FINALS | MICR_100

Staphylococci and Streptococci Staphylococcus aureus

Most significant staphylococci
GRAM POSITIVE COCCI Most virulent species; coagulase (+)
Staphylococcus aureus Halophilic bacteria (grows in high salt concentration)
- Responsible for various skin, wound, and
deep tissue infections.

Culture
REPORT AS: Heavy growth of white pin-head colonies
beta hemolytic pattern

Gold colored (Aureus): Lipochrome

Staphylococci and Micrococci
- BAP: Beta hemolytic
Both are gram positive cocci in cluster and therefore
cannot be differentiated through gram stain alone.

Staphylococci
Gram-positive cocci that belong to the family
Staphylococcaceae
- Are catalase-producing bacteria.
- Facultatively anaerobic
- (Except S. saccharolyticus: obligate anaerobe)
- Are non-motile and glucose fermenter.

Microscopy - MSA: “Old sock/stocking odor”

Spherical cells, singly, in pairs and in clusters
Staphle: greek - Bunches of grapes

Culture

BAP: Creamy, white/light gold, and “buttery looking”

Other species have gray colonies
Some species are B-hemolytic (S. aureus)
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Associated Disease and Infections Enzymes and Toxins Produced

Toxin-induced cases (Virulence Factors)
Scalded Skin Syndrome Catalase
- Bullous exfoliative dermatitis caused by A heme enzyme that catalyzes the decomposition of
staphylococcal exfoliative or epidermolytic H2O2 to water and oxygen.
toxin - Differentiates staphylococci (catalase +)
- Affects newborns from streptococci (catalase -)
Toxic Shock Syndrome
- Rare but potentially fatal
- Affects multisystem
- Characterized as a sudden onset of fever,
chills, vomiting, diarrhea, ………………
Cutaneous Infections
Folliculitis
- Inflammation of hair follicle * Bubble formation due to release of oxygen gas.
Furuncles (boils)
- Upgraded version of folliculitis
- Large, raised superficial abscesses
Carbuncles
- Group of furuncles Staphylocoagulase
- Occur when larger, more invasive lesion Coagulates fibrinogen in plasma
develop from multiple furuncles - Promotes the formation of fibrin layer around
- Has pus staphylococcal abscess thereby protecting the
Bullous Impetigo bacteria from phagocytosis.
- Pustules are larger and surrounded by the
2 Types of Staphylocoagulase
zone of erythema.
A. Cell-bound Coagulase / Clumping Factor
- Caused by staphylococci
- Bound to cell wall
Non bullous Impetigo
- Clots human, rabbit, or pig plasma
- Caused by streptococci
B. Unbound Coagulase / Free Coagulase
Treatment: IND (Incision and Drainage)
- An extracellular enzyme; free from cell wall
Other Associated disease and infections:
- Causes a clot to form when bacterial cells are
1. Bacteremia and Sepsis
incubated with plasma.
2. Osteomyelitis
3. Septic arthritis Hyaluronidase
4. Food poisoning It enhances invasion and survival in tissue
The spreading factor
- Binds cells together and renders intercellular
spaces passable to pathogens.

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Staphylokinase (Fibrinolysin) Cytolytic Toxin

Dissolves fibrin clot; has fibrinolytic activity. Destruction of cells
Causes anemia: Make iron available for microbial
Lipase (Fat-splitting enzyme)
growth
Essential for survival in sebaceous areas of the body
Important in formation of furuncles, carbuncles, and a. Alpha hemolysin
boils. - It destroys RBC, platelets and macrophages; it
causes severe tissue damage
DNAse and Phosphatase
b. Beta hemolysin
Lowers viscosity of exudates, giving pathogens more
- Hot and cold lysin (WHY?)
mobility.
- Destroys sphingomyelin and RBC around
Beta-lactamase nerves.
Breaks down penicillin and other beta-lactam drugs. - Exhibited in the CAMP test for group B
- 90% or more clinical staphylococci isolates are streptococci.
resistant to penicillin as a result of enzyme c. Gamma hemolysin
production. - Causes toxic compared to alpha and beta
Inhibits or destroys any antibiotic mode of action: lysins
inhibits cell wall synthesis. - Causes injury in RBC in culture

Enterotoxins (heat stable) d. Delta hemolysin

Stable to heating @ 100°C for 30 mins - Destroys RBCs
- Reheating contaminated food will not prevent - Associated with the Panton- Valentine
disease. Leukocidin (PVL)
- Resistant to hydrolysis by gastric and jejunal
Exfoliative Toxin A and B
enzymes.
- It causes the epidermal layer of the skin to
Examples:
slough off -stratum granulosum (surface
- A,B,D: Food Poisoning
layers of the skin) is destroyed
- B, C, G, I: Toxic Shock Syndrome
- It causes scalded skin syndrome or ritter
* Enterotoxin b: has been link to staphylococcal
disease
pseudomonas enterocolitis
Toxic Shock Syndrome Toxin-1 (TTST-1) /
Panton-Valentine Leukocidin (Cytolytic Toxin)
Enterotoxin F / Pyogenic exotoxin
Attacks and kills WBC (PMN, macrophage, and
- A chromosomal-mediated toxin
monocytes)
- Causes almost all cases of
- Often associated with community-acquired
menstruating-associated TSS.
staphylococcal infections.
Protein A
Immunologically active substance found in cell wall.
Antiphagocytic: By competing with neutrophils for
the Fc portion of specific opsonins.

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Voges-Proskauer Test
Differential Tests for Staphylococcus aureus
Differentiates S. aureus (+) from S. intermedius (-)
Coagulase Test
- Both tube coagulase positive
The best single criterion of pathogenicity of
- (+) result: pink color with acetone or
Staphylococcus aureus.
acethylmethyl carbinol production
- Reagent: Rabbit / Pig / Human plasma
qN: WHY MSA DIFFERENTIATE WHY IS A PATHOGENIC
Slide Method IS YELLOW IN COLOUR
Detects cell bound coagulase/ clumping factor on the
surface of the cell wall
- Insensitive test
(+) Result : clot/coagulum formation within 30
seconds.

Other slide coagulase (+) result

- S. Lugdunensis and S. schleiferi
Tube Method
Considered a sensitive method; definitive test
- Detects extracellular/unbound/ free
coagulase
- (+) Result: clot/coagulum formation after 1-4
hours of incubation
Other Tube (+) coagulase test:
- S. hyicus, S. intermedius, S. delphini, ………..
Mannitol Fermentation Test
Used to differentiate pathogenic from nonpathogenic
staphylococci
- MSA: 1% mannitol + 7.5% NaCl is both
selective and differential
Result + yellow color: S. aureus (.....)
Methicillin-resistant Staphylococcus aureus
Penicillin-resistant strains require treatment w/
penicillinase-resistant penicillins.
- Nafcillin or Oxacillin
- Acquired after prolonged hospital stay (ICU
and burn patients) proximity to patients with
MRSA; after receiving broad spectrum
antibiotics; nasal carriage.
Controlled by: proper isolation of organism; hand
hygiene; rapid identification of the bacteria and
control and treatment of sources
- Resistance of staphylococci to
penicillinase-resistant penicillin is due to
meCA gene which encodes an altered
penicillin binding protein 2a known as PBP2a
Detection
Specimen for testing: Nasal swab
Chromogenic test: (+) mauve-colored colonies within
24 hours and confirmed within 48 hours.

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Rapid tests: IDI-MRSA test, BD Gene Hom assay - CoNS

(results within 2 hours) - DNAse(-)
Oxacillin: Generally used for detection
Urine culture
Cefoxitin: CLSI recommended
10,000 CFU/mL (significant findings)
When an isolate shows resistance to one of the
penicillinase-resistant penicillins, it must be Antimicrobial test
considered resistant to the entire group. Resistant to 5ug Novobiocin (6-12 mm)

Staphylococcus epidermidis Staphylococcus lugdunensis

Normal flora of skin.
Contaminant of medical instrument.
Causes:
- Hospital-acquired infection (Health care
acquired UTIs)
- Contaminants in improperly collected blood
specimens.
For adherence: Poly-gamma-DL-glutamic acid
Culture
Small to medium-sized y-hemolytic, non-pigmented,
white opaque, pin-head colonies
Biochemical test
- MSA (-)
- CoNS
Antimicrobial test
Susceptible for 5 mg-ug novobiocin
Staphylococcus saprophyticus
Associated with community acquired UTI in young,
sexually active females
Adheres more effectively to the epithelial cells
lining the urogenital tract than other CoNS
CoNS by tube method
More aggressive than other CoNS in ability to be
infective; associated w/ catheter related bacteremia
and endocarditis.
Contains mecA gene which codes for oxacillin
resistance.
Resistant Gene Produced by Staphylococci
Erythromycin ribosomal methylase (erm)gene
Codes for methylation of the 23S ribosomal
ribonucleic acid (rRNA) which results in resistance to
erythromycin and inducible or constitutive resistance
to clindamycin.
Methionine sulfoxide reductase A gene
Codes for an efflux mechanism, which results to
resistance to erythromycin but susceptibility to
clindamycin

Culture
White, opaque, pin-head slightly larger colonies;
nonhemolytic

Biochemical test
- MSA (+/-)

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DIAGNOSTIC TEST TO IDENTIFY ● (-) : Do not Ferment Mannitol (Red) 8.4 pH

STAPHYLOCOCCI
1. MICROSCOPIC EXAMINATION
● Check the gram stain
● Gram positive cocci in clusters
● Two swab: (1 Gram Stain & 1 Culture)
● Aspirates is the best sample
CULTURE MEDIA: Staphylococcus aureus 5. COLISTIN NALIDIXIC ACID AGAR (CAN)
2. BLOOD AGAR PLATE: ● Inhibits gram negative bacteria.
● Differential but not selective ● To isolate Streptococci and Staphylococci
● Enrich other organism ● Colistin: disrupt cell membrane of Gram
3. CULTURE MEDIA: Staphylococcys epidermis negative
● BAP: small to medium sized, Non hemolytic ● Nalidixic: block the DNA replication of gram
gray to white negative

CULTURE MEDIA
● Staphylococcus haemolyticus: medium to 6. PHENYETHYL ALCOHOL AGAR
large with weak hemolysis ● Inhibits the growth of Gram negative bacteria.
● Disrupt gram negative cell wall.
7. CATALASE TEST
● To differentiate Staphylococcus and
Streptococcus
● Uses 3% Hydrogen Peroxide
● Aerobes and Facultative Anaerobes possess
Staphylococcus saprophyticus : slightly larger
the catalase
colonies w/ about 50% yellow pigment
● H2O2 ¨ H2O + O2*
4. MANNITOL SALT AGAR
● Procedure: Colony + 2 to 3 drops of Hydrogen
● Contains 7.5% NaCl
Peroxide
● Carbohydrate: Mannitol
● Culture in NA
● Indicator: Phenol Red
● (+): Bubbling
● Selective Medium for Staphylococcus
● (-): No Bubbling for 30 secs
● (+): Ferment Mannitol (Yellow) 6.8 pH
● False positive: when colonies at BAP

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●
8. SLIDE COAGULASE TEST
● To detect Cell bound coagulase/ Clumping
Factor
● Fibrinogen converted to Fibrin by Clumping
Factor.
● Clumping factor: plasma in EDTA tube
● POSITIVE:
o Staphylococcus aureus
o Staphylococcus lugdunensis
o Staphylococcus schleiferi
● (+): White Fibrin Clot (Agglutination)
● (-) : Smooth Suspension
9. TUBE COAGULASE TEST 10. GLUCOSE OXIDATION-FERMENTATION TEST
● Detect free bound coagulase or ● All Staphylococcus ferment glucose except S.
Staphylocoagulase. saprophyticus, S. hominis, S. xylosus, S. cohnii
● Citrate utilizing organism can leas to false • (+): Turn to Yellow
positive, EDTA must be used as a • (-): Green
anticoagulant for plasma.
• (+) : With Clot or Fibrin clot 11. NOVOBIOCIN SUSPECTIBILITY TEST
• (-): No fibrin clot ● Used 5 ug Novobiocin
● Procedure: ● Differentiate Staphylococcus saprophyticus
1. 0.5 mL plasma + 0.1 mL broth culture of organism and Coagulase Negative Staphylococcus spp.
(loopful of organism.
2. Incubate in 35 to 37 Celsius. Check every 30 mins
for 4 hours.
3. Negative after 4 hours, Incubate at room temp for
18 to 24 hrs.

May halo = susceptible to Novobiocin

12. DNAse TEST

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● Used to detect DNAse activity in aerobic
bacteria
● Toluidine blue : Blue → rose pink
● Methylene Green: Green → Colorless
• Result:
o (+) Clear zone: Staphylococcus aureus
o (-) No clearing: Staphylococcus epidermidis
15. VOGUE PROSKAUER (VP) TEST
● To detect glucose fermentation by Butanediol
● Reagent: Alpha naphthol + KOH
● (+) deep pink/red : S. aureus, S. lugdunensis,
S. haemolyticus, S. schleiferi
● (-): S. intermedius

3. PYRROLIDONYL ARYLAMIDASE TEST (PYR)

16. RAPID METHOD: LATEX AGGLUTINATION
● To detect Staphylococci producing
● BBL Staphyloslide
arylamidase enzyme
● Pyroglutamyl- B naphthylamide + p
dimethylaminocinnameldehyde ¨
L-pyrrolidone + B naphthylamine ( red)
● (+) Cherry red: S. aureus
● Straphaurex
● (-) S. lugdunensis, S. intermedius, S. schleiferi

● Bactistaph
14. MICRODASE DISK (MODIFIED OXIDASE)
● Modified oxidase test to differentiate
Micrococcus and Staphylococcus spp.
● Reagent used: 6%
tetramethylphenylenediamine hydrochloride
17. BETA LACTAMASE TEST
in dimethyl sulfoxide)
● Cephalosporinase test
● Micrococcus possess cytochrome C
● Uses cephalosporin or cefinase (with
• (+): Turn blue in 2 minutes (Micrococcus spp.)
nitrocefin)
• (-): No Color change
● (+) pink or red in 60 minutes ( 10 minutes in
other bacteria)
● Acidimetric method
● Reagent: citrate buffered penicillin
● (+) Red to yellow color
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● Iodometric method Inducible resistance to clindamycin by the D

● Reagent: Phosphate buffered penicillin and test
starch iodine Used to detect inducible clindamycin resistance in
• (+) Colorless solution staphylococci
• (-) Purple color. Performed when isolate of S. aureus is resistant to
erythromycin but susceptible to clindamycin
18. Antimicrobial test - Performed before clindamycin is reported to
Drugs for treatment of staphylococcal infections be susceptible.
- Methicillin
- Oxacillin
- Nafcillin
- Cloxacillin
- Dicloxacillin
- Penicillinase-resistant penicillin drugs
When an isolate shows resistance to one of the
penicillinase-resistant penicillins, it must be
considered resistant to the entire group.

MISSED A SLIDE

Oxacillin Screen Plate

Detects MRSA
MHA with 4% NaCl + 6 ug/mL oxacillin
After overnight incubation at 35C, growth is an
indication that the isolate is oxacillin resistant
Microdilution testing: Oxacillin

Cefoxitin Disk Diffusion Test

Recommended by CLSI
Confirmatory tests for oxacillin resistance
Broth Dilution & E Test

Borderline Oxacillin-Resistant Isolates

Unrelated to presence of mecA gene
Resistance: Caused by hyperproduction of
β-lactamase or the presence of altered, normal,
penicillin-binding proteins
Have MICs right above (or zones of inhibition right
below) the breakpoint for oxacillin susceptibility
Do NOT grow on oxacillin plates.
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Enterobacteriaceae Lactose Fermenters

Gram negative bacilli and coccobacilli Rapid Lactose Late Lactose Non-Lactose
Fermenters Fermenters Fermenter
Characteristics Exceptions
E coli Samonella arizonei Proteus
Cytochrome Plesiomonas shigelloides
oxidase negative Enterobacter Shigella sonnei Morganella

Nitrate reducers Photorhabdus Klebsiella Serratia Shigella

Xenorhabdus
Citrobacter Salmonella
Ferments glucose N/A ALL ENTEROBACTERIACEAE
MUST FERMENT GLUCOSE
Yersinia Edwardsiella

Motile at body Klebsiella (PATHOGENIC ORGANISMS; Hafnia Erwinia

not part of normal flora)
temperature Shigella
Salmonella Providensia
Yersinia

Nonencapsulated Klebsiella Capsulated: non-motile & mucoid Virulence And Antigenic Factor
Enterobacter

MacConkey Medium
Most used differential and Selective medium which
O antigen
differentiates Bacteria from lactose to non lactose
Somatic antigen
fermenter, and selective by inhibiting the growth of
- Found in the body of microorganism; heat stable
gram positive bacteria and only promotes the
Significance: Serotyping organisms
growth of gram negative bacteria.
Bile salts: Inhibits the growth of gram positive H Antigen
organisms Flagellar antigen.
- Heat labile.
K Antigen
Capsular antigen (mask); Interfering substance.
Heat labile.
- Covers somatic antigen

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Clinical Significance Escherichia coli

Opportunistic Pathogens: typically part of normal Part of the normal bowel flora of humans and
flora inhabits the female genital tract.
- Ex: Escherichia coli (E.coli) Invades enterocytes lining the large intestine
Primary Pathogens: never considered normal flora; Leading cause of nosocomial infection (hospital
infectious organisms acquired) - UTI
- Ex: SSY Salmonella, Shigella, Yersinia
Primary marker of fecal contamination in water
Antimicrobial Resistance purification.
Klebsiella pneumoniae, Klebsiella oxytoca, and many
Escherichia coli Culture
- Resistant to ampicillin because of production of a MAC
plasmid-B-lactamase known as TEM-1 or SHV-1 Flat, dry, pink colonies; agar will also turn pink
- Susceptible to extended spectrum cephalosporins - In exam it's always lactose fermenter
and aztreonam.
Treatment
Extended spectrum cephalosporins and aztreonam.
- Extended-Spectrum β-lactamases (ESBLs):
Organisms that are resistant to ESCA
Carbapenem-resistant Enterobacteriaceae (CRE)
Enterobacteriaceae develop resistance to the group of
BAP
antibiotics called carbapenems.
Beta hemolytic some strains

EMB Agar
Greenish metallic sheen

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Antigenic Determinants
O, H, and K antigens
- E. coli O groups have shown cross reactivity
with the O antigens of Shigella.

Virulence Factors
Endotoxin, common pili, K1 antigen, intimin
K1 antigen
- Identical to the capsular antigen found in
Neisseria meningitidis group B.
- Neonatal Meningitis

Biochemical Tests
(+) sex pili and adhesive fimbriae
IMViC reaction: + + – –
TSI reaction: A/A, (+) gas, (-) H2S
IMViC: Indole, Methyl red, Voges-Proskauer, Citrate
- Methyl red and Voges-Proskauer are often opposites/inverse.
TSI: Triple Sugar Iron

Triple Sugar Iron Agar (TSIA) Test

3 sugars
Glucose, Lactose, Sucrose
- Red = Acidic (Glucose or Lactose fermenter) E. coli o157:H7 colonies on selective agars
- Yellow = Alkaline (non Glucose or non Lactose - Part of EHEC; bloody diarrhea
fermenter) Sorbitol MacConkey Agar: Screening test for E. coli
- Black = hydrogen sulfide formation O157:H7
Appearance:
(-): clear/ colorless
(+): pink/deep red

A/A: Acid, Acid (Slant, Butt)

- Glucose and lactose fermenter
K/A: Alkaline, Acid (Slant, Butt)
- Glucose fermenter (NON LACTOSE FERMENTER)
* NEVER A/K OR K/K = for Enterobacteria *

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Klebsiella Biochemical Test

Found in GIT of humans/animals; indicator of fecal Neufeld Quellung Test: (+)
contamination Indole (-)
Species Capsular swelling
TSI reaction: A/A, (+) gas, (-) H2S
﹘ K. oxytoca ﹘ K. pneumoniae subsp. pneumoniae

﹘ K. ornithinolytica ﹘ K. pneumoniae subsp. ozaenae

﹘ K. planticola ﹘ K. pneumoniae subsp. rhinoscleromatis

﹘ K. terrigena

Biochemical Test IMViC reaction: - - + +

Beta lactamase KCN: (+) growth
Indole (+): K. oxytoca and K. ornithinolytica
- None produce H2S
- A few hydrolyze urea slowly
- All are methyl red (-), Voges-Proskauer (+);
With a few exceptions, no indole is produced from
tryptophan

Klebsiella pneumoniae subsp. pneumoniae

AKA “Friedlander’s bacillus”
The most common isolated species of Klebsiella
- Causative agent for community acquired
pneumonia (currant jelly-like sputum)

Culture

MAC
pink, mucoid colonies (capsulated)

Virulence factor Streptococcus pneumoniae

Polysaccharide capsule - Most common cause of CAP
- Often follows influenza; acute onset
Differential Test Klebsiella pneumoniae
String Test (+) - Ethanol abuse

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- Diabetes, immunocompromised Cronobacter sakazakii

- Aspiration Previously known as “Enterobacter sakazakii”
Yellow pigment

Enterobacter
Resembles Klebsiella when on MacConkey Agar

Culture
MAC
Pink colonies and maybe with mucoid

Significant species : Pathogen in neonates: meningitis (powdered infant

Common isolate: E. aerogenes, E.cloacae - Other formula)
species: E. gergoviae and E. hormaechei
Serratia
Biochemical Test
Resistant to a wide range of antibiotics
(+) growth on
Species
- Simon Citrate Agar
- Potassium Cyanide Broth ﹘ S. marcescens ﹘ S. plymuthica

MR: Negative ﹘ S. liquefaciens ﹘ S. ficaria

VP: positive
﹘ S. rubidaea ﹘ S. fonticola
Produces
- Ornithine decarboxylase ﹘ S. odorifera ﹘ S. entomophila
- Lysine Decarboxylase EXCEPT: E. gergoviae or
S. marcescens, S. rubidaea and S. plymuthica: have
E. cloacae
pink to red pigment (prodigiosin) at 25°C.

Pantoea agglomerans
Previously known as “Enterobacter agglomerans”

serratia
Kawasaki - yellow
Late lactose fermenter
Outbreak of septicemia due to contaminated IV fluids
Triple decarboxylase negative: AD, LD, OD Biochemical Test
A = Arginine decarboxylase Ferment lactose slowly
L = Lysine decarboxylase ONPG: (+) Late Lactose Fermenter
O= Ornithine decarboxylase They have the ability to produce extracellular Dnase

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S. odorifera: musty- pungent odor of “rotten

potato-like” odor
Biochemical Test
Hydrolyze urea & produce H2S
Serratia marcescens
Indole and ornithine decarboxylase tests
Causes bacteremic outbreaks in nurseries and cardiac
TSI: K/A
surgery and burn units.
PAD test (+)
Common contaminant in antiseptic solutions used for
joint injections causing an epidemic of septic arthritis

Biochemical test
Urease producer
Gelatinase (+)
IMViC reaction: - - + +
TSI: K/A, (+) gas, (-) H2S
- Late Lactose Fermenter
PPM
Proteus
It is isolated from urine, wound and ear infections.
Swarming organism.
It can infect the proximal kidney tubules and can
cause Acute Glomerulonephritis
- Trimethoprim: inhibitory agent
Rapid urease producer
- Urease: splits urea in urine, raises urine pH
(alkaline), and encourages renal stone
formation
Species:
Human pathogens: P. mirabilis, P. vulgaris

Culture
Burnt chocolate or burnt gunpowder. Swarming
phenomenon

Providencia
One of the causes of nosocomial outbreaks involving
burn units.
Species
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﹘ P. alcalifaciencs ﹘ P. stuartii ﹘ P. rettgeri Citrobacter

AKA Citrate-lover
﹘ P. rustigianii ﹘ P. heimbachae Producces colonies on MacConkey agar that
resembles E. coli and biochemically resembling
Biochemical Tests
Salmonella
It can cause false (+) agglutination test with
Salmonella
All species grow on Simmon citrate agar; slow urease
producers.
Pad test (+)
Culture
OGDP
Providencia rettgeri
LLF
A pathogen of the urinary tract
NLF after 24 hours, LF after 48 hours colonies are
Also causes diarrheal
light pink after 48 hrs.

Morganella
Citrobacter freundii
Same biochemical reaction with P. vulgaris except
Isolated in diarrheal stool cultures (extraintestinal
citrate (-)
pathogen)
Species
Colony morphology on primary plated media can be
- M. morganii subsp. morganii
mistaken for Salmonella when isolated from stool.
- M. sibonii
- LLF
Biochemical Tests
Differentiate citrobacter from salmonella
Urease (+)
KCN (+)
Ornithine Decarboxylase test (+)
Pad test (+)
IMViC reaction: + + – –
K/A, H2S

Edwardsiella
It has been isolated from cold/warm blooded animals
Species
- E. hoshinae, E. icatluri
- E. tarda (human pathogen)
Plesiomonas shigelloides
Biochemical Tests Opportunistic bacteria
IMVIC: + + – – (E. tarda) The only oxidase (+) member of the
TSI reaction: K/A, (+) gas, (+) H2S (E. tarda) Enterobacteriaceae
Formerly classified as vibrio
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Culture Biochemical Principles

Similar to E. coli on primary plated media Bacterial spp incapable of fermenting glucose cannot
- Opaque “apron-like colonies”- CIN utilise lactose
- (-) growth on TCBS 2 enzymes necessary for a bacterium to take up
- (+) growth on HEA lactose:
- B galactoside permease
Biochemical Tests
- B galactosidase
Positive trio decarboxylate Test
(+) cross-agglutinate with Shigella Enzymes present in Rapid Lactose Fermenter, Lactose Late
Lactose Fermenter, NonLactose Fermenter
TSI: K/A, (-) gas, (-) H2S
Antigenic structure: O and H

Take Note:
2 enzymes associated with lactose fermentation
Permease and beta-galactosidase.
Permease regulates the movement of lactose across
the bacterial cell wall. Once lactose is inside the cell, Rapid Lactose Fermenter:
it is broken down into the individual components, Late Lactose Fermenter: Lack the β-galactosidase-permease
glucose and galactose, by beta-galactosidase. enzyme
Non Lactose fermenter:

Identification of Enterobacteriaceae Culture

Culture MacConkey Agar
BAP and CAP: Large, gray, smooth colonies pH indicator: Neutral Red
- Organisms should be identified on the basis of Used to diff E. coli 0157:H7 colorless; other types of
patient history and gross description of the E. coli are with color
specimen (bloody or watery stool) Eosin Methylene Blue Agar
Carbohydrate Fermentation LF (purple to greenish Metallic sheen)
NLF (colorless)
Fermentation
Anaerobic process carried out by obligate, facultative, Other coliform
and aerotolerant anaerobes Hektoen Enteric Agar
The electron acceptor is an organic compound pH indicator: Bromothymol blue, acid fuchsin
- Indicate any type of utilization of a H2S indicator: Ferric Iron
carbohydrate-sugar-with the resulting Gram + inhibitor?
production of an acid pH LF: yellow
Respiration NLF: blue green
An efficient energy-generating process in which Other coliforms: orange-pink
molecular O2 is the final indicator
Xylose-Lysine Desoxycholate Agar
Shigella - pink to red color

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Salmonella pink to red w/ balck center

Other: yellow
Salmonella-Shigella Agar
Medium that only promotes growth of Salmonella
and Shigella
pH indicator = neutral red
H2S indicator= thiosulfate and ferric citrate
Appearance of Shigella/Salmonella
CHROMagar Salmonella
(+)Mauve color

Serotyping (Slide Agglutination Test)

Serologic identification
Common org: Salmonella, Shigella, E. coli
Preferred media for testing: 5% sheep’s BAP

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Triple Sugar Iron Reaction Sulfide Indole Motility Test

Used to determine whether a Gram-negative rod
utilizes glucose and lactose or sucrose fermentation
and forms hydrogen sulfide
Lactose:Sucrose:Glucose Ratio 10:10:1
pH Indicator: phenol Red
H2S indicators: Na thiosulfate + ferrous sulfate
Result:
a. No Fermentation
Alkaline slant/Alkaline butt (K/K)
Organisms with this reaction are not
Enterobacteriaceae
b. No lactose fermentation, glucose
fermentation
Alkaline slant/Acid butt (K/A): 18-24 hours of
incubation (final reaction/reading)
c. Lactose, sucrose, glucose fermentation
Acid slant/Acid butt: 18-24 hours of
incubation
The Enterobacteriaceae attack the simple
sugar (glucose) first and then the lactose and
sucrose
d. Hydrogen sulfide production
Indicators: Na thiosulfate + ferrous sulfate
(+) result: black ppt
The production of H2S requires an acidic
environment
H2S producers: Citrobacter, Proteus, Salmonella.
Edwardsiella
e. Gas producers
f. (+) result: formation of bubbles (Co2 and H2)
or splitting of the media in the butt or
complete displacement of the media in the
butt or complete displacement of the media
from the bottom of the tube
Gas producers:
(++) Klebsiella and Enterobacter
(+) E. coli, Edwardsiella, Citrobacter, Hafnia,
Morganella, P. mirabilis, Salmonella and Serratia
(+) Sulfide: Black Color
(+) indole: pink to wine colored ring after addition of
Kovac’s reagent
Organisms: E. coli, Edwardsiella, C. koseri, K. oxytoca,
K. ornithinolytica, P. vulgaris. Providencia, Morganella
(+) motility: spread out/movement away from the
stab line/hazy appearance
Citrate Utilization
Determines the ability of an organism to utilize
sodium citrate as its only carbon source and inorganic
ammonium salts as its only nitrogen source
(+) from green to blue colored slant
pH indicator: bromthymol blue
Organisms:
Citrate (+) Klebsiella and Enterobacter
Citrate (-) Escherichia and Edwardsiella
Lysine Iron Agar Test
Important on differentiating Salmonella (+) from
Citrobacter (-)
Helpful in differentiating Proteus, Morganella and
Providencia: this group of enterics deaminate amina
acids (LIA +)
pH indicator: bromcresol purple
LIA reactions
a. Alkaline slant/Alkaline butt (K/K)
(-) Lysine deamination
(+) Lysine decarboxylation
b. Alkaline slant/Acid butt (K/A)
(-) Lysine deamination
(-) Lysine decarboxylation
c. Red slant/Acid butt (R/A)
(+) Lysine deamination
(-) Lysine decarboxylation
REMEMBER:
1. If the organism produces lysine
decarboxylase, cadaverine (purple) is formed

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2. If the decarboxylase is not produced, the butt
remains acidic (yellow) K/A
3. If oxidative deamination of lysine occurs, it
will form burgundy (red) color on the slant
4. If deamination does not occur, the LIA slant
remains purple
Decarboxylase Test (Moeller’s Method)
Measure the enzymatic ability of an organism to
decarboxylate (hydrolyze) an amino acid to form an
amine (putrescine or cadaverine)
It is useful in the differentiation of Klebsiella and
Enterobacter
(+) result: alkaline purple color
pH indicator: crescol red and bromcrescol purple
Reactions
Lysine ----Ld---> cadaverine +CO2
Ornithine ----OD----> putrescine
Arginine ----AD---> citrulline
Orthonitrophenyl B-Galactopyranoside (ONPG)
Determines whether the organism is a slow or LLF or
true NLF
B-galactopyranoside acts on the ONPG (a colorless
compound)
Salmonella arizona is the only ONPG positive
Salmonella serotype
Reaction:
ONPG ---B galactosidase→ galactose + Phenylpyruvic acid (end product) is detected by
orthonitrophenyl
(+) result: yellow color (orthonitrophenyl) within 20
minutes
If the organism is a NLF, the compound remain
colorless
MRVP (Methyl Red Voges-Proskauer)Test
To determine the ability of an organism to produce
and maintain stable acid end products (pyruvic acid)
from glucose fermentation
VP reaction: 40% KOH and alpha naphthol
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Glucose Metabolic End Products
Methyl Red test
Add 5 or 6 drops of methyl red per 5 mL broth
(+) bright red color indicative of mixed acid
fermentation (E. coli)
(-) yellow color (Klebsiella and Enterobacter)
Voges-Proskauer Test
Add 6 drops of a-naphthol and 2 drops of 40% KOH to
1 mL broth, expose to O2 and stand for 10-15 mins
(+) Red (pink-red) color at the surface of the medium
(Klebsiella and Enterobacter)
(-) Yellow color (copper like) at the surface of the
medium (E. coli)
(+) bright red color: MR test (E.coli) at pH 4.4 or less
(+) red color: VP/Barritt’s method
Yellow color: alkaline medium
Nitrate Reduction
Reagent: sulfanilic acid + alpha-naphthylamine
Zinc dust is used to confirm a negative reaction
(+) result: red, water-soluble azo dye (E. coli)
Phenylalanine Deaminase
Differentiation of Proteus, Morganella, and
Providencia (only PAD+) from the rest of
Enterobacteriaceae
(+): green colored complex on slant
Phenylpyruvic acid + FeCl3 produces green color
adding few drops of 10% ferric chloride
Urea Hydrolysis Test (Christensen’s Method)
Reagent: urea disk dissolve in 1 mL distilled water
(+) result: change in the color of slant from orange to
magenta
Proteus and Morganella: rapid urease producers
K. pneumoniae: slant turns red
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IMViC (Indole Motility Voges-Proskauer Citrate)
Reaction
E. coli, E. tarda, M. morganii = ++--
K. pneumoniae, Enterobacter, Serratia = --++
Salmonella = -+-+
Krigler Iron Agar (KIA)/Double Sugar Iron Agar
It contains tenfold (like TSI) more lactose than
glucose, preptone, phenol red,. Sodium thiosulfate,
and ferrous ammonium sulfate
Heat-labile, coenzyme I (found in the blood
secreted by certain organisms)
- These both are paper disks
- Used for the differentiation of Haemophilus
species, including Aggregatibacter
aphrophilus
Human Pathogens
H. influenzae, H. parainfluenzae, H. haemolyticus, H.
parahaemolyticus, H. paraphrohaemolyticus, H.
pittmaniae, H. aegyptius, and H. ducreyi
- H. segnis was renamed Aggregatibacter segnis
Two other former members of the genus
Haemophilus
- H. aphrophilus and H. paraphrophilus were
also moved into the genus Aggregatibacter

Laboratory Diagnosis
1. Gram stain
Microscopic
- Small
- Gram negative
- Pale pink coccobacilli to long filaments
(pleomorphic)
It can resemble the amorphous serous material
because of its pleomorphic appearance
Fastidious Gram-Negative Bacilli H. ducreyi: School of fish, railroad tracks or
fingerprints
Haemophilus Spp.
Blood-lovers
Obligate parasites of the mucous membranes of
humans except H. ducreyi.
Pleomorphic, Capnophilic, Facultatively anaerobic
bacteria
Oxidase and catalase positive
Very susceptible to drying and extreme temperatures
Haemophilus require the X & V Factor found in the 2. Culture
blood Media
X-factor: hemin (heat-stable substances) - CAP
V-factor: nicotinamide adenine dinucleotide - NAD

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- CAP w/ Bacitracin (selective medium); inhibits

the growth of the normal flora
- CAP with 1% IsoVitaleX/ Vitox promote the
growth of (H. aegyptius)
- Nairobi Plate promotes the growth of (H.
X & V factor
ducreyi)
Satellitism
Blood Agar Plate is NOT used as a culture medium for
Phenomenon in which bacterial species grow more
Haemophilus spp. Hemin (X-Factor) is bound to red
vigorously in the immediate vicinity of colonies of
blood cells.
other unrelated species, owing to the production of
Chocolate Agar Plate is used because X-factor is into
an essential metabolite by the latter species.
the medium when red blood cells are broken up.
- Staphylococcus aureus produce NAD (nicotinamide
Horse blood is preferred rather than sheep’s blood. adenine dinucleotide) as metabolic by-product
- Sheep’s Blood contain enzymes that - Haemophilus spp. May grow on sheep blood agar
deactivate NAD very close to the colonies of Staphylococcus aureus
H. aegyptius requires 4 days of incubation while H.
ducreyi requires 7 days.
Most Haemophilus strains require X and/or V factors
in the culture medium.
X and V Factor Requirements
Traditional approach
Strips or disk
Porphyrin Test (Delta-aminolevulinic Acid Test)
Based on the ability of the enzyme to convert the
substrate delta-aminolevulinic acid into porphyrins or
porphobilinogen
- Haemophilus species that need x-factor are
unable to synthesize porphyrin from δ-ALA
*check on the diffusion of the substance to determine what
Reagent: Kovac’s reagent (PDAB)
factor is required & how to identify the organism.
End products: porphobilinogen - red color
Porphyrins - reddish-orange color (UVL 300nm)
(+) result: red color : H. parainfluenzae, H.
parahaemolyticus, H. paraphrophilus
(-) result: H. influenzae, H. hemolyticus, H.
aegypticus, H. ducreyi

Haemophilus influenzae
May be mistaken for S. pyogenes if the Gram stain is
not well decolorized.
Commonly tested for ꞵ-lactamase production.
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Main cause of meningitis in children Haemophilus ducreyi

- Main cause of meningitis in adults:
Streptococcus pneumoniae (pneumococcus).
Transmission: Person to person by contaminated
respiratory droplets
Culture
Mousy or bleach-like odor
Not part of the normal human flora
Sexually transmitted infection; high chance of
infecting STD/AIDS
Agent of chancroid or “soft chancre”
A highly communicable sexually transmitted genital
ulcer disease (GUD).

Virulence Factors HACEK Group

Capsule (most significant factor)
Haemophilus spp - Aggregatibacter aphrophilus
Serogroups: A, B, C, D, E, F
Aggregatibacter actinomycetemcomitans
Most invasive: Serotype B
Cardiobacterium hominis
- Polymer composed of ribose, ribitol, and
Eikenella corodens
phosphate(polyribitol phosphate)
Kingella spp. (K. kingae - HACEK spp.)
Not all strains of H. influenzae are encapsulated.
Part of the normal flora; opportunistic pathogens
These strains are commonly referred to as nontypable
SmallSmall non-motile, will not grow on MAC
H. influenzae (NTHi).
Fastidious and require increased CO2.
Not all strains are encapsulated - non encapsulated
They have slow growth dysoginic on BAP and CAP
strains are part of the normal microbiota of the URT.
(7-14 days) -require an additional 1 to 2 days before
Do not produce endotoxin; rapidly killed by
they can be isolated from blood cultures
phagocytes; very fastidious
They caused slow, progressive bacterial endocarditis
Immunoglobulin A (IgA) proteases (vegetation)
Fimbriae: Adherence They utilize δ-aminolevulinic acid; all are indole (-)
Outer membrane proteins and lipopolysaccharide except C. hominis
(LPS)
Aggregatibacter aphrophilus (foam loving)
Haemophilus aegyptius Most prevalent HACEK species causing endocarditis.
AKA Koch-Weeks bacillus It is isolated from dental plaque and gingival
Genetically related to H. influenzae scrapings
Observed in conjunctivitis exudes from Egyptians by V-factor dependent
Koch in 1883.
It is the etiologic agent of pinkeye conjunctivitis.

Aggregatibacter actinomycetemcomitans
Haemophilus influenzae biogroup aegyptius Recognized etiologic agent in development of
Non encapsulated periodontitis
It causes conjunctivitis primarily in pediatric
populations

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Culture May grow on gonococcal media (TMA) and may

Star shaped w/ 4-6 points in the center of colonies resemble N. gonorrhea if the isolate does not pit the
after 48 hours. agar (as many strains do)
The additions of serum into the medium is necessary Species:
to demonstrate carbohydrate - K.kingae (most pathogenic)
Urease (-) = which differentiates it from - K. oralis
Actinobacillus spp. - K. denitrificans

Cardiobacterium hominis Capnocytophaga Spp.

It infects the aortic valve more frequently than other Family Flavobacteriaceae
HACEK organisms Includes bacteria previously called DF-1 and DF-2
It shows false Gram-positive reactions in parts of the (dysgonic fermenters)
cells Fastidious, facultatively anaerobic, gram-negative
bacilli and require increased CO2 for growth and
Microscopy
isolation from blood cultures.
Rosette formation, stick-like structures - yeast
Thin and often fusiform
extract
Motility: Gliding motility.

Pasteurella Spp.
Zoonotic bacteria
- The etiologic agent of shipping fever in cattle.
Culture - A commensal in the URT of fowl and
Pitting may be seen mammals
It is isolated from animal bite (felines) or scratch
Eikenella corrodens wounds.
AKA Corroding bacilli Motility: Nonmotile
The least common isolate of the HACEK group in
Microscopy
adult infectious endocarditis
Safety pin appearance (bipolar staining)
Asaccharolytic
Causes: mixed infection (bites or clench-fist wounds) Biochemical Test
cellulitis (users of abused drugs) - Oxidase (+)
- Catalase (+)
Culture
- Indole (+) weak glucose fermenter
Pit or corrode the agar with sharp odor of bleach
Grows well on BAP and CAP but not Mac Conkey Agar

Virulence factors
Endotoxin and capsule
Kingella Spp.
They have tendency to resist decolorization

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Pasterurella multocida
It is the most frequently isolated species
It has characteristic “mushroom smell”
It grows well on BAP
Susceptibility: Penicillin
Laboratory Diagnosis
Brucella Spp. Specimens: Blood, bone marrow, tissues
AKA Bang’s Bacillus Should be handled under BSC level 3 due to aerosol
Important human and animal pathogens- infect mode of transmission.
human through contact with infected animals and B. abortus requires niacin (nicotinic acid) for growth;
animal products (milk) inhibited by thionine dye
Category B select biological agent. Isolates can be recovered after 7 days, but may
Strict aerobe; intracellular parasites. require prolonged incubation up to 30 days (culture
Motility: Nonmotile bottles may not become turbid).
Localized in tissues rich in erythritol (ex. Placental
tissue). Francisella tularensis
- Induce spontaneous abortion among animals. Category A select biologic
Recovered from blood and bone marrow. Potential bioterrorism weapon.
Very small, obligate aerobic, coccobacillus
Microscopy
Mobility: Nonmotile
Sand Appearance
Facultative intracellular parasite
Culture
Microscopy
Colonies may become brownish with age
Faint, bipolar staining
Biphasic medium is used.
Culture
Biochemical Test
Blue-gray to white, slightly mucoid colonies
(+) Catalase
(+) Oxidase Growth Factors
Rapid urease producers Cysteine/cystine, thiosulfate
Asaccharolytic
Biochemical test
Disease Catalase weakly (+)
Malta/Crimean/Mediterranean fever Oxidase (-)
Undulant fever (brucellosis) Citrulline uridase (+)
Glycerol fermenter
Species
B. abortus, B. canis, B. suis, B. melitensis (common Virulence factor
isolate) Capsule
Most virulent species: B. melitensis and B. suis

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Reservoir - Can tolerate up to 3 mg/L of chlorine; resits

Cottontail rabbit water treatment.

Microscopy
Mode of Acquisition Faintly staining and thin Gram negative bacilli
- Handling infected animal carcasses or skin
infected animals
Culture
- Through insect vector (deer flies and ticks)
Colonies appear iridescent with sticky consistency
- Being bitten by carnivores
It will not grow on routine media BAP
- Inhalation
Requires medium supplemented with L-cysteine
Diseases buffered to pH 6.9 (BCYE)
Tularemia
Zoonotic disease Legionella pneumophila
Acquired through ingestion, inhalation, arthropod Most commonly isolated human pathogen in the
bite, or contact with infected tissues. genus Legionella.
It is isolated from air conditioning ducts, cooling
Laboratory diagnosis
towers, warm water plumbing system, humidifiers,
Specimen
nebulizers(man-made facilities), ponds and creeks
Scrapings from infected ulcers, lymph node biopsies
- It has the ability to invade the
It will not grow on MAC.
bronchoalveolar macrophages producing
Other Diseases localised tissue destruction through export of
- Deer Fly fever a cytotoxic exoprotease
- Rabbit fever Colony morphology: “Ground glass” appearance
- Lemming fever (BCYE)
- Water rat trappera’s disease
Disease
Legionella Spp. Legionnaires Disease
The only genus in the family Legionellaceae AKA legionellosis
It is primarily acquired through inhalation Febrile and pneumonic illness
From the air conditioner. Pontiac fever
It can infect and multiply within some free-living Nonfatal respiratory infection
amoebae Resembles an allergic disease; pneumonia does not
Fastidious, aerobic and motile occur
Infect and multiply with some free-living amoebae
Laboratory Diagnosis
- Capable of surviving at extreme ranges of
Preferred specimens: sputum and bronchoalveolar
environmental conditions for long periods.
lavage
- Has the ability to adhere to pipes (even when
Culture is the most important test for Legionnaire’s
flushed), rubber, plastics, and sediments
disease.

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Selective medium: BYCE with L-cystine, ferric salt and Contains a protective antigen but when combined
alpha-ketoglutarate with antibody, it abolishes its infectivity.
Acid treatment (KCl-HCl) of contaminated specimens
Culture
enhances isolation of the bacteria
Mercury drops appearance
Immunofluorescence Assay (IFA): The most common
method used for serologic diagnosis of Legionnaires Growth inhibitors
- Fatty acids
Bordetella Spp. - Metal ions
Are obligate aerobic, coccobacilli; non-carbohydrate - Sulfides
fermenter - Peroxides
Motility: Nonmotile except(B. bronchiseptica) and
with bipolar metachromatic granules. Virulence factors
Replicate on ciliated respiratory epithelial cells of - Pertussis toxin
humans. - Pertactin
- Fimbriae Specimen
Culture - Nasopharyngeal swab
Smooth, glistening, silver in color becoming
whitish-gray with age Diseases
Whooping Cough
Biochemical test AKA Pertussis
Catalase (+) Highly contagious, acute infection of (URT); disease
Oxidase (+): B. bronchiseptica and B. avium of the children.
The rest are oxidase negative Acquired through the respiratory tract via inhalation
Indole (-) via the aerosol route (inhalation of the bacterium)
Are most inactivated in biochemical test systems
Laboratory Diagnosis
Growth Factors Specimen: B. pertussis
- Nicotinic acid - Nasopharyngeal swabs (Calcium alginate or
- Cysteine Dacron swab) and bronchoalveolar lavage
- Methionine Enhance visibility: 2-minute safranin or 0.2% basic
fuchsin as counterstain
Species
- B. pertussis Transport and enrichment medium
- B. parapertussis - Regan Lowe agar
- B. bronchiseptica - Bordet-Gengou potato infusion agar
- B. Avium - Modified Jones-Kendrick charcoal
Other Bordetella species are less fastidious and will
Bordetella pertussis grow on MAC or media containing blood (ex. B.
AKA Bordet Gengou bacillus bronchiseptica)
It is the etiologic agent of whooping cough

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Transport System Two species of Salmonella

Casamino broth: if the transmit time is less than 2 hrs 1. Salmonella enterica
the swab can be placed into a solution of 1% casein 2. Salmonella bongori
hydrolysate. 6 Subspecies of S. Enterica
Amies transport medium with charcoal: Appropriate 1. S. enterica ssp. enterica (I)
up to 24 hours. 2. S. enterica ssp. salamae (II)
3. S. enterica ssp. arizonae (IIIa)
4. S. enterica ssp. diarizonae (IIIb)
5. S. enterica ssp. houtenae (IV)
6. S. enterica ssp. indica (VI)

Gastrointestinal pathogens
(SSY) Never considered as normal intestinal flora K antigen: capsular
Salmonella, Shigella, Yersinia - Salmonella typhi has the K antigen.
Serotype is based on Somatic, flagellar antigen
Salmonella
Virulence factor: Fimbriae and enterotoxins
The most serious pathogenic enterobacteria for
Main etiologic agent of enteric fever
humans, causing enteric fever (typhoid fever) and
- Salmonellae organisms infect various animals that
acute gastroenteritis (food poisoning)
serve as reservoirs, and source of infections,
Acquisition:
except S. Typhi and S. Paratyphi (human carriers).
- Ingestion of contaminated animal food products
or ingestion of improperly cooked Biochemical characteristics
poultry/milk/eggs/dairy products All species are motile except S.pullorum and S.
- “Human carriers” Gallinarum
Typhoid Mary: spread Salmonella by giving free food All produce gas except S. gallinarum and S. typhi
All produce H2S except S. paratyphi A
Culture
TSI: K/A, (+) gas, (+)H2S
MAC: Clear, colorless colonies
IMViC Reaction: ‒ + ‒ +
- Non-lactose fermenter
HEA, BSA, and XLD: colored colonies with black S. typhi
centers TSI: K/A, (-) gas, (+) H2S
SSA: Colorless colonies with black centers 3 General Categories of Salmonella Infection
- Hydrogen sulfide formation Gastroenteritis
One of the most common forms of “food poisoning”
Salmonella strains associated with this infection are
found in animals, mostly S. enterica subsp. enterica.
- Sources of Infection: Poultry, milk, eggs, and egg
products
- Infective Dose: 10^6

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Salmonella that infected the Arrowhead Mills Species

peanut butter is Salmonella enterica serovar - S. dysenteriae (most virulent) - A
- S. flexneri (gay bowel syndrome) - B
Enteric Fever
- S. boydii - C
AKA “Typhoid Fever”
- S. sonnei - D
Caused by S. Typhi
- Febrile disease that results from the ingestion of
S. dysenteriae
contaminated food originated from the infected
Most virulent
individuals/carriers.
Antigenic structures: Somatic O
- Characteristic “rose spots” appear during the 2nd
Specimen: rectal swab
week of fever.
IMViC: V + - -
- Gallbladder: Site of long-term carriage of S. typhi
TSI reaction: K/A, (-) gas, (-) H2S
Complications: Necrosis in gall bladder & Peyer’s
Patches in intestines Shigella sonnei
Specimens for Salmonella identification Is unique in its ability to decarboxylate ornithine
1. Blood: 1st week - Late Lactose Fermenter
2. Stool: 2nd week - ONPG (+)
3. Urine: 3rd week Bacillary dysentery
It is most caused by S. dysenteriae type 1
Bacteremia
Clinical Feature: acute inflammatory colitis and
Characterized by primarily prolonged fever and
bloody diarrhea
intermittent bacteremia.
- It is highly communicable because of low
Serotypes most commonly associated are
infective dose (<200 bacilli)
Typhimurium, Paratyphi andhasfml.sahfo;shg’
- In young children, rectal prolapse occurs due to
Shigella excessive straining
Closely related to the genus Escherichia Source of Infection: Human carrier
Not a member of the GI normal flora
Yersinia pestis
Citrobacter and Shigella have the same serotype?
AKA “Plague bacillus”
- Nonmotile; NLF
Class A Bioterrorism Agent
- An intracellular organism that multiply within
- Nonmotile
the cells of the colon epithelium
- The only Enterobacteriaceae transmitted through
Transmission: Four Fs and water (fecal oral)
vector (Bite of infected flea: Xenopsylla cheopis)
Reservoir: human
Virulence Factor: Shiga toxin Three forms of Plague
1. Bubonic Plague
2. Pneumonic Plague
Culture 3. Septicaemic Plague
MAC: Clear, fragile, NLF colonies
It is the causative agent of bubonic plague, black
SSA: Colorless w/out black center
death or 6th century pandemic

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Virulence factors Mode of acquisition

- Endotoxin Consumption of incompletely cooked food (pork and
- Coagulase pork intestines and vacuum packed meat)
- Fibrinolysin Yersinia Yersinia pestis
Microscopy enterocolitica
Short, plump rod with “bipolar staining or closed
Indole V -
safety pin appearance”
- Visible using Wayson or methylene blue stain Methyl Red (MR) + +

VP -25°C V -

Motility

25°C + -

Culture 37°C - -
Pinpoint colonies: grows best at 25-30°C
Christens Urea + -
Stalactite pattern: broth cultures
OD + -
Yersinia enterocolitica
It is the most commonly isolated spp of Yersinia of
blood components Lab diagnosis
Causative agent of Enterocolitis-waterborne Specimen: stool, rectal swab, blood, urine
gastroenteritis. Members of Enterobacteriaceae are routinely
- Motile at 22°C but not at 35°C isolated from stool cultures; therefore identification
- Requires cold enrichment technique (4°C) should only be directed toward true intestinal
using phosphate buffered saline. pathogens
- Direct smears of stool specimens are not
Microscopy
helpful in the identification because it is rich
Coccobacilli with bipolar staining
in normal flora
Culture
CIN medium: bulls eye colonies
- Grows on routine culture media at 25-30°C

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Non Enteric Gastrointestinal Pathogen Epidemic V. cholerae 01 Biogroups

1. Classical: VP (-) Do not agglutenate chicken RBC,
Vibrio (Comma/Curved bacillus)
susceptible to polymyxin B (50ug)
Not part of the human flora
2. El tor: VP (+); agglutinate chicken RBC, resistant to
Found in brackish or estuarine water, and marine or
polymyxin B (50 uG)
saltwater
Can be isolated from algae, plankton, fish, and Cholera
shellfish It is an acute diarrheal disease that is spread mainly
Monotrichous through contaminated water
Halophilic (require NaCl for growth) except V. Hallmark of cholera: Rice-watery stool (10-30x of
cholerae and V. mimicus defecation/day)
Microscopy: gram (-) short and curved rod All antigens are poorly immunogenic, so repeat
Culture: NLF - MacConkey Agar infection occur
Biochemical Tests: Oxidase (+) and reduce nitrates to
nitrite (except V. metschnikovii) Biochemical Test
Glucose Fermenter (NLF except V. vulnificus) TSI reaction: A/A, (-) gas, (-) H2S
Indole (+)
Vibrio Cholerae Citrate (+)
LIA: Alkaline/alkaline (K/K)
Causative agent of cholera/asiatic cholera/epidemic
TCBS: Yellow colonies
cholera
Motility: Rapid darting or shooting star motility
Single flagellum covered with Lipopolysaccharide
Vibrio parahemolyticus
sheath The second most common Vibrio spp implicated
Has caused cholera epidemics (O1 and O139 strains) gastroenteritis
and seven pandemics (O1 strains) Etiologic agent of “summer diarrhea” in Japan
Potent enterotoxins: cholera toxin (CT), zot, and ace Acquisition: eating contaminated seafoods and
toxin sardines
Virulence factors: Choleragen (cholera toxin) Virulence factor: Heat-stable hemolysin
String test: (+) mucoid stringing reaction when using Pathogenicity: Kanagawa phenomenon (hemolysin
0.5% sodium deoxycholate lyses human RBCs)
Antigenic Structures: somatic O and Flagellar H (V. Selective Medium: Wagatsuma Agar (high salt
cholerae subgroups) mannitol medium)

V. cholerae subgroups: Vibrio vulnificus

V. cholerae O1V, cholerae O139, V. cholerae non-O1 Was commonly referred to as the lactose-positive
V. cholera 01 serotypes: Ogawa (A, B), Inaba (A, C), Vibrio
and Hikojima (A, B, C) Acquisition: Eating raw oysters/fish
Serological test: the best to differentiate serotypes

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Vibrio alginolyticus Aeromonas

The least pathogenic Vibrio for humans, not Found in freshwater, estuarine and chlorinated
commonly isolated water; isolated from meat products
Strict halophile: 1%-10% NaCl Not part of human flora
An occupational hazard (fishermen and sailors) - Causative agent of “red leg” disease (frogs)
- In humans it causes a nebulous syndrome
Laboratory Diagnosis
known as “Traveler’s diarrhea” similar to ETEc
Specimen: stool, rectal swab, pus, and tissue
Microscopy: Gram (-) straight rods
Cary-Blair medium: (maintains viability) media for
Culture: Bull’s eye colony - CIN )with 4 ug cefsulodin):
Vibrio species when collected and transported
LF: (Mac Conkey: A. caviae)
Culture Media: TCBS, alkaline peptone water,
Biochemical Tests: Oxidase (+), motile with single
Cary-Blair, MAC, BAP
polar flagellum (but some are nonmotile)
Growth of vibrios require media containing ).5% NaCl
Will grow in media with 0% NaCl but not in 6%
(except V. cholerae and V. mimicus)
Most common isolate: A. caviae
V. alginolyticus tolerates up to 10% NaCl
Vibriostatic O1/29 Test: resistant (aeromonas and
Alkaline peptone water with 1% NaCl (pH 8.5) can ve
plesiomonas)
inoculated at least 20 mL and incubated for 5 to 8
Inositol fermentation: (+) (Plesiomonads are
hours at 35°C before subculturing to TCBS
negative)
TCBS: Yellow colonies (Sucrose fermenters)
TSI: A. Caviae: A/A, (-) gas, (-) H2S
V. cholerae, V. alginolyticus, V. metschnikovii
A. Hydrophila & A. veronii: A/A, (+) gas, (+) H2S

Campylobacter jejuni
The most common cause of bacterial gastroenteritis
worldwide
Non Sucrose Fermenters (Green Acquired from eating contaminated chicken/turkey
colonies on TCBS) Secrets a toxin antigenically similar to cholera toxin
V. mimicus, V. vulnificus, V. (may be misdiagnosed as Vibrio)
parahaemolyticus, and V. damsela Slow growing, fastidious, asaccharolytic and
String Test: 0.5% Sodium microserophilic
deoxycholate Motility: Darting motility (single polar flagellum);
Differentiates Vibrio spp from Aeromonas spp. unable to grow in 3.5% NaCl
(+) result: lysis (vibrio) releases DNA which can then Optimum growth: 42°C
be pulled into a string (viscous string) using an Microscopy: curved, seagull-winged shaped;
inoculation loop S-shaped colony

Vibriostatic Test (Susceptibility Test) Laboratory Diagnosis

Used to separate vibrios (susceptible) from other Specimen: feces, rectal wav (less performed) and
oxidase-positive, glucose fermenters like aeromonas blood

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Blood culture: 2 weeks incubation may be needed for

detection; blind cultures may be necessary
Culture: gray, flat, glistening, irregular with a tailing
effect along the streak line or runny spreading
Selective media: Campy-BAP, Butzler agar and
Skirro’s media and charcoal cefoperazone
desoxycholate agar

Helicobacter pylori
Motility: Monopolar or multi-bipolar flagella
Microaerophilic
The most common cause of type B gastritis, peptic
ulcer and gastric carcinoma
Binds to Lewis antigen (part of the blood group
antigens) and to monosaccharide sialic acid
Microscopy: Gram (-) spiral-shaped organisms
(S-shaped) rods resembling Campylobacter
Transmission: Oral-oral and fecal-oral routes
Biochemical test: Oxidase and urease (+) (urease
may neutralize acidity of GI tract)
Primary habitat: human gastric mucosa

Laboratory Diagnosis
Specimens: tissue biopsy material (Staurt’s medium),
urine (ammonia testing), feces and dental plaque
Stains for biopsy specimens: Warthin-starry, silver
stain, or giemsa stain
Culture media: CAP, MTM, Skirrow agar, 5% sheep’s
Blood Brucella agar
Urea Breath Test: Excellent sensitivity/specificity

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Anaerobic Organism ● Superoxide anion + Hydrogen Peroxide +

Fe3+/Fe2+ 🡪 Hydroxyl Radical (OH-)
● Hydroxyl Radical
AEROBES
● the most potent biologic oxidant known.
CAPNOPHILIC ● Damaging to the cells particularly protein and
- grow best when the concentration of carbon nucleic acid
dioxide is increased to 5% to 10% in a CO2
Example: Capnocytophaga spp WHY ORGANISM IS ANAEROBES?
● They do not contain the two enzyme that
MICROAEROPHILIC protects the bacteria to superoxide anions
- require the oxygen concentration to be and toxic derivatives
reduced to 5% or less. ● Superoxide dismuthase
Example: Campylobacter spp ● Catalase

FACULTATIVE ANAEROBES
- preferentially use oxygen as an electron
acceptor if it is available but can grow well in
the absence of oxygen
Example: Enterobacteriaceae
OBLIGATE AEROBE
- require oxygen for metabolism, and they can
grow well in an ambient air incubator
ANAEROBES
OBLIGATE ANAEROBES
- Killed in the presence of Oxygen
AEROTOLERANT OR MODERATE ANAEROBES
- can survive some oxygen exposure but will
not be able to perform metabolic processes
unless placed into an anaerobic environment.
Example: Bacteroides fragilis
FREQUENTLY ISOLATED ANAEROBES SPORE
FORMING
CLOSTRIDIUM SPP.
● Obligate anaerobes, catalase negative spore
forming Gram positive bacilli
● Exogenous anaerobic infection
● Identify by where the spores is located
Terminal: C. tetani
Subterminal: C. sordellii
● Motile with peritrichous flagella except: C.
perfringens
● Have swollen sporangia except: C. perfringens
and C. bifermentans
● Single hemolytic reaction except: C.
perfringens
● Carbohydrates fermentation except: C. tetani
and C. bistolyticum

CLOSTRIDIUM PERFRINGENS (GANGRENE BACILLUS)

● associated with two types of food poisoning
WHY SOME ORGANISM ARE ANAEROBES? ● type A, a relatively mild and self-limited GI
● Toxicity of the Oxygen illness

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● type C, a more serious but rarely seen disease

and can cause enteritis necroticans
● Virulence factor: alpha toxin and enterotoxin
Microscopy: Boxcar shaped bacilli (subterminal
spores)
• Infections
• Gas gangrene (Myonecrosis)
• Enteritis necroticans

GAS GANGRENE
● LECITHINASE (Phospholipase C) the alpha BIOCHEMICAL TEST
toxin of C. perfrigens ● Lecithinase (+) using Egg Yolk Agar (EYA)
● Clinical Manifestation: Bullae (fluid-filled ● Naegler Test (+): Lecithovitellin on EYA
blisters), serous discharge, discoloration, and ● Reverse CAMP test (+): Arrow head shaped
tissue necrosis are observed towards the test organism
● Other organism: C. histolyticum, C. septicum, NAEGLER TEST
C. novyi, and C. bifermentans
DIAGNOSTICS
● Culture
● Blood Agar plate: Dome shaped and grayish
white with double zone of hemolysis
● Litmus milk
● Stormy fermentation of milk

DOUBLE ZONE HEMOLYSIS

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REVERSE CAMP TEST ● Microscopically: Drumstick or tennis racket

appearance
LAB DIAGNOSIS
● BLOOD AGAR PLATE: matte surface with
narrow zone of Beta hemolysis
● Gelatinase and indole (+)
● Lecithinase and Lipase (-)

CLOSTRIDIUM DIFFICILE
● Most common cause of antibiotic associated
and pseudomembranous colitis.
● Produce two toxin:
● Toxin A: Enterotoxin; Toxin B: cytotoxin

LABORATORY DIAGNOSIS
● Ferment fructose
CLOSTRIDIUM BOTULINUM “CANNED FOODS ● Cycloserine cefoxitin fructose agar (CCFA)
BACILLUS” ● Exhibit yellow color and ground glass
● Found in soil and acquatic sediments appearance
● Presence of subterminal spores ● Blood Agar plate
● Virulence factor: botulism toxin ● “Horse stable odor”
● extremely potent neurotoxin; it takes only a ● Non hemolytic
small amount to produce paralysis and death ● Fluorescent chartreuse in UV light

BOTULISM
GRAM-POSITIVE, NON–SPORE-FORMING
● FOOD BORNE
● cause by botulism A ANAEROBIC BACILLI
● INFANT BOTULISM ACTINOMYCES ISRAELII
● Ingesting organism thru honey or breast ● Microbiota of oral cavity
feeding ● Common cause of Actinomycosis
CLOSTRIDIUM TETANI ● a chronic, granulomatous, infectious disease
● Tack head bacillus characterized by the development of sinus
● Found in soil tracts and fistulae, which erupt to the surface
● Virulence factor: Tetanospasmin (neurotoxin) and drain pus that may contain so-called
● acts on inhibitory neurons, preventing the sulfur granules
release of neurotransmitters ● Seen in Maxillary region, neck, thorax, female
● trismus (lockjaw), risus sardonicus (distorted genital tract because of Intrauterine devices
grin), and difficulty breathing.

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LACTOBACILLUS SPP
• L. fermentum, L. vaginalis, L.
salivarius, L. Plantarum
• Lactobacillus acidophilus
● Comprises the largest portion of the vagina
● Produce Lactic acid from glycogen
● Bacterial vaginosis
● a shift in the vaginal biota occurs,
resulting in the overgrowth of other
endogenous anaerobes of the vagina
● Mobiluncus sppBacteroides spp.
● Prevotella spp., anaerobic
● gram-positive cocci, and the aerobic
bacteria Gardnerella vaginalis
● Treatment: Penicillin + Aminoglycoside
LABORATORY DIAGNOSIS
•Tomato juice agar
•Catalase, H2S, Esculin Hydrolysis (-)
ANAEROBIC GRAM-NEGATIVE BACILLI
BACTEROIDES FRAGILIS
● isolated anaerobes from blood cultures
● Beta lactamase producer
● Non Motile and saccharolytic
● Cause Intra abdominal abscesses
● Culture:
Bacteroides Bile Esculin Agar: Gray color
and growth in 20% bile, blackening of the
agar
Biochemical test
• Esculin hydrolysis positive
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PREVOTELLA, PORPHYROMONAS,
FUSOBACTERIUM
● Found in the oral cavity
● associated with mixed biota in diabetic foot
ulcers and decubitus pressure sores.
● Fusobacterium necrophorum
● Caused Lemierre disease
ANAEROBIC COCCI
• Gram Positive Anaerobic Cocci
• Peptostreptococcus
• Anaerococcus
• Pepostreptococcus
• Finegoldia
- F. magna
• Parvimonas
• Peptoniphilus
• Gram Negative Anaerobic Cocci
• Veilonella
- very small (0.3 to 0.5 µm in diameter) and
inhabit the oral cavity
• Mixed biota absceses
DIRECT EXAMINATION OF SPECIMEN
• Morphotypes might provide a presumptive
identification of organisms and serve as a
guide to media selection
• The Gram stain often reveals the presence of
leukocytes, indicating an inflammatory
response at the site of the infection.
• The Gram stain may also reveal the presence of
squamous epithelial cells that would
suggest mucosal surface contamination during
specimen collection.
• Gram stain can serve as a valuable quality control
tool.
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• Note: Chemically fixed (Methanol) GAS PAK JAR

• Pale in Safranin: To enhance the red color of Needs 30 – 45 minutes to obtain an anaerobic
gram-negative anaerobes, the use of 0.1% environment
basic fuchsin as the counterstain or extending the
counterstaining with safranin for 3 to 5
minutes is recommended

APPROPRIATE CULTURE MEDIA

• Anaerobes have special nutritional requirements
for vitamin K, hemin, and yeast
extract,
• CDC blood agar provides the best recovery

ANAEROBIC CULTIVATION
• Use special culture media with thioglycolate and
cysteine (reducing agents)
• Boiling is done to remove oxygen
• Use of Anaerobic chamber with vacuum pump and
nitrogen to remove the oxygen
• Gaspak must contain Palladium Catalyst
• Pouches and plastic bags contain Calcium
carbonate and Catalyst

ANAEROBIC CHAMBER
• Nitrogen gas
- Filler for anaerobic atmosphere

• Palladium Pellets
- Used to remove residual Oxygen
- Combine Oxygen with Hydrogen to form
Water

• Dessicant or silica gel

- Absorbed water

• Methylene blue or reazurin

- Oxygen reduction indicator
- Colorless in absence of O2
- Blue with Presence of O2

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