy is the only remedy to the chronic state of enteric carriers

Specimens for Salmonella identification:

1. Blood- 1st week of infection

2. Stool- 2nd week of infection
3. Urine- 3rd week of infection

Shigella
  It is closely related to the genus Escherichia
 Is not a member of the normal gastrointestinal flora
 It is an intracellular organism- they multiply within the cells of the colon epithelium
 It is transmitted by flies, fingers, food and feces and water by infected persons (fecal-oral
route)
 Culture: MAC- clear, fragile, NLF colonies
SSA- colorless colonies without black centers
 Virulence factor- Shiga toxin
 Species- S. dysenteriae (most virulent), S. flexneri (gay bowel syndrome), S. boydii, S. sonnei
 Antigenic structures- somatic “O”
 Specimen- rectal swab

Biochemical Characteristics:

 All species are non-motile

 All species except S. dysenteriae are mannitol fermenters
 Urease test: negative
 TSI reaction- K/A, (-) gas, (-) H2S

Notes to remember:

 Shigella is susceptible to disinfectants and high concentrations of acids and bile

 Shigella is sensitive to pH changes (susceptible to the acidic stool)- specimens should be
plated immediately after collection to increase the recovery of the organisms

Shigella sonnei

 Is unique in its ability to decarboxylate ornithine among the Shigella species; Late lactose
fermenter; (+) ONPG
 Infection from this organism is self-limiting, and usually characterized by fever and watery
diarrhea (stool without blood)

Clinical Infection

 Bacillary dysentery
o It is mostly caused by S. dysenteriae type 1
 o It is marked by penetration of intestinal epithelial cells by the organism, following
attachment of the organism to mucosal cells
o It is characterized by acute inflammatory colitis and bloody diarrhea (blood, mucus, and
WBCs in the stool)
o Its presence usually indicates improper sanitary conditions and poor personal hygiene
o Source of infection: human carrier
o Transmission- person to person, fecal-oral route, flies, fingers, and food or water
contaminated by infected persons

Yersinia
Yersinia pestis (formerly Pasteurella pestis)

 It is also known as the Plague bacillus

 It is a Class A Bioterrorism Agent
 The only Enterobacteriaceae that is transmitted to humans by the bite of an infected flea-
Xenopsylla cheopis
 It is not part of the normal flora of GIT; non-motile
 It is the causative agent of bubonic plague- “black death or 6 th century pandemic”
 It may be isolated on routine culture medium; grow best at 25ºC-30ºC
 Microscopy: short, plump rod with “bipolar staining or closed safety pin appearance” using
Wayson or methylene blue stain
 Culture: BAP- pinpoint colonies at 24 hours; rough cauliflower colonies at 48 hours
Broth- “stalactite pattern”- clump of cells adhere to one side of the tube

Clinical Infections:
 Plague
o Is a disease of the rodents transmitted to humans by fleas
o It is carried by urban and domestic rats and wild rodents
o Humans may also be infected by ingestion of contaminated animal tissues and
inhalation of contaminated airborne droplets
o Once inside the human body, the bacteria multiply in the blood and lymph
 2 forms of plague
1. Bubonic Plague
 a. It is associated with high fever and painful inflammatory swelling of axilla and groin
b. It results from the bite of an infected flea
2. Pulmonary Plague
a. It is acquired by close contact with other victims
b. It occurs secondary to the bubonic plague

Yersinia enterocolitica

 It is the most common isolated species of Yersinia

 It is the causative agent of enterocolitis- waterborne gastroenteritis
 It is motile at 22ºC but not at 35ºC
 It requires cold enrichment technique (4ºC) using phosphate buffered saline for several weeks
for isolation from fecal material
 It has the ability to survive in cold temperatures (food refrigeration)
 It has been isolated from contaminated packed RBCs (blood transfusion)
 It grows on routine culture media (BAP and Mac Conkey); grows best at 25º-30ºC
 Mode of acquisition- consumption of incompletely cooked food and dairy products and
handling pets
 Microscopy- coccobacilli with bipolar staining
 Culture: “bull’s eye colonies” (dark red centers with transparent borders) at 48 hours
 Selective medium- Cefsulodin- Irgasan Novobiocin (CIN) agar
 Reservoirs- swine, dogs, cats, rabbit and cattle

Yersinia pseudotuberculosis

 It is a pathogen of the rodents, particularly guinea pigs

 Reservoir- farm and domestic animals (birds)

Hafnia

 It is not known to cause gastroenteritis but is occasionally isolated from stool cultures

Erwinia

 Is a plant pathogen and are not significant in human infections

 It grows poorly at 37ºC and fails to grow on Mac Conkey and EMB agars, and other differential
media used for the isolation of enterobacteria
Plesiomonas

Plesiomonas shigelloides

 The only species in the genus

 The only oxidase (+) member of the Enterobacteriaceae
 It is found in fresh water especially in warmer climates; not part of the human flora
 It is widely distributed among warm and cold-blooded animals
 It cannot tolerate high salt concentration; minimum growth requirement of 8ºC
 It often cross-agglutinate with Shigella, hence the species name “shigelloides”
 Microscopy- gram-negative straight bacilli; occur singly, in pairs, short chains or filamentous
forms
 Culture- CIN- opaque “apron-like colonies”
 Biochemical test- oxidase (+)

Campylobacter

 It is motile by a single polar flagellum; non-sporeforming

 It grows in 5-10% oxygen (microaerophilic)
 It is the most recognized antecedent cause of Guillain-Barre syndrome- many patients with
GBS are tested positive for antibodies to Campylobacter
 It is also an animal pathogen causing sterility and abortion
 Microscopy- faintly staining gram-negative, small, curved or S-shaped rod
- Old culture- may appear as coccobacilli
- Long spirals or seagull- wing shaped
 Culture- gray, flat, glistening, irregular, with a “tailing effect along the streak line” or “runny
spreading” colonial growth
 Mode of acquisition- ingestion of contaminated water, poultry and dairy products handling pets
like dogs, birds and cats; sexually transmitted

Campylobacter jejuni

 It is the most common cause of bacterial gastroenteritis worldwide

 It is acquired from eating contaminated chicken and turkey
 It invades the epithelium of the small intestine, causing inflammation
  It also secretes a toxin that is antigenically similar to the cholera toxin
 It is slow growing, fastidious and asaccharolytic; darting motility; unable to grow in 3.5% NaCl
 Optimum growth- 42ºC
 Microscopy- curved or seagull-winged shaped

Campylobacter fetus subsp. Fetus

 It has been isolated most frequently from blood cultures and is rarely associated with
gastrointestinal illness

Helicobacter

 It is found in the GIT of mammals and birds

 It is motile; microaerophilic
 Most species have strong urease activity
 Culture- gray with translucent colonies

Helicobacter pylori

 It is the major cause of type B gastritis, peptic ulcer and gastric carcinoma
 Primary habitat- human gastric mucosa- mucus layer of the antrum and fundus of the stomach
but does not invade the gastric epithelium
 It binds to Lewis antigen
 Biochemical test- a strong urease producer; (+) oxidase and catalase
 Routes of transmission- oral-oral route, fecal-oral route
 Characteristics of H. pylori
o Motility allows this organism to escape acidity of the stomach
o Urease enzyme plays a significant role in the survival and growth by creating an alkaline
microenvironment
 Laboratory Diagnosis
o Specimens- tissue biopsy material (Stuart’s medium) and urine (ammonia testing),
feces and dental plague
o Tissue specimens should be maintained at 4ºC and processed within 2 hours of
collection
o Other tests-
 Urea breath test- excellent sensitivity and specificity
  H. pylori is susceptible to metronidazole

NON ENTERIC GASTROINTESTINAL PATHOGEN

Vibrio (Comma/Curved bacillus)

 It is not part of the human flora; facultatively anaerobic; monotrichous organism

 It is found in brackish, marine and salt water
 It is temperature sensitive (>20ºC) and it can be isolated from algae, plankton, fish and
shellfish
 It is halophilic organism (require the addition of sodium for growth)
 Microscopy- Gram-negative short, curved rod
 Culture- MAC- NLF (except V. vulnificus-LF)
BAP- α or β hemolysis
 Mode of acquisition- consumption of raw or undercooked seafoods
 Disease/ infection- cholera, wound infection, septicemia and necrotizing fasciitis

Biochemical Tests:

 Oxidase (+) and reduce nitrate to nitrite

 Glucose fermenter, NLF except V. vulnificus
 Motility test- broth- polar sheathed flagella
Solid media- peritrichous, unsheathed flagella

Vibrio cholera
 It is the causative agent of cholera/Asiatic cholera/epidemic cholera
 It has rapid darting or shooting- star motility
 The single flagellum is covered with lipopolysaccharide sheath
 It has caused cholera epidemics and seven pandemics
 Virulence factor- choleragen (cholera toxin)
 Culture- smooth, medium to large colonies with a greenish hue (BAP)
 Epidemic V. cholera 01 biogroups
o Classical- VP (-); do not agglutinate chicken RBC, susceptible to polymyxin B
 o El tor- VP (+); agglutinate chicken RBC; resistant to polymyxin B
 Potent enterotoxins- cholera toxin (CT), zot toxin and ace toxin
 Antigenic structures- somatic O and flagellar H
 String test- positive (mucoid “stringing” reaction)
 TSI reaction- A/A, (-) gas
 Other biochemical test- (+) oxidase

Clinical manifestations and pathogenesis

Cholera

 It is an acute diarrheal disease that is spread mainly through contaminated water

 It is acquired from ingesting improperly preserved food like seafood (shellfish), milk and ice
cream
 Hallmark of cholera- rice-watery stool (10-30X of defecation/day)
 Cause of outbreak- improperly handled and preserved seafood, milk, ice cream and meat

Vibrio parahaemolyticus

 Is the second most common Vibrio species implicated in gastroenteritis

 It is the etiologic agent of “summer diarrhea” in Japan
 Virulence factor- heat-stable hemolysin
 Pathogenicity- Kanagawa phenomenon (hemolysin lyses human RBCs)
 Selective medium- Wagatsuma agar (high salt mannitol medium)
 Strain of this organism which can lyse human RBCs is Kanagawa toxin positive

Vibrio vulnificus

 It was commonly referred to as the “lactose-positive” Vibrio

 It is second to V. cholerae in terms of producing serious type of Vibrio-associated infection
 Infections- primary septicemia and wound infections
 Mode of acquisition- eating raw oysters and fish

Vibrio alginolyticus

 It is the least pathogenic Vibrio for humans; not commonly isolated

 It is a strict halophile- 1%-10% NaCl
 It can be an occupational hazard
  Related infections- eye, ear and wound infections

Laboratory Diagnosis

 Specimen- stool, rectal swab, pus and tissue

o Vibrio species should be collected and transported only in Cary-Blair medium
 Culture media
o TCBS-, alkaline peptone water, Cary-Blair, Mac Conkey and BAP
o Growth of Vibrios requires media containing 0.5% NaCl except V. cholera and V.
mimicus
o V. alginolyticus tolerates up to 10% NaCl
o Pathogenic Vibrio grows as NLF on Mac Conkey agar
o Sucrose fermenters (yellow colonies on TCBS)- V. cholera, V. alginolyticus, V.
metschnikovii
o Non-sucrose fermenters (green colonies on TCBS)- V. mimicus, V. vulnificus, V.
parahaemolyticus and V. damsel
 String test- 0.5% Na desoxycholate
o It differentiates Vibrio spp. From Aeromonas spp.
o (+) result- lysis of cells (Vibrio) releases DNA, which can then be pulled up into a string
(viscous string) using an inoculating loop
 Vibriostatic test (Susceptibility test)
o It is used to separate Vibrios from other oxidase (+), glucose fermenters like
aeromonads
 Biochemical test
o (+) Citrate; yellow colonies on TCBS= V. cholera
o (+) Indole- V. cholera, V. mimicus and V. vulnificus
o (+) cellobiose- V. vulnificus
 Serological test
o Strains that phenotypically resemble V. cholera but fail to agglutinate in o1 antisera are
referred to as V. cholera non-O1

Aeromonas

 It is found in fresh water; isolated from meat products

  It is not part of the human flora; facultatively anaerobic
 Microscopy- gram-negative straight rods
 Biochemical test- oxidase (+) and catalase (+), glucose fermenters; motile with single polar
flagellum but some species are non-motile
 It can typically grow from 4ºC to 42ºC
 It will grow in media with 0% NaCl but not in 6% NaCl

NON-FERMENTATIVE GRAM NEGATIVE BACILLI

 Opportunistic pathogens; environmental bacteria; not usually found as normal flora of the
human body

Biochemical test

 They are motile except B. mallei; oxidase (+); NLF

 They fail to acidify oxidative- fermentative media when it is overlaid with mineral oil or fail to
acidify TSI agar- no acid production in the slant or butt of TSI or KIA
 Do not ferment CHO by enzymatic reaction but by oxidative method and produce very weak
acids as end product

Pseudomonas

 They are the most commonly isolated non-fermentative bacilli

 Their metabolism is respiratory and never fermentative
 They can be found in cosmetics, swimming pools, hot-tubs, inner soles of sneakers
 They usually grow on Mac Conkey agar
 TSI reaction- K/K, H2S (-)

Pseudomonas aeruginosa

 Is the most commonly isolated species of the genus in clinical specimens

 Is the most commonly encountered Gram- negative bacterium that is not a member of the
Enterobacteriaceae
 It has the ability to invade the vascular walls of blood vessels, which facilitates its spread in
body
 It is non-sporeforming, monotrichous organism; good growth at 42ºC
  It is acquired through ingestion of contaminated food or water, exposure to contaminated
medical devices; penetration of wounds
 It exists in distilled water and chlorinated water; hot tubs and contact lens solutions
 Culture- flat spreading pigmented colonies
BAP- fruity “grape-like” or “corn tortilla” odor; β-hemolytic
 Pigments produced by P. aeruginosa
o Pyoverdin- yellow-green or yellow-brown pigment
o Pyocyanin- blue
o Pyorubin- red
o Pyomelanin- brown or black

Clinical significance

 It is the leading cause of nosocomial respiratory tract infections

 It is the 3rd most common cause of Gram-negative bacillary bacteremia
 It is the causative agent of ecthyma gangrenosum, swimmer’s ear, hot tub or Jacuzzi
syndrome
 It causes infections of the nail beds and lung infections in cystic fibrosis patients
 It is the 3rd most common cause of hospital-acquired infection

Distinguishing Characteristics

 (+) gluconate production

 (+) arginine dihydrolase (ADH)
 Grow at 42ºC
 (+) acetamide and citrate utilization

Pseudomonas fluorescens and Pseudomonas putida

 They have been isolated from contaminated blood products, cosmetics, hospital equipment,
urine and respiratory specimens
 They can grow at 4ºC and have been linked to transfusion- associated septicemia
 They can produce acid from xylose
 Differential test- P. fluorescens- gelatin hydrolysis (+)

Acinetobacter
  Is a member of the family Moraxellaceae
 It is the 2nd frequently isolated non fermenters
 It has been isolated from hospital equipment
 They may appear as gram-positive bacilli in smears made from blood culture bottles
 Some species may be mistaken for Neisseria species, while other species have tendency to
resist alcohol decolorization
 They have optimum growth at 30-35ºC and pH between 5.5-6.0
 Microscopy- plump, gravitational coccobacilli; appear in pairs
 Biochemical test- strict aerobe and nonmotile; (+) catalase; (-) oxidase
 Related infections- UTI, pneumonia, endocarditis, meningitis and cellulitis
 Species-
o A. baumanii- glucose-oxidizing; non hemolytic strains
o A. iwoffi- glucose- non oxidizing; non hemolytic strains
o A. haemolyticus- glucose- non oxidizing; hemolytic strains

Stenotrophomonas maltophilia

 It is the 3rd most commonly isolated non fermentative gram- negative bacilli
 It has been isolated from plant materials, water, milk, frozen food and sewage
 It is associated with pseudo infections- as a result of contaminated blood collection tubes
 Maltophilia- “maltose loving” (produce acid with maltose and not glucose)

Burkholderia

 It is not considered part of normal human flora; generally non- pathogenic

 It is aerobic and non- sporeforming; motile except B. mallei

Burkholderia cepacia

 It is a low-grade nosocomial pathogen

 It has been isolated from irrigation fluids, anesthetics, nebulizers, detergents and disinfectants
 Culture- BAP- non-fluorescing and non-wrinkled yellow or yellow-green colonies
 Biochemical test- weak (+) oxidase reaction, (+) ONPG
 Related infections- pneumonia in patients with cystic fibrosis

Burkholderia mallei
  It is a potential bioterrorism agent
 It is the only non-motile member of the genus
 Oxidase production is variable
 It has variable growth on Mac Conkey agar

Burkholderia pseudomallei

 Agent of melioidosis
 It has been isolated from muddy soil, streams and surface water such as rice paddies
 It can survive within phagocytes- “Vietnamese time bomb”
 It is motile with polar tuft of flagella
 Microscopy- bipolar staining
 Culture- ashdown medium with colistin- dry, wrinkled, deep pink colonies “earthy odor”
 Mode of acquisition- through inhalation of contaminated debris or direct inoculation through
damaged skin or mucus membranes

Alcaligenes faecalis

 It is obligate aerobic gram-negative bacillus; motile by peritrichous flagella

 It grows well on Mac Conkey agar
 Infection is acquired through exposure to contaminated medical devices and solutions
 Culture- BAP- feather-edged non-pigmented colonies; “fruity odor resembling apples or
strawberries”

Oligella

 It may colonize the distal urethra

 Microscopy- small, paired gram-negative bacilli or coccobacilli
 Culture- BAP- small, opaque, whitish; non saccharolytic

Moraxella lacunata

 It is the agent of Blephanoconjunctivitis/Angular conjunctivitis

 Culture- small colonies that “pit the agar”

Chromobacterium violaceum

 Is the only member in the genus Chromobacterium

  It is an opportunistic pathogen, affecting the immunocompromised patient with neutrophil
deficits
 It is motile with polar flagella
 Culture- violet pigmented colonies (violacein pigment)

SMALL, PLEOMORPHIC GRAM-NEGATIVE BACILLI

Haemophilus

 The genus name is derived from the Greek words meaning “blood lover”
 Are obligate parasites on the mucous membranes of humans
 Are fastidious and non-motile; non-sporeforming; capnophilic and facultatively anaerobic
bacteria
 They die rapidly in clinical specimen- very susceptible to drying and extreme temperature
 Most species will not grow in BAP
 Biochemical test- catalase (+); oxidase (+)
 Exhibits Satellitism
o Can be demonstrated using the Staphylococcal streak
o Satellitism appears as the colonies of Haemophilus grow adjacent the Staphylococcus
aureus colonies. Culture of Staphylococcus supplies V factor for Haemophilus
 Can be classified according to factor X and V requirement
o X factor- hemin; heat stable factor- released after degradation of hemoglobin (during
hemolysis)
o V factor- coenzyme I/ NAD; heat labile factor
 Horse blood is preferred for blood agar plates for better production of beta hemolysis for
Haemophilus
 Porphyrin test is an alternative method for differentiating the heme-producing species of
Haemophilus
o Detects the ability of the organism to convert delta-aminolevulinic acid into porphyrins
or porphobilinogen, which are intermediates for the synthesis of X factor

Haemophilus influenzae

 It is transmitted by person to person by contaminated respiratory droplets

  It is the commonly tested organism for Beta-lactamase production
 It is the main cause of meningitis in children- the organism spreads from the nasopharynx to
the regional lymph nodes to the blood and finally to the meninges
 The only member of the genus that produce IgA protease
 Do not produce endotoxin; rapidly killed by phagocytes; very fastidious
 Culture- CAP- translucent, convex, tan mucoid colonies; “mousy or bleach-like odor”

Haemophilus ducreyi

 Is not part of human flora; only found in humans during infection

 It infects mucosal epithelium, genital and non-genital skin, and regional lymph nodes
 Is the agent of chancroid or “soft chancre”- a highly communicable sexually transmitted
genital ulcer disease
 Suppurative, enlarged, draining, inguinal lymph nodes are common in the majority of infected
patients
 Culture- CAP- transparent, small, flat, smooth, non-mucoid, tan or yellow colonies; saline
suspension- “clumpy appearance”; “schools of fish”- in gram stain
 Chancroid- genital lesions; from tender papules to painful ulcers with several satellite lesions

Haemophilus aegyptius

 It was observed in conjunctivitis exudates from Egyptians by Koch

 It is the etiologic agent of pink-eye conjunctivitis

Haemophilus influenzae biogroups aegyptius

 It causes conjunctivitis primarily in pediatric populations

 It is the etiologic agent of Brazilian Purpuric Fever

SPECIES X FACTOR V FACTOR

H. influenzae + +
H. parainfluenzae 0 +
H. haemolyticus + +
H. parahaemolyticus 0 +
H. aprophilus 0 0
H. paraprophilus 0 +
H. aegyptius + +
 H. ducreyi + 0

Other Fastidious Gram-negative bacilli

 HACEK group
o Group of capnophilic organisms associated with endocarditis
o Haemophilus aprophilus
o Aggregatibacter mycetemcomitans
o Cardiobacterium hominis
o Eikenella corrodens
o Kingella denitrificans

Eikenella corrodens

 “corroding bacterium”- capable of pitting or corroding the surface of the agar

 Normal flora of the human mouth and produces bleach-like odor
 Associated with human bite wounds or fights (clenched fist wounds), meningitis, endocarditis,
peritonitis as well as brain and intraabdominal abscesses
 Requires hemin for growth and prefers chocolate agar

Kingella spp.

 Oxidase (+), catalase (-), indole (+) and ferments glucose

Brucella spp.

 Small, non-motile, aerobic, gram-negative coccobacilli or short rods

 Culture medium- biphasic medium (Castañeda medium)
 Agents of Brucellosis, undulant fever, Malta fever
o Is characterized by normal temperatures in the morning followed by high temperatures
in the afternoon and evening
 General characteristics- oxidase(+), catalase(+), nitrate reduction(+), urease(+) within 2
hours, non-motile coccobacilli that can grow in sheep blood agar
 Specimen for isolation- blood, bone marrow
 Serum H2S Urease Enhanced Inhibition Inhibition
agglutination production growth in of growth of growth
(Patient (Lead presence of in dye; in dye;
antibodies) Acetate CO2 thionine fuchsin
test)

B. + 0 V 0 0 0
melitensis

B. abortus + + +<2hours +/0 + 0

B. suis + + +<0.5hours 0 0 +
B. canis 0 0 +<0.5hours 0 0 0

Bordetella species

 Are obligate aerobic, gram-negative fastidious coccobacilli; non-carbohydrate fermenter

 Are non-motile; with bipolar metachromatic granules
 They replicate on ciliated respiratory epithelial cells of humans
 Are mostly inactive in biochemical test systems
 B. parapertussis is also citrate positive

Bordetella pertussis

 The agent of whooping cough or pertussis

 Pertussis is highly communicable disease that can be prevented by vaccine
 Infection has three phases:
o Catarrhal phase- symptoms are insidious and non-specific
o Paroxysmal phase- sudden onset of severe, repetitive coughing followed by the
characteristic “whoop” at the end of the coughing spell
o Convalescent phase- decrease in frequency and severity of the cough spells
 Ideal specimen- nasopharyngeal swab
 General characteristics of B. pertussis
o Regan Lowe (charcoal cephalexin agar) or Bordet Gengou (potato blood glycerol agar)
o B. pertussis produces mercury droplet colonies in Regan-Lowe
 o Fastidious, does not grow in BAP and MAC
o Non-motile, oxidase(+), urease(-)

Bordetella parapertussis

 Causes a disease similar to pertussis but with milder symptoms

Bordetella bronchiseptica

 Respiratory pathogen to animals

Legionella pneumophila

 Agent of Legionnaire’s disease (febrile disease with pneumonia) or Pontiac fever (febrile
disease without pulmonary involvement)
 Isolated in air conditioning towers and heating systems
 Stains poorly with gram stain; stained using silver impregnation (Dieterle)
 Medium for culture- Buffered charcoal yeast extract agar (BCYE)- grayish-white or blue-green,
glistening convex colonies central portion has “ground-glass” appearance

Francisella tularensis

 It is considered as a potential bioterrorism weapon- is easily disseminated with high mortality

rate and posing a risk to national security
 Is a very small, obligate aerobic, coccobacillus; extremely invasive
 Vector- deerflies and ticks
 Microscopy- gram-negative bacilli with “faint bipolar staining”
 Biochemical test- catalase weakly (+); oxidase(-); citrulline uridase; glycerol fermenter
 Disease- tularemia/ deerfly fever/ rabbit fever/ lemming fever/ water rat trapper’s disease
 Culture medium- blood-cystine-glucose-agar

Capnocytophaga

 Fastidious, capnophilic, fusiform or filamentous bacilli

 Ferments sucrose, maltose, glucose and lactose
 Indole (-), nitrate reduction(+), esculin hydrolysis(+)
 Flagella is usually absent but they exhibit characteristic gliding motility

Pasteurella multocida
  It is the etiologic agent of “shipping fever” in cattle
 it is isolated from animal bite(felines) or scratch wounds
 it is facultatively anaerobic; non-motile
 it has characteristic “mushroom smell”
 bipolar staining (safety pin appearance when poles of the cells are more intensely stained)

AEROBIC GRAM POSITIVE BACILLI

Bacillus

 Are sporeforming, rod-shaped organisms; can be isolated from soil

 They can be aerobic or facultative anaerobes- but only form endospores aerobically
 It is motile (peritrichous) except B. anthracis
 They can survive in temperatures as low as -5ºC and as high as 75ºC
 They can survive in extreme environmental conditions due to endospores
 Catalase positive
 Species:
o B. subtilis- considered as the most common laboratory contaminant; used in Guthrie
test- microbiologic test for phenylketonuria
o B. stearothermophilus- used as a biological indicator for autoclave
o B. anthracis
o B. cereus

Bacillus anthracis

 Characteristics:
 Bamboo pole or fishing rod arrangement on Gram stain
 Cell wall contains poly-d-glutamic acid
 Encapsulated, with oval centrally-located spores; non-motile; non-hemolytic
 Biochemical reactions:
o Produces acid from glucose, sucrose and maltose
 o Mostly lecithinase positive; starch hydrolysis positive
 Culture:
o Medusa head colonies in sheep blood agar
o Beaten egg-white consistency when lifted with inoculating needle
o Strings of pearls in Mueller-Hinton agar with 10U penicillin disk
 Forms of anthrax
o Cutaneous form
 Most common, but less severe
 Organism enters through a cut
 Lesion occurs at the site of entry and develops into a black necrotic area known
as eschar
o Respiratory/Pulmonary form
 Woolsorter’s disease; inhalational anthrax
 Inhalation of spores during shearing or sorting of animal hair
o Gastrointestinal form
 Most severe form
 Results from ingestion of the bacilli or spores in contaminated food
 Agent of bioterrorism

Bacillus cereus

 “fried rice bacilli”

 Commonly associated with food poisoning in rice, cereal, vegetables and milk
 Forms of infection:
o Emetic form
 Caused by production of a heat-stable emetic toxin
 Vomiting occurs within 6 hours after ingestion of contaminated food
o Diarrheal form
 Caused by production of a heat-labile enterotoxin
 Toxin is produced when foods are left at room temperature
o Septicemia, pneumonia, meningitis and peritonitis
o Eye infections associated with trauma
 Characteristics:
 o Motile; hemolytic (wide zone of beta hemolysis)
o Grayish to lavender color on blood agar
o Produces acid from glucose, maltose and salicin
o Lecithinase positive

Bacillus subtilis

 It is a halophilic organism; source of bacitracin antibiotic

 It may cause eye infection among heroin addict

Corynebacterium

 Gram positive, non-motile rods with palisading, picket fence and Chinese letter appearance
 Pleomorphic due to irregular snapping during cell division
 Produces metachromatic granules (Babes-Ernst or volutin granules)
 Clinically significant organisms are those that produce the diphtheria toxin

Corynebacterium diphtheria

 Also known as Klebs-Loeffler bacillus

 Mode of transmission: droplets to susceptible individuals
 Conditions associated:
o Cutaneous diphtheria
o Pseudo-membrane in the tonsils, pharynx and larynx due to production of exotoxin,
which can eventually cause respiratory obstruction
 Childhood immunization: DPT (diphtheria, pertussis, tetanus)
 Biochemical reactions:
o Catalase positive
o Oxidase positive
 Culture:
o Culture Media:
 Blood agar plate- colonies have narrow zone of beta hemolysis
 Loeffler’s serum slant- enhances metachromatic granule formation
 Cystine tellurite blood agar- gray to black colonies
 Potassium tellurite medium- gray to black colonies
  Tinsdale medium- also known as cystine-sodium-thiosulfate-tellurite medium;
black colonies with brown halo
 Loeffler’s serum slant and Pai’s coagulated medium can enhance pleomorphism
and metachromatic granule formation
o Colonial types of Corynebacterium diphtheria
 Gravis type- 1-2 mm colonies on blood agar; largest colonial type
 Mitis type- fried egg appearance on blood agar (clear colonies with white
centers); bleach-like odor on tellurite medium
 Intermedius type- small colonies (0.5mm) on blood agar; black colonies with
gray borders on tellurite medium
 Toxin demonstration
o In vitro toxigenicity test is known as modified Elek test
 Filter paper strip impregnated with antitoxin is placed in a medium containing
rabbit serum, potassium tellurite and prepared agar
 Plate is inoculated with the toxigenic strain of C. diphtheria, with the streak being
in a right angle to the strip of antitoxin
 A negative control is streaked on the same manner
 The unknown culture is streaked parallel to the positive and the negative
controls
 Incubate at 35ºC for 24-48 hours
 Plate is observed for lines of precipitation (positive result)
o In vivo toxigenicity test in the form of animal (guinea pig) inoculation test
 Diphtheroids
o Corynebacterium jeikeium- associated with endocarditis, pneumonia and peritonitis;
pinpoint white colonies in SBA after incubation at increased CO 2 at 30ºC
o Corynebacterium ulcerans- produces diphtheria-like toxin and diphtheria-like infection
o Corynebacterium pseudotuberculosis- formerly known as C. ovix
o Corynebacterium xerosis- normal flora of skin, nasopharynx and conjunctiva
o Corynebacterium pseudodipthericum- normal flora of oropharynx/throat

Listeria monocytogenes
  Described as gram-positive to gram-variable coccobacillus, can be mistaken as Streptococcus
agalactiae
 Found in the environment (soil, water, decaying vegetation)
 Major source of infection: contaminated foods, such as cabbage, raw fruit, dairy products
 Conditions associated:
o Meningitis, endocarditis, conjunctivitis
o Spontaneous abortion or stillbirth
 Encapsulated, gram-positive bacilli
 Motile (tumbling end-over-end motility) when incubated at RT for 1-2 hours and in hanging-
drop technique
 Biochemical characteristics:
o Bile esculin hydrolysis positive
o Catalase positive and oxidase negative
o Indole negative and Voges-Proskauer positive, H2S negative
o CAMP test positive
o Ferments glucose, trehalose and salicin, but not mannitol
o Motility and salicin test can differentiate Listeria monocytogenes from Corynebacterium
 Culture:
o Facultative anaerobe; can survive even at 4ºC
o Specimen: blood, CSF and tissues
o Smooth, clear to gray colonies with narrow band of beta hemolysis
o Umbrella-shaped or inverted Christmas tree pattern after overnight incubation at RT in
a semi-solid medium
o Refrigeration of the specimen for several months may enhance isolation
 Pathogenicity (animal inoculation) test: Ocular test of Anton
o Isolate is suspended in sterile water
o 2 or 3 drops of the suspension are inoculated in the conjunctival sac of a rabbit’s eye
o Positive test is indicated by the development of purulent conjunctivitis
o The other eye serves as a negative control

Erysiphelothrix rhusiopathiae

 Indole negative, H2S positive; nitrate negative; catalase negative; non-motile

  Ferments glucose but not mannitol and salicin
 Culture:
o Specimen- skin biopsies, tissue aspirates, blood
o Alpha hemolytic or nonhemolytic in blood agar plate
o Test tube brush growth in semi-solid medium
 Conditions associated:
o Butcher’s disease
 An occupational hazard to individuals handling meat and poultry
 Seal finger/ whale finger- swelling at the site of organism’s inoculation
 Erysipeloid- red, spreading in nature

Lactobacillus acidophilus

 Normal flora of the mouth, gastrointestinal tract and vaginal canal

 Non-pathogenic and has little clinical significance
 Tolerates highly acidic environment; can grow at pH 3.0-4.0; cultured on tomato juice agar
 Catalase negative, pleomorphic, non-motile

Aerobic Actinomycetes

Nocardia asteroids

 Causes chronic pulmonary infections in immunocompromised patients

 Long, thin, branching, gram positive bacilli
 Stained using Modified Kinyoun’s stain; partially acid fast
 Grown in Saboraud Dextrose agar, inhibited by chloramphenicol
 Catalase positive; urease positive; digests paraffin
 Can grow in any media that does not contain antibiotics

Other aerobic Actinomyctes

 Include Nocardiopsis, Streptomyces, Actinomadura, Dermatophilus

 Associated with chronic granulomas of the skin and subcutaneous tissues
 Causes actinomycetoma characterized by a swelling draining lesion of the extremities and can
be acquired through inoculation from the soil
 GRAM POSITIVE ANAEROBIC SPOREFORMING BACILLI

Clostridia

 Is an obligate anaerobic; gram-positive sporeforming rod; catalase negative

 Is frequently encountered in exogenous anaerobic infections or intoxications
 Its toxins usually gain access to the body through ingestion or via open wounds that have
been contaminated with soil
 Contribute to virulence- collagenase, hyaluronidase, lecithinase and phospholipase
 C. septicum- is a marker for a malignancy in the GI tract; “smooth swarming” growth on
plated media

General Characteristics of Clostridia

 They form endospores anaerobically

 They are motile with peritrichous flagella except C. perfringens, C. ranosum and C. innocum
 They have swollen sporangia
 They are non-encapsulated except C. perfringens
 They have a single hemolytic reaction except C. perfringens

Clostridium tetani

 Causes lock jaw or tetanus with characteristic backward arching of the back muscles (risus
sardonicus)
 Introduced into the body through an exogenous wound (puncture wound, gunshot, burn,
animal bite)
 Produces neurotoxin (tetanospasmin) which disrupts nerve impulses to muscles
 Drumstick appearance (round, terminal spores)
 Motile, gelatinase positive and indole positive
 Lecithinase negative and lipase negative
 Unable to ferment most carbohydrates
 Treatment: administration of anti-toxin
 Prevention: tetanus toxoid
Clostridium perfringens

 Most common species isolated from clinical specimens

 Conditions associated:
o Gas gangrene or myonecrosis
o Food poisoning after ingestion of large amounts of organism
 Virulence factors:

o Alpha toxin
o Theta toxin
o Beta toxin
o Hemolysins
o Lecithinase
o Cardiotoxin
o Enterotoxin
o DNase
o RNase
o Protease
o collagenase
  Characteristics
o Non-motile, box car shaped bacilli
o Round, subterminal spores
o Double zone of hemolysis in anaerobic blood agar
 Beta-hemolytic inner zone of hemolysis due to theta toxin
 Alpha-hemolytic outer zone of hemolysis due to alpha toxin and lecithinase
o Positive lecithinase production detected using egg yolk agar- where it produces opaque
yellow halo
o Ferments glucose, lactose, maltose and fructose
o Reverse CAMP test positive
 Suspected clostridial species is streaked on blood agar plate
 S. agalactiae is streaked at right angle to the first streak
 After incubation, positive reaction is indicated by the formation of an arrowhead
at the intersection of the two streaks
o Nagler plate
 One-half of the surface of the plate is streaked with few drops of C. perfringens
type A antitoxin
 Suspected culture is streaked across the plate at a right angle to the antitoxin
 A zone of precipitation around the colonies on the side without antitoxin, with
little or no precipitation around the colonies on the side with anti-toxin indicates
a positive lecithinase test

Clostridium botulinum

 Canned good bacilli

 Botulism toxin- the most potent toxin
o A powerful neurotoxin that binds to the synapse of the cholinergic nerve fibers
o Leads to acute and flaccid paralysis, involving first the muscle of the face, head and
throat and later those of the thorax, diaphragm, arms and legs
o Death may occur due to respiratory paralysis
 Forms of botulism
 o Food botulism- ingestion of food contaminated with preformed toxin in home-canned
foods
o Wound botulism- organism produces toxin at the site of wound infection
o Infant botulism- most common type; colonization of the gastrointestinal tract of infants,
and produces toxin; involves ingestion of spores from soil, dust or honey that germinate
in the gastrointestinal tract
 Characteristics:
o Oval, subterminal spores
o Motile
o Lipase positive, lecithinase negative, indole negative
o Ferments glucose, but not lactose or xylose
 Diagnosis is done by detecting neurotoxin in serum, feces, gastric contents, vomitus or food

Clostridium difficile

 Conditions associated:
o Antibiotic-associated Pseudomembranous colitis
 Found in patients who have received one or more broad-spectrum antibiotics
 Antibiotics decreases the normal flora in the gut
 Organism takes this as an opportunity to establish itself in the gut
 Produces toxin, leading to diarrhea
 Toxins:
o Toxin A- enterotoxin
o Toxin B- cytotoxin
 Toxins can be demonstrated using EIA, latex agglutination or tissue culture
 Tissue culture is the gold standard for toxin identification
 Characteristics:
o Gelatinase positive, lecithinase negative, lipase negative, indole negative
o Ferments glucose and fructose
o Does not ferment lactose, maltose or xylose
o Oval, subterminal spores
o Motile, horse stable odor and fluoresces under UV light
o Produces yellow colonies in CCFA (Cycloserine-Cefoxitin-Fructose Agar)
Other Notable Clostridia

Clostridium ranosum

 Normal flora of the large bowel

 Can cause intra-abdominal infections following trauma
 Round to oval terminal spores
 Grows well in 20% bile and esculin hydrolysis positive
 Indole negative, lecithinase negative, catalase negative, lipase negative
 Ferments glucose, lactose, maltose, fructose and mannitol

Clostridium septicum

 Causes bacteremia associated with malignancies such as colon cancer, breast cancer and
leukemia
 Oval, subterminal spores
 Gelatinase positive, lecithinase negative, lipase negative, indole negative
 Ferments glucose, lactose, maltose and fructose
 Does not ferment mannitol

ANAEROBIC NONSPOREFORMING BACILLI AND COCCI

Some of the clinically significant anaerobes

Anaerobic gram positive cocci

 Peptococcus  Ruminococcus
 Peptostreptococcus  Coprococcus

Anaerobic gram positive bacilli

 Actinomyces  Eubacterium
 Bifidobacterium  Propionibacterium

Anaerobic gram negative cocci

 Veilonella  Acidaminococcus
 Megasphera
Anaerobic gram negative bacilli

 Bacteroides
 Fusobacterium
 Prevotella
 Porphyromonas
Things to remember about anaerobes

 Anaerobic gram-negative bacilli are the major normal flora of the colon, outnumbering aerobes
by 1000:1
 Specimen of choice: needle and syringe aspirate
 Anaerobic medium
o Anaerobic blood agar
o Anaerobic phenylethyl alcohol blood agar
o Anaerobic Kanamycin-Vancomycin blood agar
o Anaerobic paromomycin- Vancomycin laked blood agar
o Thioglycollate
o Bacteroides bile-esculin

Peptococcus

 Anaerobic Staphylococcus
 Appears in clusters; catalase positive

Peptostreptococcus

 Anaerobic Streptococci

Veilonella

 Gram negative cocci in pairs, short chains or irregular clumps

 Highly sensitive to oxygen
 Produces brick red fluorescence under UV light

Actinomyces israelii

 Agent of actinomycoses
 Characterized by presence of sulfur granules in the exudate of infection
 Branching, filamentous, anaerobic gram positive bacilli
 Rough and white colonies with molar tooth appearance
 Associated with lumpy jaw

Bifidobacterium

 Normal flora of the GI tract; rare cause of pulmonary infection

Propionibacterium acnes

 Normal flora of the skin, nasopharynx, oral cavity and GI tract

 Often found as a skin contaminant in blood cultures
 Anaerobic diphtheroids since they resemble the Chinese letter appearance of Corynebacterium

Fusobacterium

 Appear as long, thin, filamentous, gram negative bacilli with tapered ends (spindle shape)
 Most common isolate is Fusobacterium nucleatum
 Colonial morphology: opalescent speckles

Prevotella

 Pigmented, saccharolytic, gram- negative bacilli

 Produces brown to black pigment on anaerobic blood agar
 Susceptible to rifampin; resistant to kanamycin

Porphyromonas

 Pigmented, asaccharolytic, gram negative bacilli

 Inhibited by 20% bile; indole positive

Bacteroides fragilis

 Non-motile, gram negative bacilli

 Non-hemolytic, gray colonies in blood agar plate
 Growth is enhanced by 20% bile
 Positive to bile esculin hydrolysis

MISCELLANEOUS BACTERIA

Streptococcus monoliformis

 It is the etiologic agent of rat-bite fever and Haverhill fever in humans

 Is a Gram-negative bacillus that requires media containing blood, serum or ascites fluid
 It is normally found in the oropharynx of wild and laboratory rats.
 It is facultatively anaerobic, nonmotile, non-encapsulated and nonhemolytic.
 It is known to spontaneously develop "L forms".
 Dienes stain is required to demonstrate the L-form colonies.
  It has extreme pleomorphism with long, looped, filamentous forms, chains and swollen cells
 Microscopy: "necklace-like arrangement or string-of-beads" - chains of bacilli with prominent
yeast like swelling; "fried egg" appearance with a dark center and a flattened, lacy edge
 Culture: broth culture - "fluff balls or bread crumbs"
 Biochemical Test: (-) catalase, oxidase, nitrate, urea, indole and LDC
 Transmitted by 2 routes:
o Rat bite or by direct contact with rats.
o Ingestion of contaminated food like unpasteurized milk or milk products and water.

Notes to Remember:

 After collecting blood and joint fluids, it is mixed with equal volumes of 2.5% citrate to prevent
clotting, then inoculated to BHl-cysteine broth supplemented with Panmade (a papain digest of
ox liver)
 The organism may be cultured from blood or aspirates from infected joints, lymph nodes or
lesions.
 Acridine orange stain also reveals the bacteria when gram stain falls due to lack of cell wall
constituents.

Spirillum minus/ minor

 It causes rat-bite fever in human referred to as Sodoku

 It cannot be grown in synthetic culture media; and strictly aerobic.
 It is closely related to Neisseria.
 Direct visualization of specimens (blood, exudates or lymph node tissues) using Giemsa stains,
Wright stains, or dark-field microscopy is recommended.
 Microscopy: Gram-negative bacilli, thick and helical
o 2 to 3 coils and polytrichous polar flagella
Gardnerella vaginalis

 It is part of the normal flora of anorectal adult.

 Is a small, pleomorphic, gram- positive coccobacillus or rods; may stain Gram-variable;
 It’s the etiologic agent of bacterial vaginosis, considered a sexually transmitted disease.
 Culture: small and -hemolytic on media containing rabbit or human blood
 Biochemical test: (+) hippurate hydrolysis; (-) oxidase and catalase
  Bacterial vaginosis
 Is characterized by a malodorous discharge and vaginal pH > 4.5.
 It results from a reduction in the lactobacillus population in the vagina, followed by a rise in
vaginal pH
 Laboratory Diagnosis:
o Specimen: vaginal secretions
o Culture media: CAP, CAN and Human Blood Bilayer Tween (HBT) agar
o HBT is for isolation from female genital tract.
o It often takes longer than 24 hours to develop visible colonies.
o It will not grow in blood culture broths because it is inhibited by SPS.
 Diagnostic Tests:
o Direct saline wet mount of vaginal secretions
o (+) "clue cells" - large, squamous epithelial cells with numerous attached small rods
o Whiff Test or KOH test
o (+) "fishy amine odor" (after adding one drop of 10% KOH) and vaginal pH > 4.5

RICKETTSIACEAE

 The simplest bacterial form and considered transitional organism between bacteria and viruses
 This group of organism infects wild animals, with humans acting as accidental hosts
 Most are transmitted between animals by an insect vector
 The best means of prevention for rickettsial and ehrlichial infection is to avoid contact with the
respective vectors
 Mode of acquisition: humans become infected following the bite of an infected arthropod
vector
 Microscopy: small, non-motile, pleomorphic gram-negative bacilli
 Culture: they have not been grown in cell-free media- all species require a living cells for
growth except Bartonella Quintana

Rickettsia

 Are fastidious bacteria, obligate and intracellular parasite

  Are small organisms, pleomorphic; that have gram-negative cell wall and multiply by binary
fission in the cytoplasm of host cells (the release of mature Rickettsiae results in the lysis of
the host cell)
 They reside and only multiply in the cytosol of the host cell
 They survive briefly outside of a host
 They do not undergo any type of intracellular development cycle
 Vector: ticks

Transmission and spread of Rickettsia

 When humans contact the infected ticks, the organism is deposited on the skin and then
rubbed or scratched into the skin or deposited into the skin as the tick feeds
 They are spread by way of the bloodstream and infect the endothelial cells of blood vessels
resulting to vasculitis
 Following infection, organisms escape the vacuole, becoming free in the cytoplasm then
multiply, causing cell injury and death

Orientia tsutsugamushi

 It was placed into a separate genus due to absence of LPS and peptidoglycan, and the
presence of 54-58 kDa major surface protein
 The gram-negative cell wall has an ultrastructurally thicker outer leaflet and thinner inner
leaflet of the outer envelope
 They grow in the cytoplasm of host cell and released via “pinching off”
 Humans and rats are accidental, nonessential dead-end hosts
 It infects endothelial cells causing vascular injury
 Is transovarilly maintained in mites
 Vector: Chigger

Ehrlichia

 Are small, gram-negative coccobacilli and undergo intracellular development cycle following
infection of circulating WBC
 Natural hosts: dogs, deer and humans
 Primary vector: lone star tick
  Microscopy: Wright-Giemsa staining of intravacuolar microcolony resembles “mulberry”-
morula

Coxiella burnetii

 Is the only species in the genus; smaller than Rickettsia species

 Is the causative agent of Q (query) fever- systemic infection that affects the lungs
 Is strictly intracellular organism- it develops within the phagolysosome of infected cells; the
target cells for infection are the macrophages
 It can survive extracellularly because of its endosporelike body
 It can be grown only in lung cells
 Is highly contagious and can be considered as a potential bioterrorism agent
 It can infect fish, birds, rodents and livestock- organisms are shed in urine, feces, milk and
birth products
 It is not transmitted by arthropod vector- humans are infected by the inhalation of
contaminated aerosols from dried animal feces and ingestion of unpasteurized milk
 In contrast to rickettsial infection, a rash does not develop among person following Coxiella
infection
 Animal reservoir: cattle, sheep and goats

Laboratory Test

 It does not multiply in bacteriologic culture media

 It is more resistant to chemical and physical agents, dessication and sunlight
 Biopsy specimens has not resulted in any laboratory acquired infections
 Shell vial assay with human lung fibroblasts is used to isolate the organism from buffy coat
 IFA- method of choice
 B. henselae and B. quintana create cross-reactivity

Bartonella

 Are facultative intracellular gram-negative bacilli; do not belong to the order Rickettsiales
 They do not synthesize acid from carbohydrates
 They lived within red blood cells in their natural mammalian hosts
 They can be cultivated in CAP with 5% CO2 or charcoal yeast extract agar
  B. bacilliformis and B. henselae: (-) catalase, oxidase and urease have “twitching motility” in
wet mounts
 Species:
o B. Quintana- causative agent of Trench fever/Louse-borne disease
o B. henselae- causative agent of Cat scratch disease
o B. elizabetheae- causative agent of Infective endocarditis
o B. bacilliformis- causative agent of Oroya fever (chronic verruga peruana) and febrile
acute hemolytic anemia; a sandfly transmitted bacteria
o B. clarridgeiae- is a suspected second agent of cat scratch disease
 Trench fever- is transmitted from person to louse (Pediculus humanus corporis) to another
person

Differential Characteristics of Rickettsiaceae

ORGANISM DISEASE/S VECTOR/MODE OF

TRANSMISSION
SPOTTED FEVER GROUP
A. Rickettsia conorii Boutonneuse fever/ Mediterranean Ticks
Spotter fever
B. Rickettsia rickettsii Rocky Mountain Spotted fever Wood ticks and Dog
ticks
C. Rickettsia akari Rickettsialpox Mouse mite
TYPHUS GROUP
A. Rickettsia prowazekii Epidemic Typhus/ Brill-Zinsser Louse, Squirrel flea and
Disease/ Recrudescent Typhus/ Flying Squirrel louse
squirrel typhus
B. Rickettsia typhi Endemic Murine Typhus Rat fleas
SCRUB TYPHUS GROUP
A. Orientia tsutsugamushi Scrub Typhus Chigger bite
EHRLICHIA
A. Ehrlichia chaffeensis Human MonocyticEhrlichiosis Lone star tick
B. Ehrlichia ewingii Ehrlichiosis ewingii Tick bite
 C. Anaplasma Human Granulocytotropic Deer bite
phagocytophila Anasplasmosis
Coxiella burnetti Q Fever Inhalation of aerosol
from infected animals
Bartonella quintana Trench Fever Feces of Pediculus
humanus corporis
Bartonella henselae Cat scratch disease; Bacillary Kitten scratch or bite
angiomatosis
Bartonella bacilliformis Oroya Fever, Verruga Peruana Sandfly bite

Spotted Fever Group

 Rocky mountain spotted fever is the most serious rickettsial infection

 In RMSF, humans are the accidental hosts and ticks are the principal vector and reservoir


CHLAMYDIACEAE

 Gram negative organism that were previously mistaken as viruses

 Have DNA and RNA and divide by binary fission; have affinity to columnar cells
 Obligate intracellular parasites; energy parasites
 2 forms
o Elementary bodies- infectious form; capable of limited extracellular survival
o Reticulate bodies- intracellular, metabolically active form; incapable of surviving outside
the cell
 Diagnosis:
o Specimen: scrapings and swab
o Stained with iodine or Giemsa for demonstration of inclusions
o Cultured using McCoy’s heterophloid murine cell pretreated with cycloheximide

Chlamydia trachomatis
  Agent of lymphogranuloma venereum, endemic trachoma, non-gonococcal urethritis and infant
pneumonitis
 Serotypes A,B,Ba and C are associated with endemic trachoma, the leading cause of blindness
 Serotypes D,E,FG,H,I,J and K are associated with venereal disease, neonatal pneumonitis and
inclusion conjunctivitis
 Serotypes L1,L2 and L3 are associated with LGV through venereal route
 TRIC conjunctivitis includes: trachoma and inclusion conjunctivitis
 Organism contains inclusion bodies that contain glycogen, which can be stained using iodine
or Periodic Acid Schiff (PAS)
 Serologic test: Frei test: direct fluorescence assay using monoclonal antibodies (for genital
smear specimen)
 Treatment: tetracycline, erythromycin or fluoroquinones
 Chlamydia trachomatis is the only species sensitive sulfonamides

Chlamydia psittaci

 Agent of psittacosis or ornithosis

 A disease of the psittacine birds such as parrots, parakeets and cockatoos
 Humans acquire infection through inhalation

Chlamydophila pneumoniae

 Also known as TWAR, which came from two initial isolates TW-183 and AR-39
 Pear shaped with large periplasmic space and round elementary bodies

MYCOPLASMA

Mycoplasma

 Smallest free-living organism known

 Pleomorphic in appearance due to lack of cell wall, and therefore, would not be affected by
antibiotics the inhibit cell wall synthesis
 Contains DNA and RNA
 Can grow on artificial media
 Includes: Mycoplasma pneumoniae and Ureaplasma urealyticum
Mycoplasma pneumoniae

 Also known as Eaton’s agent

 Causes community-acquired pneumonia and tracheobronchitis
 Causes primary atypical pneumonia or walking pneumonia
 Diagnosis:
o Culture:
 E-agar or Shepard’s A7 B-agar
 Mycoplasma pneumoniae produces fried egg appearance- dense center and
translucent periphery
o Best method for the identification of Mycoplasma species- inhibition of growth by
specific anti-sera
o Serological test
 Test for cold agglutinins- non specific
 Cold agglutinins are IgM (with anti-I specificity) that agglutinate group O RBCs at
4ºC but not at 37ºC
 Titer of 1:128 is diagnostic for current infection

Ureaplasma urealyticum

 Ureaplasma is positive for urease test which is indicated by a brown halo surrounding the
colonies
 Genital mycoplasma; initially called T-strain mycoplasma (T-tiny)
 Causes non-gonococcal urethritis
 Does not produce haze in a broth culture

SPIROCHETES

Spirochetes

 Slender, flexuous, helically coiled, unicellular bacteria that is motile via periplasmic flagella
 Characteristically gram-negative but difficult to gram stain
 Includes: Treponema, Leptospira, Borrelia

Treponema pallidum

o Causes syphilis, a sexually transmitted infection

o History:
o Historical theories suggest Christopher Columbus and his sailors brought the disease to
Europe from America
o Syphilis is a sexually transmitted disease
o Treponema pallidum is not recovered in blood, serum or plasma stored at 4ºC for more
than 48 hours
o Part of ToRCHeS (Toxoplasmosis, Rubella, CMV, Herpes simplex virus, Syphilis)- which
are vertically-transmitted infections (i.e. mother to baby)
o Other Treponema species:
o T. pallidum subspecies pertenue- yaws
o T. pallidum subspecies endemicum- non-venereal endemic syphilis or bejel
o T. carateum- pinta
o T. cuniculi- rabbit syphilis
o Forms of Syphilis
o Acquired syphilis (characterized by hard chancres, condyloma, gummas)
o Congenital syphilis- transmitted from mother to offspring
o Stages of syphilis
o Primary syphilis- characterized by hard chancres
o Secondary syphilis- is the result of disseminated organisms, with the appearance of
multiple, widespread lesions on mucus membranes or on skin
o Latent syphilis- asymptomatic
o Tertiary/Late syphilis- manifest as (1) gummas/gummatas, (2) cardiovascular syphilis or
(3) neurosyphilis
o Diagnostic tests for syphilis
o Culture:
 Treponema cannot be cultured on artificial medium
 Can be cultured in the testicles of rabbit
o Direct demonstration of Treponemes
 Darkfield microscopy- can be used to demonstrate corkscrew motility
 Levaditi’s silver impregnation method- since they do not stain well using gram
stain; Treponemes appear black
  Direct fluorescent-antibody staining for T. pallidum
o Non-treponemal tests for syphilis

 Unheated serum reagin

 Toluidine red unheated
serum test
 Wasserman complement
fixation test
 Kahn flocculation test
 Regain screen test
 Automated reagin test
 Plasmacrit
 VDRL and RPR
o Characteristics:
 Detects anti-cardiolipin antibodies (also known as reagin or antibodies to the
Wasserman antigen or anti-lipoidal antibodies)
 Non-specific; often used as screening procedures, not as confirmatory
 Reaction between reagin and cardiolipin is known as flocculation
 Flocculation is a physical process of contact and adhesions wherein the
aggregates form larger-size clusters called flocs or flakes being excluded from
suspension
o VDRL- Venereal Disease Research Laboratories
 Principles: qualitative or quantitative slide flocculation test
 Can be used in both serum and CSF specimens
 Requires serum inactivation
 Inactivation is done by heating serum for 56ºC for 30 minutes
 Inactivated serum must be used within 4 hours
 If more than 4 hours have elapsed, serum must be reinactivated by
heating it at 56ºC for 10 minutes
 Reagents/antigen:
 Alcoholic solution of cardiolipin-cholesterol-lecithin
 0.03% cardiolipin- phospholipid isolated from beef heart; responsible for
reactivity
 0.9% cholesterol- serves as center for absorption of tissue lipids to
increase the size of antigen
 0.21% lecithin- to produce standard reactivity
 Antigen suspension may be used only on the day of preparation
 Gauge numbers of needles of the Hamilton syringe used to deliver antigen
suspension
 Serum-antigen mixture is placed in rotator for 4 minutes at 180 RPM
 Test is performed at RT
 Results are read microscopically using LPO
 Results are reported as:
 Reactive- medium to large clumps
 Weakly-reactive- small clumps
  Non-reactive- no clumping or slight roughness as compared to antigen
control
 Positive VDRL test on spinal fluid is diagnostic of neurosyphilis
o RPR- Rapid Plasma Reagin
 Antigen:
 Cardiolipin, lecithin, cholesterol
 EDTA, Na2HPO4, thimerosal, charcoal, choline chloride- stabilizes the
antigen and inactivates complement
 Serum-antigen mixture is placed in rotator for 8-minutes at 100 RPM
 Uses plastic-coated disposable cards (10-18mm circles) instead of glass slide
 Advantages:
 No requirement for heat inactivation
 Antigen suspension more stable, can be stored
 Charcoal is added to make reaction more visible; results are read
macroscopically
 Test can be automated, more sensitive and rapid than VDRL
 Cannot be used in CSF
o Treponemal test for syphilis
o Characteristics:
 More specific than non-treponemal tests; may be used as confirmatory test
 Detects antibodies directed towards Treponema pallidum
o TPI (Treponema pallidum Immobilization Test)
 Treponemal test of reference but too cumbersome for routine testing
 First test developed for detection of specific anti-treponemal antibody
 Specimen: serum
 Reagent: live, motile Treponema pallidum from experimental rabbits, guinea pig
complement
 Interpretation of results:
 Positive:>50% immobilized
 Negative:<20% immobilized
 Doubtful:20-50% immobilized
o FTA-Abs (Fluorescent Treponema pallidum Antibody-Absorbed Test)
  Specificity of test enhanced by pre-absorption of serum with Reiter Treponemes
 Reagents:
o Antigen: dead Treponema pallidum in slide (Nichols virulent strain)
o Label: FITC-AHG
o Sorbent: Reiter strain of Treponemes (non-pathogenic) to remove
antibody against commensal spirochete
 Antibodies present in patient’s specimen binds the antigen. FITC-AHG is then
added and fluorescence is viewed using ultraviolet light
o Agglutination Test
 TPHA (Treponema pallidum hemagglutination)
 Confirmatory test for syphilis; an indirect hemagglutination test
 Uses tanned sheep red cells coated with antigen from the Nichol’s strain
 MHA-TP (Microhemagglutination- Treponema pallidum)
 Similar to TPHA, except that MHA-TP is a microtechnique (performed in
microtiter plates)
 Uses formalinized, tanned sheep erythrocytes sensitized with Treponema
pallidum antigen (Nichol’s strain)
 HATTS (Hemagglutination treponemal test for syphilis)
 Automated conversion of TPHA
 Uses glutaraldehyde-stabilized turkey RBCs
 TP-PA (Treponema pallidum Particle Agglutination)
 Uses gelatin particles (instead of RBCs) sensitized with T. pallidum
antigens
 An indirect agglutination test

Leptospira

 Aerobic, tightly-coiled, thin, flexible spirochetes, with ends that are bent or hooked
 Species include: Leptospira interrogans, Leptospira biflexa

Leptospira interrogans

 Has specific serovars such as icterohaemorrhagiae, canicola and pomona

 Pathogenic species that usually infects rodents, cattle, dogs, cats raccoons and bats
 Shed in the urine of the animals
  Mode of transmission:
o Organism enters human through breaks in skin and mucus membranes
o Acquired through direct contact with the urine or blood of animals who carry the
organism
o Acquired by indirect contact with the urine of animals- i.e. when urine or blood from
animal with Leptospira contaminates the soil or water
 Causes leptospirosis, and the rare, severe form known as Weil’s disease
 Leptospirosis affects the kidneys, liver and central nervous system
 Diagnosis:
o Specimen:
 Cultures of blood and CSF are done during the first 10 days of illness
 Second week of illness, urine is the sample
o Culture:
 Grows best under aerobic conditions at 28-30ºC
 Requires rabbit or bovine serum albumin for growth; as well as 10% serum plus
fatty acids
 Culture is the definitive test
 Culture medium includes:
 Fletcher’s semisolid medium
 EMJH (Ellinghausen-McCullough-Johnson-Harris) medium
o Serologic test
o Fourfold increase in antibody
o 2 specimens must be collected; acute and convalescent specimen

Leptospira biflexa

 Non-pathogenic species
 Found in water and soil

Borrelia

 Microaerophilic, arthropod-borne helically-coiled bacteria that stain well with Giemsa

 Include: Borrelia burgdorferi and Borrelia recurrentis

Borrelia burgdorferi
  Causes Lyme disease
 Transmitted by deer ticks Ixodes dammini, Ixodes pacificus, Ixodes ricinus
 Has the same vector as the parasite Babesia microti
 Stages:
o 1st stage- erythema chronicum migrans- characterized by red bull’s eye rashes
o 2nd stage- dissemination occurs through the blood; carditis and meningitis
o 3rd stage- chronic stage- includes chronic neurologic abnormalities, arthritis and skin
lesions
 Morphology:
o Loosely coiled spirochete
o Silver impregnation technique can be used to demonstrate the organism in the lesions
 Culture Media:
o Barbour-Stoenner-Kelly medium
 Serologic testing
o Fluorescent immunoassay, indirect immunofluorescence, enzyme-linked immunoassay
o EIA
o Western blot (gold standard)

Borrelia recurrentis

 Transmitted by louse, Pediculus humanus subspecies humanus

 Causes relapsing fever
o Organism enters blood and cause multiple lesions in the spleen, liver and kidneys
o High fever, muscle and bone pain
o Fever persists for 2-6 days and then the patient appears to recover, only to relapse
days or weeks later
o Relapses are due to ability of Borrelia to alter its antigenicity, thereby requiring the host
to develop new immunity to each altered strain
o Diagnostic test:
 Blood film evaluation
 Increased yield of the organism on blood is found during the febrile
episode
 Giemsa or Wright stain can be used, and Borrelia appears blue in color
 MYCOBACTERIUM

Mycobacterium

 Obligate aerobes
 Contains cord factor wax D and mycolic acid
 Gram-positive, more commonly referred to as “gram ghost” since the crystal violet cannot
penetrate the cell wall of the organism due to high levels of mycolic acid
 The cell wall of Mycobacteria binds to alkaline stains such as carbolfuchsin, and are referred to
as acid-fast bacilli
 Can be spread through inhalation or droplets
 Mycobacterium tuberculosis complex includes: M. tuberculosis, M. bovis and M. africanum
 Identification of Mycobacteria includes biochemical reaction, pigment production and growth
rate evaluation
 Runyon classified the mycobacteria other than tuberculosis (MOTT) into four groups:
o GROUP I- Photochromogens= develop yellow pigment when exposed to constant light
source; nonpigmented in dark
o GROUP II- Scotochromogens= pigmented yellow to orange in dark; pigment intensifies
to orange or red when exposed to constant light source for 2 weeks
o GROUP III- Non-photochromogens= white to tan in color; cannot develop pigment on
exposure to light
o GROUP IV- Rapid growers= grow in 3-5 days in culture media; saprophytes

Runyon’s classification Color produced Growth rate Species

Photochromogens Not pigmented unless 10-21 days M. kansasii
exposed to light M. asiaticum
-cream or buff M. marinum
-orange/yellow M. simiae
Scotochromogens Pigmented both in the 10-21 days M. szulgai
dark and light M. gordonae
-yellow to orange M. scrofulaceum
colonies M. flavescens
 M. xenopi
M. thermoresistible
Non-photochromogens Non-pigmented both in 10-21 days M. avium complex
the dark and light M. ulcerans
M. terrae complex
M. gastri
M. haemophilum
Rapid growers Pigment variation 3-7 days M. fortuitum
M. chelonae
M. smegmatis
M. phlei
M. abscessus
M. mucogenicum

Laboratory test for Mycobacterium spp

1. Culture characteristics
a. Photoreactivity(pigment production)
b. One tube of Lowenstein Jensen medium is incubated covered with foil and one tube is
incubated uncovered. Tubes are observed when growth appears on the uncovered tube
c. Growth rate
i. Hold cultures of Mycobacterium for 8 weeks before reporting as negative
ii. Mycobacterial cultures should be incubated in 5% carbon dioxide
d. Growth temperature
i. 30-32ºC- M. haemophilum, M. ulcerans and M. marinum
ii. 42ºC- M. xenopi
iii. 52ºC- M. thermoresistible
2. Niacin accumulation
 All Mycobacterium species produce niacin and most possess an enzyme that converts
free niacin to niacin ribonucleotide
 Positive- yellow (M. tuberculosis, M. simiae, and rare strains of M. bovis, M. marinum
and M. chelonei
3. Nitrate reduction
  Detects for production if nitroreductase, which converts nitrate into nitrite
 Positive: M. tuberculosis, M. kansasii, M. szulgai, M. fortuitum
4. Catalase and heat stable catalase test
 Most Mycobacterium are positive for catalase, but not all of them are positive to heat-
stable catalase
 Heat-stable catalase is a catalase that is resistant to heat at 68ºC
5. Arylsulfatase
 Detects ability of organism to produce Arylsulfatase
 Positive result: pink color (M. fortuitum-chelonei)
6. Pyrazinamidase
 Detects production of enzyme Pyrazinamidase
 Positive result: production of red pigment (M. tuberculosis, M. marinum)
7. Urease
 Positive result: pink color (M. bovis, M. scrofulaceum, M. gastri)
8. Tween 80 hydrolysis
 Positive result is change in color from amber to pink (M. kansasii, M. gordonae)
9. Iron uptake test
 Detects ability of organism to grow in 20% ferric citrate and the ability to convert ferric
ammonium citrate into iron oxide
 Positive result: rusty-brown colonies
10.5% NaCl tolerance
 Most Mycobacteria cannot grow in 5% sodium chloride
 Positive result: M. triviae, M. flavescens
11.Tellurite reduction
 Ability to reduce colorless potassium tellurite into black metallic tellurium
 Positive: M. avium complex
12.NAP (p-nitroacetylamino-beta-hydroxypropiophenone)
 NAP inhibits M. tuberculosis complex
13.T2H (thiophene-2-carboxylic acid hydrazide)
 Distinguish M. tuberculosis (resistant) from M. bovis (susceptible)
14.Growth on MacConkey agar without crystal violet
 M. fortuitum-chelonei complex can grow on MacConkey agar without crystal violet
Mycobacterium tuberculosis

 Previously known as Bacterium tuberculosis, commonly known as Koch’s bacillus

 Causes tuberculosis which is a chronic infectious disease that primarily targets the
lungs, but can also infect the kidneys and the CNS
 Treatment:
o Anti-TB drugs
 Primary drugs- Rifampin (rifampicin), Isoniazid, Pyrazinamide, Ethambutol,
Streptomycin
 Secondary drugs-
 Injectable- amikacin, capreomycin, kanamycin
 Fluoroquinolones- ciprofloxacin, ofloxacin, levofloxacin,
moxifloxacin
 Treatment for TB is a long-term process (6 months) since Mycobacterium tuberculosis is
a slow-grower and has long generation (doubling) time
 Susceptibility tests (skin tests) for TB
o Uses tuberculin purified protein derivative (PPD)
o PPD is an extract of M. tuberculosis, heat-killed, filtered and precipitated with
ammonium sulfate
o PPD is injected intradermally in the forearm
o It is used to test if a person has been exposed to tuberculin protein, either from
previous tuberculosis vaccination, or from environmental exposure to the
bacteria
o Presence of hard, dense, raised wheal that is 10mm or larger after 48 hours
indicates previous exposure to tuberculin protein
 Specimen: sputum, urine CSF
o 3 sputum specimens collected on 3 consecutive days are recommended to
increase isolation
 Culture:
o Buff-colored colonies are often characteristics of M. tuberculosis
o Egg-based
 Lowenstein-Jensen
 Petragnani
  Gruft modified LJ medium
 Selective LJ medium
o Agar-based
 Middlebrook 7H9, 7H10, 7H11, 7H12, 7H13
 Mitchison’s selective 7H11

Mycobacterium leprae

 Causes Hansen’s disease or leprosy

 Leprosy is an infection of the skin, mucous membranes and peripheral nerves
 2 major forms of leprosy

Tuberculoid leprosy Lepromatous leprosy

Skin lesions and nerve involvement that can Skin lesions and progressive, symmetric nerve
produce areas with loss of sensation damage
Lesions in the mucous membranes may lead
to destruction of the cartilaginous septum
resulting in nasal and facial deformities
Patients exhibit an effective cell-mediated Patients do not produce an effective cell-
immune response mediated immune response
Organisms are extremely rare and may not be Acid-fast bacilli are abundant on specimen
detected in skin scrapings or biopsy
specimens

 Mode of transmission: inhalation or contact with infected skin

 Specimen for diagnosis: tissue juice
 Acid fast bacilli in a specimen from nasal mucosa are not diagnostic of Mycobacterium
leprae
 Cannot be cultured in vitro; cultured on foot pads of armadillo
 Lepra cells are macrophages containing acid-fast bacilli inside
 Sulfone dapsone is one of the drugs that is used for the treatment of leprosy

Other Mycobacteria spp.

M. ulcerans
  Causes cutaneous lesions known as Buruli ulcers, which appear as lumps under the skin that
do not heal
 Biochemically inert but positive for heat-stable catalase

M. bovis

 Slow growing, unbranched acid fast rod that is nitrate negative and niacin negative
 Causes tuberculosis in cattle
 Found in Bacillus of Calmette and Guerin (BCG)- vaccine for tuberculosis
 Growth resembles “water droplets’ in Middlebrook media

M. kansasii

 Also known as “yellow bacillus”

M. marinum

 “of the sea”

 Causes skin infections occurring as red to blue lesions and swimming pool granuloma

M. simiae

 First isolated from Macaca rhesus monkey

M. scrofulaceum

 Smooth, buttery, yellow to orange colonies

M. asiaticum

 Similar to simiae but negative for niacin

M. szulgai

 Photochromogen at 25ºC but scotochromogen at 37ºC

M. gordonae

 “tap water bacillus”

M. xenopi

 Optimally grows at 42ºC

  Branching colonies with aerial hyphae on cornmeal agar, described to be “bird’s nest”

Mycobacterium avium-intracellulare

 “battey bacillus”

ANTIMICROBIAL SUSCEPTIBILITY TESTING

 Schlichter Test is a measure of the activity of the antibiotic in the patient’s own serum against
the pathogen
 In-vitro methods to determine Bacterial Susceptibility to antibiotics”
o Tube dilution method and microplate dilution method
 Quantitative test in which serial dilutions of antibiotics are prepared and standard
concentration of bacteria are added
 Standard concentration of bacteria in broth microdilution has a final
concentration of 5.0 x 105 CFU/mL
 Incubated at 35ºC or 16-20 hours
 Can be used to determine MIC and MBC
 Valuable in case that: organism is from blood culture; organism fails to respond
to antibiotic therapy; patients who relapse while on therapy
o Agar dilution method
 Similar to tube dilution method
 Specific volumes of antimicrobial solutions are dispensed into premeasured
volumes of molten and cooled agar
 The antibiotic-agar mixture is then poured into petri dishes
 Reference method for testing of anaerobes and N. gonorrhoeae
 Disadvantage
 Not helpful in organisms that tend to spread, such as Proteus and
Pseudomonas
 Agar dilution plates have a shelf-life of only 1 week because plates are
stored at 2-8ºC, a temperature at which many drugs are labile with
 Plate preparation is laborious
o Disk method (Kirby-Bauer Disk Diffusion)
 Procedure:
  Performed by inoculating a standardized amount of organism into agar,
followed by adding antibiotic disks
 Diameter of zones of inhibition is measured around antibiotic disks and
evaluates as to whether the organism is resistant, susceptible or
intermediate
 Standardized amount of bacteria
 Adjusts the turbidity of the inoculum (bacterial suspension)
 Do not use growth from plates more than 1 day old
 Inoculum in NSS is compared against 0.5 McFarland Turbidity standard,
which is equivalent to approximately 1.5 x 108 CFU/mL
 Standard should be verified- it should have an absorbance of 0.08-0.1 at
625nm using spectrophotometer
 Standard is prepared using 0.5mL of 1.175% BaCl2 and 99.5mL H2SO4
 Standardized inoculum should be used within 15 minutes after
standardization
 Standard inoculum is spread using a swab all throughout the MHA plate
 Standard Medium for Disk Diffusion: Mueller-Hinton agar
 pH of the medium should be between 7.2-7.4
 standard agar depth is 4mm
 calcium and magnesium content of agar for Pseudomonas should be
monitored
o Standard level of Calcium: 25mg/L
o Standard level of Magnesium: 12.5mg/L
 Increased concentrations result in decreased activity of aminoglycosides
against P. aeruginosa. Decreased concentration will have opposite effect.
 Increased concentrations lead to decreased activity of tetracyclines
against all organisms. Decreased concentration will have opposite effect
 Thymidine content must be minimal. Excessive concentrations can cause
false resistance to sulfonamides and trimethoprim.
 A standard petri dish plate (150mm in diameter) can accommodate as
many as 12 different antimicrobial disks
 Antibiotics
  Antibiotics are placed on the medium inoculated with the organism, and
then allowed to diffuse for 3-5 minutes but not more than 15 minutes
before turning the plate upside-down
 Antibiotic disks are stored in:
o A non-frost free freezer at -20ºC or below (long term storage)
o A refrigerator at 2-8ºC (short term storage; for working supply) for
at least 1 week
 Disks must be stored on tightly sealed container
 Antibiotics must be allowed to warm to room temperature before it is
opened
 Penicillin and methicillin are best indicators of poor storage
 For quality control when monitoring the reagent antibiotic disks, the
antibiotic disks must be checked when container is first opened, and once
each week of use
 Incubation requirements:
 Plates are incubated at 35ºC for 18-24 hours in humidified ambient air
 Some methicillin-resistant S. aureus may go undetected in temperatures
>35ºC
 Capnophilic incubation decreases pH which can lead to decreased activity
of aminoglycosides, erythromycin and clindamycin; it will lead to an
increased activity of tetracycline
 Plates should not be stacked more than 5 plates high
 Interpretation
 Quality control plates must be checked prior to reading results of patient
isolates
 Zones of inhibition are measured using a ruler, template or caliper. It is
done using unaided eye, using the underside of the plate
 Plates are placed a few inches above a black, non-reflecting surface,
zones are examined from the back side of the plate illuminated with
reflected light
 Presence of zone of inhibition may indicate susceptibility
 Absence of zone of inhibition may indicate resistance
  Tiny colonies at the zone edge and the swarm growth into the zone that
often occurs with swarming is ignored
 When there are two concentric zones around the disk during sulfonamide
testing, the zone is measured using the diameter of the outer zone
 Diameter of the zones are not to be interpreted quantitatively. Results are
reported as resistant, susceptible or intermediate
 False susceptible
 Too thin agar
 Too light inoculum
 Low temperatures
 Too dry agar
 False resistant
 Too thick agar
 Too heavy inoculum
 Too much moisture on agar