GRAM POSITIVE COCCI

1 FAMILY MICROCOCCACEAE

Genera
✓ Staphylococcus, Planococcus, Micrococcus and Stomatococcus
General Characteristics
✓ Catalase (+), non-motile, NSF, Glucose Fermenters
Differential tests between Staphylococci and Micrococci

TESTS Staphylococci Micrococci

Gram staining reaction In clusters, grapelike Tetrads, sarcinae
Growth on BAM Creamy white, pinhead Yellow colonies; non-hemolytic
colonies; Beta-hemolytic
Bacitracin/ Taxo-A Disk test Resistant Susceptible (>10 mm)
Furazolidone Susceptibility test Susceptible Resistant
Modified Oxidase test Negative Positive
Lysostaphin Sensitivity test Sensitive Resistant
Furoxone-Tween 80 Oil Red O agar No growth With Growth
Acid production from Glycerol Positive Negative
Oxygen Requirement Facultative anaerobic Obligate Aerobe
Glucose Utilization (OF) test Fermenter Oxidizer

Staphylococcus aureus

✓ Skin, wound and deep tissue infections: Gastroenteritis, TSS, SSS, Bacteremia, Sepsis, UTI, Endocarditis, Boils,
Furuncles, Folliculitis, Impetigo, Osteomyelitis
✓ Virulence Factors: Catalase, Coagulase, Hyaluronidase, Staphylokinase, Lipase, DNAse, Beta-Lactamase,
Enterotoxins, Leukocidin (PVL), Hemolysin, Exfoliatin, Pyogenic exotoxin and protein A.
Coagulase Negative Staphylococci (CoNS)
S. epidermidis: Contaminants of instruments for catheterization and prosthetic heart valve implantation>
bacterial endocarditis
S. saprophyticus: community-acquired UTI in sexually active females

Differential tests for Staphylococcus aureus

CATALASE TEST
Purpose Principle Result
Differentiate from Streptococcus (-) Release of water and Oxygen from (+): effervescence/ rapid bubble
Hydrogen Peroxide formation
Reagent: 3% H2O2 (-): absence of bubble formation
COAGULASE TEST
Distinguish Staphylococcus aureus Conversion of fibrinogen into fibrin Slide coagulase test:
from the S. epidermidis and S. 2 forms: (+): white fibrin clots in plasma
saprophyticus a. Bound Coagulase (-): smooth suspension
Clumping factor; slide test (If negative, confirm)
b. Free Coagulase
CRF; Tube coagulase test Tube Coagulase test:
Reagent: Rabbit Plasma (+): fibrin clot formation
(-): no clot formed

Mannitol Fermentation Test

Differentiate pathogenic from MSA (1% mannitol + 7.5% NaCl) (+): yellow color: S. aureus
nonpathogenic Staphylococci Phenol red as indicator (-): pink colonies: S. epidermidis
and S. saprophyticus
Differential Test for CoNS
Novobiocin Test
Differentiate the Coagulase Negative Reagent: 5 ug Novobiocin Susceptible: zone > 16 mm
Staphylococci S. epidermidis (16-27 mm)

Resistant: zone</= 16 mm
S. saprophyticus
 Classification:

1. Bergey’s: able to grow in different temperatures

a. Pyogenic: S. pyogenes; neither grow on 10°C/ 45°C, only @ 37°C, produce pus, mostly B-hemolytic
b. Viridans: S. mutans, S. salivarius, S. mitis, S. milleri, S. sanguis; (-) 10°C, (+) 35 and 37°C, alpha/non-
hemolytic
c. Enterococcus: E. faecalis; growth @ 10°C, 37°C and 45°C, normal flora of human intestine
d. Lactic group: S. lactis; (+) growth @ 10°C and 37°C, negative @45°C; non-hemolytic; found in dairy
products
2. Smith and Brown’s: based on hemolytic reactions
a. Alpha: S. pneumoniae, Viridans Strep: partial/ incomplete/green hemolysis/ discoloration
b. Beta: S. pyogenes, S. agalactiae; complete hemolysis/ clear area/zone
c. Gamma: E. faecalis (Group D); no lysis, non-hemolytic
d. Alpha Prime: S. zooepidemicus
3. Lancefield (Ag sero-grouping): based on the antigenic structure of cell wall carbohydrates- C polysaccharide

Lancefield Classification Species Hemolytic Reaction

GROUP A Streptococcus pyogenes Beta
GROUP B Streptococcus agalactiae Beta
GROUP C S. equisimilis Beta
S. zooepidemicus
S. equi
S. dysagalactiae
GROUP D Enterococci: Beta
E. faecalis
E.faecium
E. durans
E. avium

Non-enterococci
S. bovis
S. equinis
None S. pneumoniae Alpha
None Viridans Streptococci Alpha and Gamma

2 FAMILY STREPTOCOCCACEAE

GENERAL CHARACTERISTICS

✓ G (+) cocci in pairs/ in chains, non-motile, NSF, Catalase (-), Facultative anaerobes, Oxidase (-), CHO fermenters,
growth is enhanced by blood, serum, or glucose

GROUP A STREPTOCOCCI: STREPTOCOCCUS PYOGENES

✓ Pathogenic to man, acquired through droplets, “flesh-eating bacteria”
✓ Virulence Factors: M protein, Protein F, Lipoteichoic Acid
✓ Enzymes and Toxins produced: Hemolysins, Streptolysin O and S, Deoxyribonuclease, Streptokinase,
Hyaluronidase, Pyogenic toxins
✓ Infections and Diseases: Pharyngitis (Strep Throat), Scarlet fever, Cellulitis, Erysipelas, ARF, AGN, Necrotizing
fasciitis

Group B Streptococci: Streptococcus agalactiae

✓ NF of the female genital urinary tract, nosocomial transmitted, infects fetuses and infants
✓ Virulence factors: Capsule, hemolysin, CAMP factor, neuraminidase, DNAse, hyaluronidase and protease
✓ Infections and diseases: Pneumonia, meningitis, neonatal sepsis, post-partum infections, Osteomyelitis, UTI,
endocarditis

Group C Streptococci

✓ Animal pathogen, streptokinase

Group D Streptococci

✓ Infections and Diseases: Wound infections, UTI, Endocarditis, Septicemia

Viridans Streptococci: S. mutans, S. salivarius, S. mitis, S. constelatus

✓ Oropharyngeal commensals, opportunistic pathogens of low virulence

 ✓ Infections and Diseases: Subacute endocarditis, dental carries (S. mutans)

Streptococcus pneumoniae: Diplococcus/ Pneumococcus

✓ Lancet/ oval, bullet shaped bacilli

✓ Common isolate both as a pathogen and as member of the normal RT, NF (25-50%) of the URT of preschoolers
✓ Colonial characteristics: “dome shaped”, “coin with a raised rim”
✓ Diseases and infections: Bacterial lobar pneumonia, bacterial meningitis in adults, otitis media, bacteremia,
endocarditis
✓ Virulence factors: capsular polysaccharide, hemolysin, IgA protease, neuraminidase and hyaluronidase

Nutritionally Variant Streptococci (Abiotrophia and Granulicatella)

✓ Part of the human oral and GI microbiota

✓ Opportunistic pathogen of low virulence
✓ Infections and diseases: Endocarditis, Otitis Media, Endophthalmitis, Chronic sinusitis, Septic Arthritis,
bacteremia
✓ Need Cysteine or pyridoxal (Vitamin B6), thiol requiring streptococci

IDENTIFICATION OF BETA-HEMOLYTIC STREPTOCOCCI

BACITRACIN AND SULFAMETHOXAZOLE-TRIMETHOPRIM SUSCEPTIBILITY TEST

Group Bacitracin Susceptibility SXT Susceptibility
A (S. pyogenes) Susceptible Resistant
B (S. agalactiae) Resistant Susceptible
C Resistant Susceptible
D Enterococcus Resistant Resistant
D Non-Enterococcus Resistant Resistant
Christie-Atkins-Munch-Peterson (CAMP test)
Group Principle Result
A (S. pyogenes) “Arrowhead” hemolytic pattern Negative; no zone of hemolysis
B (S. agalactiae) results when the organism is Positive; “arrowhead hemolysis”
streaked perpendicular to B-
hemolytic S. aureus
Hippurate Hydrolysis Test
Group Principle Result
A (S. pyogenes) Hippuric acid is converted to glycine Negative; yellow pink or colorless
B (S. agalactiae) and benzoic acid in the presence of Positive; deep purple color
hippuricase. Ninhydrin added to
glycine produces a purple color.
Identification of Alpha Hemolytic Streptococci:

Tests Pneumococci Viridans Streptococci

Mouse Virulence Test Death w/in 16-24 hours NEGATIVE
Inulin Fermentation POSITIVE NEGATIVE
Bile Solubility SOLUBLE, colony disintegrates INSOLUBLE; Intact colonies
Optochin (Taxo P) test: Susceptible: zone of inhibition at RESISTANT; growth up to the disk,
Ethylhydrocupreine hydrochloride least 14 mm (10ug disk); 10 mm zone of inhibition <14 mm; <10 mm
(6ug disk)
Neufeld-Quellung Reaction Positive NEGATIVE
IDENTIFICATION OF GROUP D ENTEROCOCCUS FROM GROUP D NON-ENTEROCOCCUS

BILE ESCULIN TEST SALT TOLERANCE TEST PENICILLIN PYRROLIDONYL

(GROWTH IN 6.5%) SUSCEPTIBILITY TEST ARYLAMIDASE (PYR)
GROUP D POSITIVE ; BLACKENING POSITIVE RESISTANT POSITIVE ; *S.
ENTEROCOCCUS OF THE MEDIUM PYOGENES IS ALSO
POSITIVE
GROUP D NON- POSITIVE NEGATIVE SUSCEPTIBLE NEGATIVE
ENTEROCOCCUS

GRAM NEGATIVE COCCI

1 NEISSERIA

General Characteristics

✓ Obligate aerobes, g (-) diplococci resembling coffee beans or kidney beans (except for N. elongate: rod shaped)
 ✓ Oxidase (+), Catalase (+) except N. elongate, non-motile, capnophilic (2-8% CO2)
✓ Fastidious: blood, serum, cholesterol or oleic acid, inhibited by fatty acid
✓ Pathogenic Neisseria grows at 37°

ORGANISM GENERAL CHARACTERISTICS DISEASES AND INFECTIONS

N. gonorrhoeae “gonococci” Genital infections
Man is the only known host, ✓ Gonorrhea
transmitted via sexual contact, ✓ Purulent Urethritis is males
vertically (mother to newborn), ✓ Cervicitis in females
found in the urogenital tract, -if left untreated: sterility and perihepatitis (fits-Hugh-
anorectal area, oropharynx, and Curtis Syndrome)
conjunctiva Disseminated Infections
Primary virulence factor: pili/ ✓ Pelvic inflammatory Disease, bacteremia, gonococcal
fimbriae arthritis, pharyngitis, anorectal infections,
conjunctivitis (Ophthalmia neonatorum: treated via
the Creed’s method- 1% silver nitrate), erythromycin,
tetracycline

N. meningitidis “meningococci” Epidemic meningococcal meningitis/ meningococcemia

Colonizes the nasopharynx and ✓ Petechiae (rash), DIC, thrombocytopenia, Septic
the oropharynx; transmitted via shock and hemorrhage in adrenal glands
person-person spread by (Waterhouse-Friedrichsen syndrome)
contaminated respiratory
droplets, encapsulated strains
are mucoid (A, B, C, Y and W-135)
Primary virulence factor:
Lipopolysaccharide-endotoxin
complex

2 MORAXELLA (BRANHAMELLA)

General Characteristics

✓ Aerobic, non-motile, Catalase (+), Oxidase (+), DNase (+), Nonfermenters, grows on BAM

Moraxella catarrhalis (Branhamella catarrhalis, Neisseria catarrhalis)

✓ Fastidious, encapsulated with pili (adhesins), nonmotile, oxidase (+), catalase (+),
✓ NF of the URT, causative agent of pneumonia, obstructive pulmonary disease in elderly patients and sinusitis
and otitis media in children

Laboratory Identification of Pathogenic Neisseria spp and M. catarrhalis

N. gonorrhoeae N. meningitidis M. catarrhalis

Colonial Morphology Translucent, raised, moist, Glistening, convex, Non-pigmented or gray,
grayish white nonpigmented, blue-gray, opaque, smooth
mucoid
Superoxol test: 30% H2O2 + - -
Growth on MTM, ML, NYC + + V
Growth on NA @ 25°C - - V
Growth on NA @ 35°C - - +
CHO utilization test: Cystine Acid production with Acid production with Non-fermenter
Trypticase Agar (CTA) Glucose glucose and maltose
DNase - - +
Nitrate reduction - - +
Tributyrin Hydrolysis - - +
B-galactosidase - - -

THAYER MARTIN MODIFIED MARTIN LEWIS NEW YORK CITY AGAR

THAYER MARTIN
VANCOMYCIN + + + +
COLISTIN + + + +
NYSTATIN + +
TRIMETHOPRIM LACTATE + + +
ANISOMYCIN +
AMPHOTERICIN B +
 GRAM NEGATIVE BACILLI

1 ENTEROBACTERIACEAE

GENERAL CHARACTERISTICS Two groups:

✓ Gram (-), facultative anaerobic, NSF, glucose fermenters, oxidase (-) 1. Overt Pathogens
✓ Motile with peritrichous flagella (except: Klebsiella, Shigella, Yersinia) a. Salmonella Typhi
✓ Non-encapsulated (except: Klebsiella and Enterobacter) b. Shigella spp.
✓ Catalase (+) (except: Salmonella dysenteriae) c. Yersinia pestis
✓ Nitrate reducers (except: Erwinia and Pantoea agglomerans) 2. Opportunistic pathogens
✓ Often with Gas production (except: Shigella, Salmonella, Proteus and a. Citrobacter spp.
Providencia) b. Enterobacter spp
✓ Commensal flora in the intestinal tract (except: Salmonella, Shigella c. Klebsiella spp.
and Yersinia) d. Proteus spp
✓ Nonhemolytic except for E. coli e. Serratia spp.
ANTIGENIC STRUCTURE

Location Heat Stability Antibodies Comments

K Antigen Capsular Ag Labile Salmonella spp: Vi Ag
O Antigen Somatic Ag Stable IgM
H Antigen Flagellar Ag Labile IgG Denatured by alcohol

LABORATORY DIAGNOSIS: GROWTH ON MEDIA: EMB/MAC/HEA/SSA

EOSIN METHYLENE BLUE MACCONKEY AGAR HEKTOEN ENTERIC AGAR

AGAR
CHO Lactose Lactose Lactose, Sucrose and Salicin
INHIBITOR Crystal Violet; Bile Bile salts, Bromthymol blue, acid fuchsin,
salts
pH INDICATOR Neutral Red Bromthymol Blue
H2S INDICATOR Ferric Ammonium Citrate
LACTOSE Pink to Purple colonies Pink Colonies H2s (+): yellow colonies with black centers
FERMENTER *E. coli: pink to purple
with greenish metallic
sheen
H2s (-): yellow colonies without a black
Klebsiella: pink mucoid
center
colonies
Enterobacter: pink
colonies with a dark
center
NON-LACTOSE Colorless H2s (+): green colonies with black centers
FERMENTER
H2s (-): green colonies without a black center

SALMONELLA SHIGELLA AGAR

SALMONELLA SHIGELLA
LACTOSE FERMENTATION NON-LACTOSE FERMENTER NON-LACTOSE FERMENTER
pH indicator: Neutral Red
Inhibitor: Brilliant green and bile salts
H2S PRODUCTION H2S POSITIVE H2S NEGATIVE
INDICATOR: Ferric Citrate
COLONIAL CHARACTERISTIC COLORLESS COLONIES W/ COLORLESS COLONIES WITHOUT
BLACK CENTERS A BLACK CENTER

RAPID LACTOSE FERMENTERS: Escherichia coli, Klebsiella spp., Enterobacter spp.

LATE LACTOSE FERMENTERS: Salmonella arizonae, Shigella sonnei, Serratia spp, Hafnia spp, Yersinia spp, Citrobacter
NON-LACTOSE FERMENTERS: Salmonella spp except for S. arizonae, Shigella except S. sonnei, Proteus,
Providencia,Morganella, Edwardsiella
H2S PRODUCERS: Salmonella, Proteus, Arizonae, Citrobacter, Edwardsiella
 IDENTIFICATION TESTS

a. TRIPLE SUGAR IRON AGAR (TSI)

✓ Sugars (10:10:1) Lactose, Sucrose, Glucose
✓ pH indicator: Phenol Red
✓ H2s indicator: Sodium thiosulfate is reduced to Sulfur Dioxide and Hydrogen Sulfide which reacts with
Ferrous Sulfate to produce black ferric sulfide

PURPOSE PRINCIPLE RESULTS

Initial step in the identification of The breakdown of carbohydrate substrate results in (+): yellow, gas, H2s
Enterobacteriaceae the acidification of the medium with or without gas (-): Red
formation

Iron Salts (Ferrous sulfate and ferric ammonium

citrate) reacts with H2s to produce an insoluble
precipitate (Ferrous sulfate)

b. INDOLE TEST

PURPOSE PRINCIPLE RESULTS

✓ Identify LF of The enzyme tryptophanase is capable of hydrolyzing (+): red ring at the
Enterobacteriaceae and deaminating tryptophan with the production of interface of the
✓ Distinguish E. coli (+) indole, pyruvic acid and ammonia reagent and broth
from K. pneumoniae (-) (-): no color
✓ Speciate Proteus: A red complex is formed when indole reacts with the development
-Proteus mirabilis (-) aldehyde group of p-dimethylaminobenzaldehyde,
-Proteus vulgaris (+) the active chemical in Kovac’s and Erlich’s reagent. (+/-): orange red:
✓ Useful in screening skatole production
bacteria that are prompt Rapid spot test: filter paper impregnated with p-
indole producers aminocinnamaldehyde reagent

c. MRVP TEST
✓ Medium: MR/VP broth/Peptone glucose broth
✓ Note: Most Enterobacteriaceae give OPPOSITE MR and VP reactions

PURPOSE PRINCIPLE RESULTS

To identify the LF In the first pathway, mixed acid products (lactic, acetic, (+): red color at the surface
METHYL RED TEST

Enterobacteriaceae such formic and succinic) result, leading to a decrease in the of the medium
as E. coli (MR+, VP-) pH of the medium and a positive MR test.
whereas most members (-): yellow color at the
of the Klebsiella- The pH must drop to 4.4 or less for the MR indicator to surface of the medium
Enterobacter-Serratia - take on its acidic red color.
Hafnia group are (MR-
and VP+) In the second pathway, acetylmethyl carbinol (acetoin) (+): pink red color at the
VOGES-PROSKAUER TEST

is an intermediate product to butylene glycol. It is the surface of the medium

To determine the ability neutral product detected in the VP reaction.
of an organism to (-): yellow color at the
produce and maintain In the presence of Oxygen and 40% potassium surface of the medium
stable acid end products Hydroxide, acetoin is converted to the diacetyl form,
from glucose which results in a red color in the presence of alpha-
fermentation. napthol.

d. CITRATE UTILIZATION TEST

PURPOSE PRINCIPLE RESULT
To determine if the m/o is capable of Sodium citrate is the only carbon source in (+): blue
utilizing citrate as its sole source of carbon. Simmons Citrate Agar.
If the m/o can utilize citrate, sodium (-): green
Useful in the identification of LF: E. coli (-), citrate is converted to ammonia, which is
Enterobacter and Klebsiella (+) then converted to ammonium hydroxide.
The alkalinity of the compound formed
raises the pH of the medium, and the
bromthymol blue indicator takes on its
blue alkaline color.
 e. ACETATE UTILIZATION TEST
PURPOSE PRINCIPLE RESULTS
To determine the ability of an organism to Breakdown of the sodium acetate (+): blue
use acetate as the sole source of carbon causes the pH of the medium to
shift towards the alkaline range (-): green
turning the indicator from green to
blue.

f. UREASE TEST

PURPOSE PRINCIPLE RESULT

To identify the rapid urease Urease splits urea into ammonia and Christensen’s Agar/Stuart Broth
producers (Proteus and Morganella) water. Ammonia reacts in solution to (+): red
and weak urease producers form an alkaline compound, (-): yellow
(Klebsiella pneumoniae and ammonium carbonate, which results
Enterobacter spp.) in an increased pH of the medium Rapid urease producers w/in 2-4 hrs:
and a color change in the indicator to Proteus, Providencia, Morganella
pink red Slow urease producers @ 4 hours
Citrobacter, Klebsiella,
Enterobacter, Yersinia and Serratia

g. ONPG (B-GALACTOSIDASE TEST)

PURPOSE PRINCIPLE RESULT
To determine the presence of late or slow ONPG (o-nitrophenyl-beta-D- (+): yellow color within 20 mins to
LF. galactopyranoside) is useful in 24 hrs
To detect the late LF strains of E. coli detecting late LF since the ONPG is
To distinguish some Citrobacter species and structurally similar to lactose. (-): no color change, colorless
Arizonae subspp (ONPG +) from Salmonella In the presence of galactosidase, after 24 hours.
subspp (ONPG -) ONPG (colorless) is converted into
To speciate Shigella, since Shigella sonnei is galactose and 0-nitrophenyl, which
the only ONPG (+) Shigella spp. is a yellow alkaline end product
chromogen.

h. LYSINE IRON AGAR

PURPOSE REACTIONS
To determine if a g- rod decarboxylates or Alkaline slant/ alkaline butt (K/K) (-) lysine deamination
deaminates lysine and forms h2s. (+) lysine decarboxylation
Alkaline slant/acid butt (K/A) (-) lysine deamination
Differentiates Salmonella (+) from Citrobacter (-) (-) lysine decarboxylation
Red slant/acid butt (R/A) (+) lysine deamination
Differentiates Proteus, Morganella and (-) lysine decarboxylation
Providencia: deaminate amino acids.
INTERPRETATION
If oxidative deamination of lysine occurs, it will form a burgundy (red) color on the slant in the presence of ferric
ammonium citrate and flavin mononucleotide
If deamination does not occur, the LIA slant remains purple
When glucose is fermented, the butt of the medium becomes acidic (yellow)
If the organism produces lysine decarboxylase, cadaverine (purple) is formed.
If the decarboxylase is not produced, the butt remains acidic (yellow).

i. DECARBOXYLASE-DIHYDROLASE REACTIONS
PURPOSE PRINCIPLE RESULTS
Measures the enzymatic ability of an Decarboxylase removes carboxyl groups from the (+): purple
organism to decarboxylate or hydrolyze and amino acid’s lysine and ornithine.
amino acid to form an amine.
Dihydrolase enzyme removes a carboxyl group from
arginine
Lysine→cadaverine
Ornithine→ putrescine
Arginine→ citrulline
Ornithine→ Putrescine
 j. PHENYLALANINE DEAMINASE (PAD)
PURPOSE PRINCIPLE RESULTS
To determine the deaminase activity using Deamination of the amino acid (+): intense green color (green
the amino acids phenylalanine or results in a colored compound with slant and fluid)
tryptophan. Only Proteus, Providencia and the addition of 10% ferric chloride (-): no color change
Morganella spp. Possess the deaminase
enzyme. Phenylalanine→ phenylpyruvic
acid +10% FeCl3

k. GELATIN LIQUEFACTION
✓ Determine if an organism can breakdown gelatin into amino acid
✓ Positive result: Liquefaction

l. MALONATE UTILIZATION
✓ Determines if an organism can utilize malonate as its sole source of carbon
✓ Differentiates Salmonella (+) from Shigella (-)
✓ Positive result: Blue
✓ Negative Result: green or Yellow

SUMMARY OF BIOCHEMICAL REACTIONS FOR FAMILY ENTEROBACTERIACEAE

TSIA Gas H2S Ind MR VP CIT MOT LYS ARG ORN UREASE PAD ONP
G
Escherichia coli A/A + - + + - - + + -/+ -/+ - - +
Shigella group A, B, K/A - - -/+ + - - - - - - - - -
C
Shigella sonnei K/A - - - + - - - - - + - - +

Edwardsiella tarda K/A + + + + - - + + - + - - -

Salmonella K/A + + - + - + + + -/+ + - - -

Citrobacter freundii A/A + + - + - + + _ -/+ -/+ -/+ - +

K/A
Citrobacter koseri K/A + - + + - + + _ -/+ + -/+ - +

Klebsiella A/A ++ - - - + + - + - - + - +
pneumoniae
Klebsiella oxytoca A/A ++ - + - + + - + - - + - +
Enterobacter A/A ++ - - - + + + + - + - - +
aerogenes
Enterobacter A/A ++ - - - + + + - + + -/+ - +
cloacae
Hafnia alvei K/A + - - -/+ + - + + - + - - +
Pantoea A/A -/+ - -/+ -/+ -/+ -/+ + _ - - -/+ -/+ +
agglomerans K/A
Serratia marcescens K/A + - - -/+ + + + + - + - - +

Proteus vulgaris K/A -/+ + + + - -/+ + - - - + + -

Proteus mirabilis K/A + + - + -/+ -/+ + - - + + + -
Morganella K/A + - + + - - + - - + + + -
Morgagni

Providencia rettgeri K/A - - + + - + + - - - + + -

Providencia stuartii K/A - - + + - + -/+ - - - -/+ + -
Providencia K/A -/+ - + + - + + - - - - + -
alcalifaciens

Yersinia K/A - - -/+ + - - -/+ - - + -/+ - +

enterocolitica
Escherichia coli

STRAINS OF DIARRHEAGENIC E. COLI INFECTION

Enterotoxigenic E. coli (ETEC) Montezuma’s revenge/turista, traveller’s and childhood
-produces heat labile (LT) and heat stable (ST) diarrhea characterized by profuse watery stool
enterotoxins.
LT: closely related in structure and function to the
Cholera toxin
ST: results in net intestinal fluid secretion by stimulating
guanylate cyclase
EnteroInvasive E. coli (EIEC) Dysentery-like/Shigella-like infection
-invades the intestinal epithelium -necrosis, ulceration and inflammation of the large bowel
usually in young children living in areas with poor
sanitation
-Stool with RBCs, neutrophils and mucus
Enteropathogenic E. coli (EPEC) Infantil diarrhea
-non-invasive, no toxin production, nosocomial, seen in -watery diarrhea with mucus but no blood
infants and newborns
EnteroHemorrhagic E. coli (EHEC)/ Verotoxic E. coli Hemmorhagic Collitis
(VTEC)/ EHEC serotype 0157:H7 Hemolytic Uremic syndrome
Produces verotoxin that is named for its cytotoxic effect
on Vero cells (African Green Monkey Kidney cells)
MacConkey Sorbitol agar (SMAC): Selective medium for E.
coli 0157:H7; Colonies are colorless

EnteroAggregative E. coli (EAEC) Watery Diarrhea

-adheres to epithelial cells forming clumps of bacteria
(“Stacked brick” appearance)

Klebsiella

✓ Exhibit mucoid growth, large polysaccharide capsule

✓ K. pneumoniae: Friedlander’s Bacillus, community acquired pneumonia
✓ K. ozanae: purulent sinus infection
✓ K. rhinoscleromatis: granuloma of the nose and oropharynx

Enterobacter

✓ Opportunistic infections: UTI, RT and wound infections

✓ Most Predominant isolate: E. cloacae
✓ Produces yellow pigmentation that intensifies @ 25°C: E. sakazakii (Cronobacter sakazakii)
✓ Intense producer of Urease: E. gergoviae

Serratia

✓ Biochemical test: DNAse, Lipase, and Gelatinase, ONPG (+)

✓ S. marcescens and S. rubidea: red, non-diffusible pigment @ 25°C: Prodigiosin
✓ S. odorifera: rancid potato-like odor
✓ Common opportunistic pathogen in hospitalized patients

Hafnia

✓ It is not known to cause gastroenteritis but is occasionally isolated from stool cultures
✓ Species: Hafnia alvei (Enterobacter alvei)
✓ Distinct feature: Delayed Citrate reaction
(Citrate utilization on Simmon’s citrate agar is negative @ 35 but often delayed positive (3-4 days)@ 22-25°C)

Pantoea

✓ Pantoea agglomerans: formerly known as Enterobacter agglomerans, causes nosocomial outbreak of septicemia
due to contaminated IV fluids; triple decarboxylase negative reaction

Salmonella

✓ Sources of infection: water, milk, dairy products, shellfish, eggs, meat and meat products and household pets
✓ Causes enteritis, systemic infection and enteric fever (Typhoid Fever)
✓ Enterocolitis/gastroenteritis: most common manifestation of salmonella infection: S. Typhimurium
o Widal’s test: tube dilution agglutination test
 ▪ At least 2 serum specimens obtained at intervals of 7-10 ✓ Lab diagnosis:
days; prove rise in Ab titers o 1st week:
▪ Serial dilutions of unknown serum are tested against Blood
antigens from representative salmonellae o 2nd week:
▪ High or rising titer or O>/=1:160 suggests active infection; Urine/ Stool
IgM o 3rd week:
▪ High titer of H>/=1:160 suggests past infection or Serologic
immunization; IgG tests
▪ High Titer of Ab to Vi Ag occurs in some carriers.

BIOCHEMICAL DIFFERENTIATION OF SELECTED SALMONELLA SPECIES

BIOCHEMICAL TEST S. Typhi S. Paratyphi S. Cholerasuis Other serotypes

Citrate Utilization - - V +
Gas Production - + + -
H2s Production + - V +
Lysine Decarboxylase + - + +
Ornithine - + + +
Decarboxylase
Arabinose - + _ +
Fermentation
Rhamnose - + + +
Fermentation
Trehalose + + _ +
Fermentation

Bismuth Sulfite agar

PURPOSE COMMENTS
Selective medium for the isolation of Salmonella Bismuth sulfite and brilliant green inhibit most others. S.
Typhi colonies are black, surrounded by metallic sheen.
Others are light green.

Shigella

✓ Most Inert (Biochemical reactions are mostly negative)

✓ Natural habitat is limited to the intestinal tracts of humans and other primates where they produce bacillary
dysentery
PRESENT DESIGNATION GROUP TYPE CATALASE ONPG MANNITOL
S. dysenteriae (JAPAN) A - - -
S. flexnerii (PHILIPPINES) B + - +
S. boydii (BRITAIN) C + - +
S. sonnei (USA) D + + +

Citrobacter

✓ Causes UTI and sepsis

✓ Organisms resemble Salmonella but are ONPG positive and LDC negative
✓ Species:
o Citrobacter freundii: endocarditis in IV Drug Users
o Citrobacter koseri (C. diversus): causes nursery outbreaks of neonatal meningitis and brain abscesses

BIOCHEMICAL TEST Citrobacter freundii Citrobacter koseri

Indole - +
H2s production + -
KCN growth + -
TSI K/A, G(+), H2S(+); A/A G(+), H2S(+) K/A G (+), H2S (+)
IMVIC -+-+ ++-+
ONPG + -

Proteus

✓ Opportunistic
✓ UTI, bacteremia, pneumonia and nosocomial infections
✓ P. mirabilis and P. vulgaris: exhibit swarming motility
Providencia

✓ Normal intestinal flora

✓ UTI and occasionally other infections and are often resistant to AMT
✓ Providencia rettgerri: pathogen of the UT, also causes disease among travelers
✓ Providencia stuartii: isolated from nosocomial outbreaks in burn units
BIOCHEMICAL TEST Providencia rettgerri Providencia stuartii
Christensen’s Urea + -
IMVIC ++-+ ++-+
TSI K/A, G (-), H2S (-) K/A G (-), H2S (-)
PAD + +

Edwardsiella

✓ Isolated from the environment and many cold-blooded and warm-blooded animals including reptiles, freshwater
and aquarium fish, frogs and turtles
✓ E. tarda: most human species
✓ To differentiate from E. coli: Non-lactose fermenter and H2s Positive

Yersinia

a. Yersinia pestis
✓ Plague: infection of wild rodents transmitted from one rodent to another and occasionally by the bite of
fleas
✓ Common vector: Xenopsylla cheopsis (Rat flea)
✓ Produced pandemics of black death with millions of fatalities.
✓ Broth cultures: “stalactite pattern”
✓ Inclusions: Bipolar Bodies
✓ Wayson stain: “Safety Pin appearance”

b. Yersinia enterocolitica
✓ Enterocolitis: fever, diarrhea and abdominal pain
✓ Bacteremia
✓ Culture on CIN (cefsulodin-Irgasan-Novobiocin): BULL’S EYE COLONIES

BIOCHEMICAL TEST Yersinia pestis Yersinia enterocolitica

INDOLE - V
METHYL RED + +
VOGES-PROSKAUER - V
CITRATE - -
MOTILITY
25°C - +
35°C - -
UREASE - +
ORINITHINE DECARBOXYLASE - +
B-GALACTOSIDASE + +
LACTOSE FERMENTATION - -
SUCROSE FERMENTATION - +

NONFERMENTATIVE GRAM-NEGATIVE BACILLI

GENERAL CHARACTERISTICS

✓ Opportunistic pathogens
✓ Motile, Oxidase (+): does not ferment CHO by enzymatic reactions but by oxidative means

1 PSEUDOMONAS

✓ Obligate aerobe, straight and slender g- rods, NSF

✓ Catalase and oxidase (+), NLF, able to survive with little nutrients (acetate and glucose)
✓ Metabolism is respiratory and never fermentative
✓ Species: P. aeruginosa, P. fluorescens, P. putida, P. monteilii
 Pseudomonas aeruginosa

✓ Obligate aerobe, motile (monotrichous), non-

MUST KNOW!!!
encapsulated, NSF
*Flat, serrated eggs with confluent growth on
✓ On BAP: beta-hemolytic, sweet or grape-like or corn-
BAP
taco like odor: 2-aminoacetophenone; mucoid colonies
*Most common isolated organism in the ICU
seen in patients with CF
*Most common isolate in hospital ventilators
✓ Grows well at 37-42, growth @42 will distinguish it
*Used as a control for the acetamide test
from other spp
✓ Oxidase (+), TSI: K/K G-, H2S-
✓ Most commonly encountered g (-) species that is not a member of the Enterobacteriaceae
✓ Agent of “blue pus”: pyocyanin and pyoverdine; other pigments: prorubrin, pyomelanin
✓ Acquired through ingestion of contaminated food or water, exposure to contaminated medical devices;
penetration of wounds
✓ 3rd most common cause of hospital-acquired infections
✓ Diseases and Infections: Ecthyma gangrenosum, Swimmer’s ear, meningitis, UTI, Pneumonia, Sepsis and
respiratory infections in CF pxns.
✓ Resistant to a number of disinfectants and has been responsible for serious nosocomial infections

2 BURKHOLDERIA SPP.

GENERAL CHARACTERISTICS:

✓ Not considered part of normal human flora; generally non-pathogenic

✓ Catalase and Oxidase (+), motile except B. mallei
✓ Aerobic, non-sporeforming, medium-sized straight g (-) rods
✓ Involves human contact with heavily contaminated medical devices
✓ Species: B. cepacia, B. mallei, B. pseudomallei

Burkholderia cepacia

✓ Survives well in hospital environments, disinfectants, soil, water and plants

✓ Binds mucin form patients with CF
✓ Yellow or yellow green-colored colonies that do no fluoresce

Burkholderia mallei

✓ Agent of Glander’s disease and Farcy disease: mainly infecting horses, mules, donkeys
✓ Occupational human disease that may be fatal, beginning as ulcers of the skin and mucous membranes followed
by lymphangitis and sepsis

Burkholderia pseudomallei

✓ Whitmore’s bacillus
✓ Agent of Melioidosis: glander’s like disease
✓ Environmental inhabitant (soil, streams, surface water: rice paddies)
✓ Human acquisition: inhalation, ingestion, inoculation
✓ Motile with polar tuft flagella
✓ Bacilli can survive within phagocytes: “Vietnamese-time bomb”

3 STENOTROPHOMONAS MALTOPHILIA

✓ Maltophilia: produce acids with maltose and not glucose

✓ Short to medium size, g(-) straight rods, motile with peritrichous flagella
✓ Oxidase (-/weakly +), DNAse (+), NLF
✓ Strictly aerobic, lavender green to light purple pigment with ammonia smell
✓ Not part of the NF, colonizes the skin of burnt patients and RT of hospital (ICU) pxns
✓ Produces brown pigment on BHI with tyrosine

4 ACINETOBACTER

✓ Plump, g(-) rods, strict aerobic and nonmotile

✓ Catalase (+), oxidase (-)
✓ Tendency to resist alcohol decolorization
 ✓ May be mistaken for Neisseria spp.
✓ Acquired through contaminated medical instruments: IV/ urinary catheters
✓ A. baumanii: glucose-oxidizing, nonhemolytic strains
✓ A. lwoffi: contaminant; glucose-non utilizing, non-hemolytic strains

5 ALCALIGENES FECALIS

✓ Medium to long straight rods, motile with peritrichous flagella

✓ Oxidase (+), NLF
✓ Feather-edged colonies surrounded by zones of green discoloration
✓ Alpha-hemolytic: “fruity-odor resembling apples or strawberries”
✓ Isolated in soil and water, including moist hospital environments
✓ Infection is acquired through exposure to contaminated medical devices and solutions

6 MORAXELLA LACUNATA

✓ Morax-Axemfeld bacillus
✓ Agent of Blephanoconjunctivitis/ angular conjunctivitis
✓ Catalase (+), coccobacillus or medium sized rods
✓ BAM: small colonies that pit the agar
✓ Mac: no growth
✓ Normal human flora that inhabit mucous membranes-URT, conjunctiva

NONENTERIC GASTROINTESTINAL PATHOGEN

7 VIBRIO SPP

✓ Comma/ curved bacillus

✓ Found in brackish, marine waters: seafood: shellfish and raw fish
✓ Facultatively anaerobic, monotrichous
✓ Halophilic except: V. cholerae and V. mmicus
✓ Oxidase (+), nitrate reducers except: V. metchnikovii
✓ Glucose fermenters, NLF except V. vulnificus
✓ Species: V. cholerae, V. vulnificus, V. parahemolyticus, V. alginolyticus, V. metschnikovii, V. hollisae

Vibrio cholerae
Classical El Tor
✓ Causative agent of Cholera: Asiatic cholera, epidemic cholera Voges- - +
✓ Rapid darting or shooting star motility proskauer
✓ Single flagellum is covered with lipopolysaccharide sheath Chicken RBC - +
✓ On BAM: smooth, medium to large colonies with a greenish hue agglutination
✓ Has caused epidemics and seven pandemics (somatic antigens O1 Polymixin B Susceptible Resistant
and O139) Red cell - +
✓ Vibrio cholerae 01 serotypes: Ogawa, Inaba and Hikojima hemolysis
✓ V. cholerae 01 biotypes: Classical and El Tor

Clinical infection: Cholera

✓ Acute diarrheal disease spread mainly through contaminated water

✓ Acquired from ingestion of improperly preserved food: seafood, dairy
✓ Characterized by 10-30x of defecation/day (Rice-watery stool: hallmark of cholera)
*choleragen: composed on enzymatic A and B (intestinal receptor-binding) subunits

-A subunit enters the intestinal epithelial cells and activates the enzyme adenylate cyclase by the
addition of an ADP-ribosyl group in a way similar to that employed by the diphtheria toxin. As a result, choleragen
stimulates hypersecretion of water and chloride ions while inhibiting absorption of sodium ions, leading to massive fluid
loss and electrolytes.

✓ All antigens are poorly immunogenic, so repeat infection occurs

✓ Motility and chemotaxis mediate the distribution of V. cholerae
✓ Culture on APW and TCBS (yellow colonies)
MEDIA PURPOSE COMMENTS
ALKALINE PEPTONE WATER Enrichment medium for the recovery Alkaline pH suppresses commensals→
of Vibrio and Aeromonas in stool subcultured to TCBS
THIOSULFATE CITRATE BILE Selective media for Vibrios Alkaline pH inhibits most bacteria
SALTS SUCROSE AGAR V. cholerae ferments sucrose and produces
yellow colonies. V. parahemolyticus and V.
vulnificus do not ferment sucrose; blue-green
colonies

Vibrio alginolyticus
Biochemical tests:
✓ Yellow colonies on TCBS: sucrose fermentation (+) TSI: A/A G(-),H2S(-): V. cholerae
✓ Wound and ear infections associated with marine environment LIA:K/K
✓ Halophilic (+) indole: V. cholerae, V. mimicus and V.
vulnificus
Vibrio parahemolyticus V. cholerae and V. mimicus: do not
✓ Etiologic agent of “summer diarrhea in Japan” require salt for growth
✓ Gastroenteritis; usually associated with contaminated seafood
✓ Associated with the Kanagawa phenomenon
✓ Halophilic
✓ Sucrose fermentation (-)

Vibrio mimicus

✓ Gastroenteritis and ear infections associated with marine environment

✓ Non-halophilic
✓ Sucrose fermentation (-)

Vibrio vulnificus

✓ “lactose-positive” vibrio
✓ Septicemia and infections associated with consumption of raw oysters

8 AEROMONAS

✓ Found in fresh water and chlorinated water, not a part of the human flora
✓ Causative agent of “red leg disease” in frogs
✓ g(-) rod, Oxidase and Catalase (+), Facultatively anaerobic, motile with polar flagellum
✓ glucose fermenters and nitrate reducers, LF, DNAse (+), gas producers
✓ “Bull’s eye colonies” on CIN
✓ Colonies resemble Pseudomonas spp with greenish ground glass appearance and fruity odor, B-hemolytic on
BAP
✓ Resistant to 0129 test
✓ Species: A. hydrophila and A. liquifaciens
✓ A. hydrophila: “water loving” causes gastroenteritis and cellulitis

9 PLESIOMONAS

Plesiomonas shigelloides

✓ Found in fresh water, especially in warmer climates, not part of the normal flora
✓ G (-) rod, oxidase and catalase (+), facultatively anaerobic
✓ Motile with polar flagellum
✓ On BAM: shiny, opaque, smooth and non-hemolytic
✓ Glucose fermenters, genetically related to Proteus
✓ Decarboxylate lysine, ornithine and arginine
✓ (-) gas production, growth in 6% NaCl and TCBS, (+) indole
✓ Minimum growth requirement of 8C

10 CAMPYLOBACTER

✓ Small, curved, motile, NSF, G (-) bacilli

✓ Grows in 5-10% oxygen (microaerophilic)
✓ Species: C. jejuni, C. coli, C. lari, C. fetus subspp. Fetus

Campylobacter jejuni

✓ Slow-growing, fastidious, asaccharolytic, unable to grow in 3.5% NaCl

✓ Motility: single polar flagellum (darting motility)
 ✓ Catalase and oxidase (+), optimum growth @ 42C
✓ Small, curved, seagull-winged, S-shaped faintly staining g(-) rods
✓ Acquired from contaminated drinking water, improperly pasteurized milk, and contaminated poultry
✓ Most common cause of gastroenteritis
✓ Most recognized antecedent cause of GBS

11 HELICOBACTER

✓ Spiral shaped organisms resembling campylobacter

✓ Motile, curved, microaerophilic, g(-) rods with most species having strong urease activity
✓ Species: H. pylori, H. cinaedi, H. fennelliae, H. rappini (formerly Flexispira rapini)

Helicobacter pylori

✓ Catalase and oxidase (+), strong urease producer

✓ Causes type B gastritis, peptic ulcer and gastric carcinoma
✓ Binds to Lewis antigen and to the monosaccharide sialic acid
✓ Primary habitat: human gastric mucosa-mucus layer of the antrum and fundus of the stomach but does not
invade the epithelium
✓ Histology staining (Warthin-starry, Silver, Giemsa stains) and culture of stomach/duodenal biopsies are
recommended for identification

BIOCHEMICAL/SUSCEPTIBILITY Campylobacter jejuni Helicobacter pylori

CATALASE TEST + -
NITRATE REDUCTION + V
UREASE - +
H2S PRODUCTION - -
HIPPURATE HYDROLYSIS + -
INDOXYL ACETATE HYDROLYSIS + -
SUSCEPTIBILITY TO:
CEPHALOTHIN (30 mcg) Resistant Susceptible
NALIDIXIC ACID (30 mcg) Susceptible Resistant
GROWTH @:
15C - -
25C - -
42C + V

SMALL PLEOMORPHIC GRAM-NEGATIVE BACILLI

12 HAEMOPHILUS

✓ “Blood lover”
✓ Normally inhabit the URT of humans except H. ducreyi
✓ Obligate parasites on the mucous membranes of humans
✓ Small, pleomorphic, fastidious coccobacilli or rods
✓ Nonmotile, NSF, aerobic or facultatively anaerobic and requires 5-10% CO2 @35-37c
✓ Oxidase and Catalase (+)
✓ Die rapidly in clinical specimens-very susceptible to drying and extreme temperatures
SPECIES X V B- D-ALA/ COMMON DISTINGUISHING
FACTOR FACTOR HEMOLYSIS PORPHYRIN TEST NAME CHARACTERISTIC
H. influenzae Pfeiffer’s Mousy/ bleach like
Y Y - -
bacillus odor
H. parainfluenzae N Y - + Mannose fermenter
H. hemolyticus Y Y + -
H. parahemolyticus Tannish and dry
N Y + +
colonies
H. aegypticus Koch-week’s
Y Y - -
bacillus
H. aphrophilus N N - +
H. paraaphrophilus Lactose and mannose
N Y - +
fermenter
H. ducreyi Ducrey’s School of fish
Y N - -
bacillus
Haemophilus influenzae

✓ Transmitted person to person spread by contaminated droplets

✓ Encapsulated strains are pathogeniv
✓ Main cause of meningitis in children <5 years old
2 categories
a. Typeable: based on capsular characteristics, capsule is composed of polyribitol phosphate, types a-f
(capsular serotypes), type b: cause serious infections in humans, most frequently encountered,
resists phagocytosis
b. Non-typeable: non encapsulated, normal inhabitants of the URT, causes otitis media and acute
bronchitis

Haemophilus aegypticus

✓ Closely resembles H. influenzae biotype III

✓ Causes pink eye (conjunctivitis)

Haemophilus ducreyi

✓ Agent of STD: chancroid or soft chancre: genital lesions, from tender papules to painful ulcers with several
satellite lesions
✓ Not part of the human normal flora, only found in humans during infections
✓ Smallest pathogenic bacteria (vs. largest: B. subtilis)

GRAM STAIN
H. influenzae Pale pink coccobacilli or small rods
H. influenzae biotype aegypticus Long slender rods
H. ducreyi Slender or coccobacillary in pairs or rows, “school of fish” arrangement
H. aprophilus Very short coccobacilli and occasionally as filamentous forms
H. paraprohilus
CULTURE
Chocolate agar media Contains X and V factors, thioglycolate, BHI
*Most strains will not grow on 5% SBA (hemin only)
Rabbit or horse blood: for detecting hemolysis
Horse blood> sheep blood: growth inhibiting factor
S. aureus produces NAD: satellitism growth pattern of H. influenzae
MAC No growth
Horse Blood Bacitracin agar Selective medium for H. influenzae for respiratory secretions of patients
with CF
*MH-based chocolate agar with 1% isovitalex and 3 ug/ml vancomycin
Heart infusion-based agar with 10% fetal bovine serum +3 ug/ml vancomycin
H. ducreyi: may require 7 days of incubation
Purpose Comments
Porphyrin test Means of establishing the Haemophilus spp that need x-factor
organism’s x factor dependence and are unable to synthesize
eliminates the potential problem for protoporphyrin from D-ALA.
carryover (+) result: red fluorescence
Detects the presence of enzymes
that convert D-aminolevulinic acid
into prophyrins or protoporphyrins

HACEK GROUP

✓ Haemophilus aphrophilus, Actinobacillus actinomycetemcomitans, Cardiobacterium hominis, Eikenella Corodens

and Kingella spp.
✓ Small, NSF, will not grow on MAC
✓ Slow growth on BAM and CAM (7-14 days)
✓ Causes slow, progressive bacterial endocarditis
✓ Utilize D-ALA; indole (+) except C. hominis

Eikenella corrodens

✓ Corroding bacilli
✓ Causes mixed infection from bites or clenched-fist wounds
✓ May pit or corrode the agar surface; “sharp bleach odor”
✓ Oxidase (+), capnophilic, requires 2-3 days for growth
13 BORDETELLA

✓ Nonmotile except B. bronchiseptica

✓ Obligate aerobe

Bordetella pertussis

✓ Agent of Pertussis “Whooping cough”

✓ 3 stages:
o Catarrhal: mucous membrane inflammation, mild cold and cough: general flu-like symptoms
o Paroxysmal: severe and violent coughing (15-25x/24 hours), associated with vomiting and whooping
(hurried deep respiration) may last for 6 weeks: repetitive coughing episodes
o Convalescent: symptoms slowly decrease, can lasts for as; long as 6 months after infection: recovery
phase
✓ Ideal specimen: Nasopharyngeal swab (calcium alginate or Dacron) and aspirates
✓ Colonies: Mercury droplet colonies

MEDIA PURPOSE COMMENTS

BORDET-GENGOU Selective enrichment medium Potato-glycerol based medium enriched with blood;
for the isolation of B. pertussis contaminants inhibited by methicillin, “cough plate”
REGAN-LOWE Selective for B. pertussis Charcoal agar supplemented with 10% horse blood,
*Preferred cephalexin and amphotericin B
MODIFIED JONES- Selective for B. pertussis Supplemented with yeast extract and cephalexin
KENDRICK CHARCOAL
Bordetella parapertussis: pertussis-like syndrome

Brodetella bronchiseptica: kennel’s cough (inhabits respiratory tract of canines)

Urease Motility Nitrate reduction Oxidase

B. pertussis - - - +
B. parapertussis + - - -
B. bronchiseptica + + + +

14 BRUCELLA

✓ Bang bacillus
✓ Small, coccobacillary, strict aerobes. Intracellular parasites, class III pathogens
✓ Appear singly, in pairs or short chains, “sand appearance”
✓ Nonmotile, non-encapsulated and some species require CO2 for growth
✓ Catalase and Oxidase (+), nonsaccharolytic
✓ Normal flora of the Urinary Tract and gastrointestinal tract of sheep/goats (B. melitensis), cattle (B. abortus),
pigs (B. suis), and dogs (B. canis)
✓ Acquired through the ingestion of contaminated animal products, inhalation, penetration of ocular or oral
mucosa, direct inoculation in the bloodstream via abrasions in the skin (occupational)
✓ Induce spontaneous abortion among animals
✓ Localized in tissues rich in erythritol (placental tissues)
✓ Agent of Brucellosis, Undulant fever and Malta fever
✓ specimen Blood and Bone Marrow
✓ Culture: Castañeda Bottles
o Double medium (solid/liquid)
o Bottle contains Bile broth/agar slant
o For subculture the bottle is merely tilted

Carbon Dioxide Hydrogen Sulfide Growth in the presence of

Thionine Basic Fuchsin
B. abortus + + Inhibited Grows
B. melitensis - - Grows Grows
B. suis - - Grows Inhibited
B. canis - - Grows inhibited

15 LEGIONELLA

✓ Facultative intracellular pathogen

✓ Faintly staining g(-), majority are motile
 ✓ Colonies appear iridescent with a sticky consistency
✓ Catalase and oxidase: weakly (+), gelatinase (+)
✓ Will not grow on primary plating media
✓ Requires a medium supplemented with L-cysteine buffered to pH 6.9
✓ Can infect and multiply within some free-living amoebae (Hartmanella, Acanthamoeba and Naegleria spp),
Ciliated protozoa and biofilms.
✓ Specimen: Bronchial washing, expectorated sputum
✓ Stain: Dieterle’s stain (Dark brown to black)

Culture media Purpose Comments

Buffered Charcoal Enrichment medium Yeast extract and L-cysteine enhance growth of Legionella, Charcoal
Yeast Extract Agar for isolation absorbs toxic compounds
(BCYE)
Feeley-Gorman Non-selective Casein enzymatic hydrolysate and Beef extract provide organic
enrichment and nitrogen, carbon compounds and other growth factors. L-cysteine
isolation hydrochloride: reducing agent and source of sulfur containing amino
acids. Ferric pyrophosphate source of iron ions.
Legionella pneumophila

✓ Nutritionally fastidious aerobic g- rod

✓ Part of the natural microbial community of soil and aquatic ecosystems-isolated from lakes, rivers, marine and
potable waters and man-made facilities
✓ Isolated in air conditioning ducts, colling towers, water-plumbing systems, humidifiers and nebulizers
✓ Causative agent of Legionnaire’s Disease/legionellosis, a febrile and pneumonic illness and Pontiac Fever, a
nonfatal respiratory infection in which pneumonia does not occur.

L. micdadei: Pittsburgh pneumonia

L. bozemanii: Wiga’s agent of pneumonia

16 PASTEURELLA

✓ Small, straight g(-) rods, facultatively anaerobic, nonmotile, non-hemolytic

✓ Glucose fermenter, nitrate reducers, most strains will not grow on MAC
✓ Oxidase and catalase (+) except P. bettyae, indole (+)
✓ Part of the oral, respiratory and gastrointestinal animal flora; transmitted through cat/dog bites and scratch
wounds
✓ Agent of shipping fever in cattles, a hemorrhagic septicemia
✓ Susceptible to penicillin

Pasteurella multocida

✓ “Mushroom smell”
✓ (+) oxidase, ornithine decarboxylase and indole, (-) urease and ONPG
✓ Causative agent of Pasteurellosis

17 FRANCISELLA

Francisella tularencis

✓ Very small, obligate aerobic, coccobacillus, extremely invasive

✓ Nonmotile, facultatively intracellular parasite, weakly catalase (+) and oxidase (-)
✓ Requires cystine, cysteine and iron for growth
✓ BSL 2 pathogen which requires a BSL 3 for culture
✓ Infects rodents and cottontail rabbits
✓ Acquired by handling the carcasses of skin or infected animals through insect vectors (deerflies and ticks), being
bitten or via inhalation
✓ Considered a potential bioterrorism weapon
✓ Agent of Tularemia/ deerfly fever/ rabbit fever
✓ Culture media: Blood-glucose-Cystine Medium
 AEROBIC GRAM POSITIVE BACILLI

1 BACILLUS

✓ Form spores aerobically, catalase (+), glucose fermenters

✓ Sporeformers: able to survive in extreme environmental conditions

Bacillus anthracis

✓ “Anthrax bacillus”
✓ Most virulentnt, causative agent of Anthrax
✓ Large, encapsulated, square-ended rod; non-motile, aerobic or Types of Anthrax:
facultative anaerobic Cutaneous: via inoculation: skin cuts,
✓ Unstained central spore: Bamboo-Fishing rod appearance abrasions; “black eschar”
Pulmonary: via inhalation of spores;
✓ On BAM: non-hemolytic, “medusa-head colonies”, “beaten egg
white colonies” “woolsorter’s disease”
✓ String of pearls (MHA with penicillin) Gastrointestinal: via ingestion of
✓ Gelatin medium: “inverted pine tree” ; liquefaction (+) spores
✓ Centerpiece for counter terrorism and used as weapon for mass
destruction

Bacillus cereus
Diarrheal Type Emetic Type
✓ Fried Rice Bacillus Meat, poultry, Rice
✓ Food poisoning: Diarrheal and Emetic type vegetables
✓ On BAM: large, feathery, spreading, frosted glass appearance IP:8-16 hours IP:6 hours
colonies; B-hemolytic Heat-labile Heat-stable
✓ Best specimen for testing: suspected food enterotoxin enterotoxin

CHARACTERISTICS B. anthracis B. cereus

Motility Non-motile Motile
Capsule Encapsulated Non-encapsulated
Hemolysis Non-hemolytic Beta-hemolytic
Growth @45C No growth/ slight growth Rapid Growth
Penicillin susceptibility Susceptible Resistant
“String of Pearls” Reaction + -
Salicin Fermentation - +
Gelatin Hydrolysis - +
Growth on PEA - +
Lecithinase Production + +

Bacillus subtilis

✓ “Hay bacillus”
✓ Common laboratory contaminant
✓ On BAM; large, flat, dull with ground glass appearance, may be pigmented (pink, yellow, orange or brown), may
be hemolytic
✓ Halophilic organism (7% NaCl)
✓ Source of bacitracin antibiotic

ANAEROBIC GRAM-POSITIVE BACILLI

CLOSTRIDIUM

✓ Form spores anaerobically

✓ Catalase (-)

Clostridium perfringens/ C. welchii/ Bacillus aerogenes

✓ Myonecrosis (gas gangrene) and food poisoning

✓ In solid media and thioglycolate broth: Spores are absent
✓ Double zone hemolysis
o Inner: complete due to theta toxin
 o Outer: incomplete due to alpha toxin
✓ Lecithinase, Reverse CAMP and Nagler Reaction: (+)

Clostridium botulinum

✓ Agent of food, wound and infant botulism

✓ Botulism results from the liberation of the botulism toxin
✓ Spores: Oval and Subterminal
✓ Lipase (+)

Clostridium tetani

✓ Etiologic agent of Tetanus: resulting from entry of the organism/ spores from puncture wounds
✓ Produces tetanospasmin, associated with spastic contraction/lock jaw/devil’s grin/Risus Sardonicus
✓ Spores: Round and terminal (Tackhead bacillus/drumstick/lollipop)

Clostridium difficile

✓ Important cause of Antibiotic (clindamycin)- Associated Pseudomembranous Colitis and Diarrhea

✓ Spores: oval and subterminal
✓ Culture on CCFA: yellow colonies

MOTILITY LECITHINASE LIPASE LACTOSE GLUCOSE

C. perfringens - + - + +
C. botulinum + - + - +
C. tetani + - - - -
C. difficile + - - - +

Disease Toxin Spore Hemolytic Reverse Nagler

location pattern Camp test Reaction
C. perfringens Gas gangrene and Necrotizing Subterminal Double zone of + +
food poisoning toxin hemolysis
C. botulinum Botulism Botulinum Subterminal - - -
Toxin
C. tetani Tetanus Tetanospasmin Terminal - - -
C. difficile Pseudomembranous Toxin A and B Subterminal - - -
colitis

NON-SPORE FORMING GRAM POSITIVE BACILLI

1 CORYNEBACTERIUM

✓ Non-motile, NSF, non-encapsulated

✓ Pleomorphic: palisades, in pairs forming X, V, Y, L; Chinese letters, club shaped, beaded forms
✓ Catalase (+)
✓ Glucose and maltose fermenters except C. urealyticum

Corynebacterium diphtheriae

✓ Diphtheria bacillus/ Kleb-loeffler’s bacillys

✓ Aerobic or facultative anaerobic, grows best under aerobic conditions
✓ Inhabits the human nasopharynx but only in the carrier state; not part of the normal human flora
✓ Enriched medium is recommended (serum, cystine, and K tellurite)
✓ Agent of Diphtheria: acute, contagious disease characterized by the production of a toxin and a false membrane
lining: 2 forms: Respiratory and Cutaneous form

Corynebacterium pseudodiphthericum

✓ Hoffman’s Bacillus
✓ Normal flora of the human nasopharynx
✓ Respiratory, UTI and wound infections in immunocompromised pxns

Corynebacterium jeikeium

✓ Pleomorphic, club-shaped, arranged in V forms

✓ On BAM supplemented with 1% tween 80: large colonies
✓ Proliferate as skin flora of hospitalized patients (inguinal, axillary, rectal sites)
 ✓ Lipophilic species
✓ Common cause of diphtheroid prosthetic valve endocarditis
✓ Multiple antibiotic resistance

Corynebacterium ulcerans

✓ Associated with diphtheria-like sore throat

✓ Mastitis in cattles
✓ On BAM: small, dry, waxy, gray to white colonies

Corynebacterium urealyticum

✓ UTI and wound infections in compromised patients

✓ Lipophilic, multiple antibiotic resistance
✓ Arranged in V forms or palisades
✓ On BAM: small, pinpoint, white, smooth colonies; non-hemolytic
✓ Does not ferment glucose and maltose

Corynebacterium minutissimum

✓ causative agent of erythrasma: coral (brick red) fluorescence when exposed to Wood’s light (long-wave UV
radiation, also called black light) due to porphyrin.

Laboratory Diagnosis

✓ Culture
o Christensen Urea Slant: Urease production: C. urealyticum
o Loeffler Serum Agar/Pai’s coagulated egg: (+) poached egg colonies, Babes-Ernst granules
o Modified Tinsdale Agar: (+) black colonies with brown halo: C. diphtheriae
o Cystine Tellurite Blood Agar (CTBA): preferred medium; (+) black/ gray colonies after 48 hours
▪ Biotypes of C. diphtheriae
• Mitis: small, black, convex colonies, “bleach-like odor”, “fried-egg appearance”,
hemolytic
• Intermedius: very small, flat, dry, grayish black, non-hemolytic
• Gravis: large, flat, dark gray, “daisy head colonies”, non-hemolytic
✓ Toxigenicity test
o In vivo: Animal inoculation (guinea pig): Diphtheria anti-toxin
o In vitro: Elek’s test: positive: fine precipitin lines at a 45 angle
✓ Schick’s test: Susceptibility test: skin test for C. diphtheriae

Biochemical test

Organism Urease Nitrate Esculin Gelatinase Reverse

reduction Hydrolysis CAMP
C. diphtheriae - + - - -
C. ulcerans + - - + +
C. pseudotuberculosis + V - - +

2 LISTERIA

Listeria monocytogenes

✓ Found in the environment, soil, water, sewage, decaying vegetation and in feces of humans, swine and poultry
✓ Major source on infection: contaminated food, grows @4C and in high salt concentration
✓ Meningitis, pneumonia, abortion, stillbirth, endocarditis, conjunctivitis and urethritis
✓ Perinatal human listeriosis: granulomatosis infanseptica
✓ Motility
o Hanging drop: tumbling @ RT
o Semisolid: Umbrella-like growth/inverted christmas tree@ 25C but not @ 35C
✓ Cultured on Mcbride medium
✓ Ocular test/Anton Test
✓ Glucose fermenter, (+) catalase, Camp test, VP, bile esculin hydrolysis and growth in 6.5% NaCl
✓ (-) H2s production, nitrated reduction and Urease

3 ERYSIPELOTHRIX

Erysipelothrix rhusiopathiae
 ✓ only catalase (-), g (+) NSF rod that produces H2s
✓ aerobic or facultatively anaerobic, nonhemolytic, nonmotile, pleomorphic
✓ Occupational exposure IN HUMANS: Erysipeloid: seal/whale finger
✓ Erysipelas in Animals
✓ Gelatin stab: “pipe-cleaner/”test-tube brush pattern” @ 22C
✓ (-) catalase, oxidase, bile esculin hydrolysis, Nitrate reduction, VP and urease

DISEASE CATALASE MOTILITY ESCULIN NITRATE UREASE H2S

HYDROLYSIS REDUCTION
Corynebacterium DIPHTHERIA + - - V V -
L. monocytogenes LISTERIOSIS + + + - - -
E. rhusiopathiae ERYSIPELOID - - - - - +

4 LACTOBACILLUS

✓ square ends; arranged in pairs and in chains.

✓ anaerobes that can be aerotolerant and α-hemolytic; catalase (-)
✓ majority are vancomycin resistant
✓ major members of the normal microbiota of the vagina.

Lactobacillus acidophilus

✓ Doderlein bacilli
✓ Normal flora of the mouth, GIT and vaginal canal
✓ Non-pathogenic and has little clinical significance
✓ Cultured on Tomato juice agar

MYCOBACTERIUM SPECIES

Mycobacterium tuberculosis

✓ “Koch’s Bacillus”
✓ Slender, beaded rods with X, V, Y, L formations (microscopically)
✓ Colonies are buff in color, nonchromogenic and nonphotoreactive
✓ Catalase, nitrate reduction, niacin accumulation (+)
✓ Causative agent of Tuberculosis: usually begins in the middle or lower lung. Other areas for dissemination
include the spine (Pott’s disease), long bones, meninges and genitourinary system.
✓ Macrophages phagocytize the bacteria to form multi-nucleated cells, which are eventually surrounded by fibroid
cells to form granulomatous lesions called tubercles which can be seen on chest x-rays. The lesions calcify at
which point they are called Ghon Complexes.
✓ Methods for decontamination and digestion of Mycobacteria
o N-acetyl-L-cystein (NACL) and 2-4% NaOH
▪ NACL: digesting/mucolytic agent
▪ NaOH: decontaminating agent
o Trisodium phosphate and benzalkonium chloride (Zephiran)
o Dithiothreitol and NaOH
✓ Specimens:
o Sputum
▪ Ideal specimen
▪ Grossly thick
▪ Microscopically:
• <10 squamous epithelium cells, >25 leukocytes: presence of alveolar macrophages
o Secretions obtained from bronchoscopy, Blood, Urine, CSF, Pleural, Peritoneal and Pericardial Fluids
✓ Media (culture is maintained for 8 weeks)
o Lowenstein-Jensen(LJ)
▪ Egg-based medium
▪ Incorporated with Malachite green (inhibits NF)
▪ Colonies: Cauliflower-like: small, warty, yellow; “buff, rough and tough”
o Petragnani
o American Thoracic Society
o Middlebrook 7H11 and 7H10
o Clear agar: examine colonies (tan to buff in color)
o Middle brook Media
 ▪ Agar based
▪ Contains 2% glycerol which supports the growth of MAC
▪ Growth of colonies is observed several days before the egg-based
▪ It can be selective by incorporating antibacterial agents to suppress the growth of
contaminating flora
o Middlebrook 7H9 broth
o Used to maintain stock culture
o Use to prepare isolates for biochemical tests
✓ Staining
o Gram-stain: slender, poorly stained, beaded g (+) rods, gram neutral/ gram ghosts
o Ziehl-neelsen/Hot Stain Method
o Kinyoun/ Cold Stain Method
o Fite-Faraco’s
o Spergler’s
o Pappenheim’s stain
o Baumgarten’s stain
o Auramine-Rhodamine (truant’s)
▪ Fluorescent organisms on black
background Number of AFB seen Report
(OIO)
*view a minimum of 300 fields before a slide is deemed 0 No AFB seen
negative. 1-2/300 fields Doubtful, request another
specimen
✓ Skin test for TB (Purified Protein Derivative): heat- 1-9/100 fields 1+
killed ammonium sulfate precipitated organism is 1-9/10 fields 2+
injected intadermally 1-9/field 3+
o Tuberculin Test >9/field 4+
▪ Aka “Purified Protein Derivative test
or Mantoux Test”
▪ Type IV hypersensitivity reaction
▪ Intradermal injection of small amounts of antigen
▪ Induration and redness usually develop and reaches a peak in 24-72 hours
✓ Antituberculous Agents
o Primary drugs: Streptomycin, Isoniazid, Rifampicin, Ethambutol, Pyrazinamide (PRISE)
o Secondary: ethionamide, Capreomycin, Ciproflaxin, Ofloxavin, Kanamycin, Cycloserine, Rifabutin

Mycobacterium bovis

✓ Produces TB in cattles, dogs, cats, swine, parrots and humans

✓ Attenuated strain is used for vaccination (Bacille-Calmette-Guerin BCG)
✓ Acquired by ingestion of contaminated milk from infected cows or by exposure to infected animals and their
carcasses

Mycobacterium africanum

✓ Associated with human cases of TB in Africa

Mycobacterium canetti

✓ Smooth strain of M. tuberculosis

✓ Niacin and Nitrate (+), grows more rapidly than M. tb
✓ First human isolate was from a cervical lymph node (Somatic child), also isolated from an AIDS patient with
mesenteric TB

Mycobacterium ulcerans

✓ Inert bacillus, mostly negative reactions

Nontuberculous Mycobacteria

✓ Mycobacteria other than the mammalian tubercle bacilli, which occasionally cause human disease
✓ Atypical, anonymous, unclassified, and Mycobacteria other than M. tb (MOTT)
✓ Usually not transmitted from person to person
✓ Present everywhere in the environment; sometimes colonize healthy individuals, skin, respiratory and GIT
✓ RUNYOUN Classification (1959): based on their growth rate and pigment production or photoreactivity
o Growth rate
▪ Rapid growers: Within 1 week, 3-7 days, weakly pathogenic
▪ Slow growers: 10-21 days, most commonly pathogenic
 o Photo reactivity
▪ Photochromogenic: yellow to orange pigment only when exposed to light
▪ Scotochromogenic: yellow to orange pigment when growing in the dark and in the light
▪ Non-Chromogenic: does not produce pigment

Runyoun Group Group Name Description Species

Classification
I Photochromogens Develop pigment(cream or buff; M. kansasii
orange/yellow) following exposure to light M. asiaticum
after being grown in the dark and take more M. marinum
than 7 days to appear in solid media M. simiae
II Scotochromogens Develop pigment (yellow to orange) in the M. szulgai
dark or light and take more than 7 days to M. gordonae
appear on solid media M. scrofulaceum
M. flavescens
M. xenopi
III Nonphotochromogens Nonpigmented regardless of whether they are M. avium-complex
grown in the dark or light and take more than M. ulcerans
7 days to appear in solid media M. terrae-complex
M. triviale
M. gastri
IV Rapid growers Colonies appear on solid media in less than 7 M. fortuitum
days M. chelonae
M. smegmatis
M. phlei

Mycobacterium kansasii

✓ Yellow bacillus
✓ Common cause on NTM pulmonary disease
✓ Potentially pathogenic, long rods, shepherd’s crook

Mycobacterium marinum

✓ Diseases of fish and has been isolated in aquariums

✓ Swimming pool granuloma: cutaneous infection in humans

Mycobacterium szulgai

✓ Causes pulmonary disease in middle-aged men and bursitis

✓ Scotochromogen @ 35 C and non-pigmented @ 25-30C

Mycobacterium scrofulaceum

✓ Isolated from raw milk, water and other dairy products

✓ Causes cervical lymphadenitis in children

Mycobacterium gordonae

✓ Tap water bacillus

✓ Contaminates tap water used by patients in rinsing their mouths prior to the aerosolized saline sol’n

Mycobacterium Avium Complex

✓ M. avium (Battey bacillus), M. intracellulare, M. paratuberculosis, M. lepraemurium, M. avium subsp. Silvaticum

(wood pigeon bacillus)
✓ Important pathogen in immunocompromised and immunocompetent populations
✓ Causes a pulmonary infection in humans similar to Mtb
o M. paratuberculosis: causative agent of Johne’s disease (inflammatory bowel disease)

Mycobacterium xenopi

✓ Recovered from hot and cold water taps, water storage tanks of hospitals and from birds
✓ First isolated from an African Toad
✓ Causes pulmonary infection in adults, potential pathogen
✓ Optimal growth @ 42C than 37C

Mycobacterium terrae-complex

✓ M. terrae (Raddish bacillus), M. triviale, M. nonchromogenicum

 ✓ M. triviale: (+) growth in 5% NaCl

Mycobacterium fortuitum

✓ Isolated from water, soil and dust

✓ Skin and soft tissue infections

Mycobacterium chelonae

✓ Infections of the skin, lungs and bone

✓ Exhibits more antimicrobial resistance than M. fortuitum
✓ Outbreaks of infection is associated with administration of DPT-polio vaccines, histamine and lidocaine
injections

Mycobacterium Smegmatis

✓ Rare cases of pulmonary, skin, soft tissue and bone infections

Biochemical tests
HEAT STABLE CATALASE TEST
Reagent: 30% H2O2 The reagent and medium (+)result: column of When heated to 68C, the
Medium: tween 80 are added to a 2-week old bubbles: >45 mm (height of catalase of the MTB
(polyoxythelene sorbitan subculture on LJ and placed the bubbles) complex and M. ulcerans is
monooleate) upright for 5 minutes inactivated.
NIACIN (NICOTINIC ACID) TEST
Reagent: cyanogen Plays a role in the redox (+) result: yellow color Mtb, M. bovis and M.
bromide and aniline reaction during reaction simiae are positive
reagent mycobacterial metabolism,
detects the deficiency of
the enzyme niacin
ribonucleotide that will
hydrolyze the niacin on the
medium; detects slow
growers
NITRATE REDUCTION TEST
Reagents: sulfanilamide Influenced by the age of (+) result: red color MTB, M. kansasii, M.
and N-naphthlenediamine the colonies, temp, pH and szulgai, M. fortuitum are
enzyme inhibitors positive

ARYLSULFATASE TEST
Reagent: potassium This enzyme breaksdown (+) result: red color M. fortuitum and M.
phenolphthalein SO4 and phenolphthalein disulfate chelonae are positive.
Na HCO3 into phenolphthalein
TWEEN 80 HYDROLYSIS
Useful for separating species of photochromogens, (+) result: pink color M. gordonae; M. kansasii is
nonchromogens and scotochromogens positive within 6 hours,
Detects the ability of lipase to hydrolyze tween 80 into
oleic acid and polyoxyethylated sorbitol
PYRAZINAMIDASE TEST
Pyrazinamide is comverted to pyrazinoic acid (+) MTB and M. marinum (-) M.bovis and M. kansasii

IRON UPTAKE TEST

(+) growth in 20% ferric (+) M. fortuitum
chloride (-) M. chelonae
Urease (+): Mtb, M. kansasii, M. marinum, M. scrofulaceum, M. bovis

Mycobacterium leprae

✓ Hansen’s bacillus
✓ Non-cultivatable NTM, not cultivated in vitro
✓ Cultivated in footpads on mice and armadillos
✓ Rod shaped exhibiting: “cigar pocket/pocket fence arrangement”
✓ Causative agent of leprosy/ Hansen’s Disease
o Chronic disease of skin, mucous membranes and nerve tissus
o Transmitted through inhalation or contact with infected skin
▪ 2 forms
 • Tuberculoid: localized, benign, nonprogressive, no immune defect
▪ Associated with delayed type hypersensitivity reaction to Ags
▪ Characterized by skin lesions and damaged nerves
▪ (+) lepromin test
• Lepromatous: disseminated, malignant, progressive
▪ Loss of facial features, fingers, toes and other anatomical structures
▪ (-) lepromin test

FUNGUS-LIKE BACTERIA

1 ACTINOMYCES

✓ Gram (+) but neither acid fast nor stained with fungus stain; anaerobic
✓ Causes Chronic Suppurative Granulomatous disease
✓ Agent of Lumpy Jaw
✓ Molar tooth colonies

2 NOCARDIA

✓ Gram (+), partially acid fast, aerobic

✓ N. asteroides: most clinically relevant
✓ Primary pulmonary infections resembling tuberculosis
✓ Other species: N. brasiliensis and N. ottidiscavarium

MISCELLANEOUS BACTERIA

1 TROPHERYMA WHIPPLEI

✓ Gram (+) actinomycete that is not closely related to any other genus known to cause infection
✓ Whipple’s disease: found primarily in middle-aged men, characterized by the presence of PAS-staining
macrophages( indicating mucopolysaccharide or glycoprotein in almost every organ of the body)

2 SPIRILLUM MINUS/MINOR

✓ Gram (-), helical, strictly aerobic organism

✓ RAT-bite fever in humans: SODOKU
✓ Clinical signs and symptoms similar to those caused by S. monoliformis except that arthritis is rarely seen in pxns
with sodoku and swollen lymph nodes are prominent, febrile episodes are also more predictable in SODOKU.

3 GARDNERELLA

Gardnerella vaginalis

✓ Gram-variable to g (-), Haemophilus vaginalis and Corynebacterium vaginalis

✓ Bacterial vaginosis: foul-smelling grayish vaginal discharge
o Amsel and Nugent scoring system
o Cytology/Pap’s: clue cells
o Whiff or Sniff test: Vaginal discharge + 10% KOH: (+) fishy-amine like odor

CULTURE MEDIA PURPOSE COMMENTS

Vaginalis Agar (V agar) Nonselective enrichment medium for Columbia agar with human blood,
isolation incubation: increased CO2 for 24-72
hrs

G. vaginalis: diffuse beta-hemolysis

Human Blood Tween Agar (HBT) Semi-selective media Incubate in increased CO2 for 48
hours

4 STREPTOBACILLUS

Streptobacillus monoliformis
 ✓ Normal flora of rodents
✓ Agents of Rat-bite and Haverhill fever

5 CALYMMATOBACTERIUM

Calymmatobacterium granulomatis

✓ Agent of Granuloma Inguinale/Donovanosis

✓ Encapsulated, pleomorphic g(-) bacillus usually observed in vacuoles in large mononuclear cells
✓ Donovan Body: Groups of organisms seen within mononuclear cells
✓ Based on DNA hybridization, name be changed to Klebsiella granulomatis

CHLAMYDIA, MYCOPLASMA AND RICKETTSIAE

1 CHLAMYDIA

✓ Obligate intracellular
✓ Elementary Bodies (EB): Infective particle
✓ Reticulate Bodies (RB): metabolically inactive

Chlamydophila psittaci

✓ Agent of Psittacosis/ornithosis
✓ Pathogen of birds
✓ Acquired via inhalation of contaminated aerosols

Chlamydophila pneumoniae (TWAR strain)

✓ Associated with mild respiratory tract infections

Chlamydia trachomatis

Subtypes Clinical syndrome

A, B, Ba, C Endemic trachoma (multiple or persistent infection, may lead to blindness if left untreated)
D-K Urethritis, Cervicitis, PID, infant pneumonia and conjunctivitis
L1, L2, L3 Lymphogranuloma venereum

2 MYCOPLASMA

✓ Smallest, free living organism

✓ Lack cell wall, pleomorphic in appearance
✓ Pleuropneumonia like organisms PPLOs

Mycoplasma pneumoniae

✓ Eaton’s Agent: frequent cause of community acquired pneumonia

✓ Primary/Atypical Pneumonia

Genital Mycoplasmas: M. hominis and U. Urealyticum

✓ Cause of non-gonococcal urethritis in males

✓ M. hominis: agent of salpingitis and post-partum fever in females
3 RICKETTSIAE

✓ includes: Rickettsia, Ehrlichia, Coxiella and Rochalimea

✓ G- obligate intracellular bacteria
✓ Infections are spread through insect vectors: lice, ticks and fleas
✓ All rickettsia, except Coxiella cannot survive outside the animal host or insect vector

GROUP SPECIES INFECTION TRANSMISSION

Spotted Fever Rickettsia rickettsia Rocky Mountain Spotted Fever Ticks
R. akari Rickettsial Pox Mites
R. australis Australian/Queensland Tick Typhus Ticks
Typhus R. conorii Boutonneuse fever Ticks
Mediterranean and Israeli spotted
fevers; Indian Tick typhus, Kenya Tick
typhus
R. prowazekii Epidemic typhus Lice

Sporadic typhus Flying squirrels

Brill-Zinser typhus Reactivation of latent

transmission
R. typhi Murine Typhus Fleas
Scrub Typhus R. tsutsugamushi Scrub typus Mites, chiggers
Q fever Coxiella burnetti Q fever Ticks, aerosols
Erlichiosis E. chaffeensis Human monocyte erlichiosis Ticks
E. phagocytophila Human granulocyte erlichiosis Ticks
E. ewingii
Neorickettsia sennetsu Sennetsu fever Ticks
Rochalimeae Rochalimea quintana Trench fever Lice

Bartonella

✓ Originally grouped with members of the family Rickettsiales

✓ Short, g(-) rod shaped, fastidious organisms that are oxidase negative and grow best on blood enriched media or
cell culture systems

ORGANISM HABITAT MODE OF TRANSMISSION CLINICAL MANIFESTATION

B. quintana Uncertain, possibly small Human body louse Trench fever
rodents, humans Chronic bacteremia,
endocarditis, bacillary
angiomatosis, chronic
lymphadenopathy,
pericarditis
Carrion’s disease
B. baciliiformis Uncertain, humans Sand flies Cat-Scratch disease
B. henselae Domestic cats Cat scratch and bites, cat Primary causative agent of
fleas Cat scratch Disease
Bacteremia, endocarditis,
bacillary angiomatosis,
peliosis. Hepatitis and
neuroretinitis
B. clarridgeiae Domestic cats Cat scratch and bites Bacteremia, CSD
B. elizabethae Rats fleas Endocarditis