 Enterobacteriaceae: Glucose Fermenters, Oxidase negative

 Non-Enterobacteriaceae: Glucose Fermenters, Oxidase 

Gram Negative Bacilli  Non-Fermentative Organisms
 Fastidious Organisms
Culture Media

Medium Reagents Fermenter NonFermenter

MacConkey CHO: Lactose
Inhibitor: Crystal Violet, Bile Salts Pink Colorless
Indicator: Neutral Red
Salmonella Shigella Agar CHO: Lactose
(SSA) Inhibitor: Bile Salts Red Colorless
Indicator: Neutral Red
Thiosulfate Citrate Bile salt CHO: Sucrose
Sucrose (TCBS Agar) Inhibitor: Bile Salts Yellow Green
Indicator: Thymol Blue, Bromthymol Blue
Eosin Methylene Blue CHO: Lactose
(EMB) Inhibitor / Indicator: Pink-purple Colorless
Eosin Y, Methylene Blue
Xylose Lysine CHO: Lactose
Deoxycholate (XLD) Inhibitor: Xylose, Lactose, Sucrose Yellow Red
Indicator: Phenol Red
Hektoen Enteric Agar CHO: Salicin, Lactose, Sucrose
Inhibitor: Bile Salts Yellow Green
Indicator: Bromthymol Blue

Biochemical Testing for Enterobacteriaceae

 Oxidase Test  Nitrate Reduction Test
 screening for Gram negative organisms o Principle: NO3  NO2
o Cytochrome Oxidase o Reagent: Sulfanilic Acid, -napthylamine
o  = bluish purple; (-) = no color change o  Red Color
o Enterobacteriaceae are Oxidase (-) o (-) = colorless; to confirm: Zinc Powder
o Control Organisms:  True  = no color change
  = Pseudomonas aeruginosa  True (-) = red
 (-) = E. coli

 O-nitrophenyl-beta-D-galactophyranoside  Decarboxylase test (LOA)

(ONPG) test o Lysine  Cadaverine
o Detects Late Lactose Fermenters o Ornithine  Putrescine
o ONPG  Orthonitrophenol (yellow) in o Arginine  Citrulline
presence of beta galactosidase* o Bromcresol purple: pH indicator
 -galactosidase: for fermentation of o Glucose: CHO source
lactose (LF, LLF) o Medium: Moeller’s Broth
 -galactoside permease: for o Add mineral oil to all 3 tubes (+ 1 control
organisms’s absorption of lactose tube)
(LF, not in LLF) o  = purple; (-) = yellow

 Triple Sugar Iron Test A/A Gas  H2S  = Proteus, Citrobacter freundii
o Reagents: A/A Gas  H2S neg = EKE (E.coli, Klebsiella,
 Phenol red: pH indicator Enterobacter)
 Ferrous sulfate: H2S indicator ( = black) A/A Gas (-) H2S neg = Shigella, Yersinia
 Triple Sugar: Lactose, Glucose, Sucrose K/A Gas  H2S neg = SPACEd (Salmonella, Proteus,
o Reactions: Arizona, Citrobacter, Edwardsiella)
 A/A (Lactose Fermenters) K/A Gas (-) H2S neg = SPS (Shigella, Providencia,
 K/A (Non-Lactose Fermenters) Serratia)
 K/K (Nonfermenters) K/K Gas (-) H2S neg = Pseudomonas, NFOs
  Gas , H2S 
 Lysine Iron Agar o Control Organisms:
o Detects Decarboxylation & Deamination   K. pneumoniae
o Reagents:  (-) E.cloacae
 Bromcresol Purple: pH Indicator  K/A H2S  = Salmonella
 Ferric Ammonium Citrate: H2S indicator  K/A H2S (-) = Shigella
o Slant: Deamination  R/A H2S (-) = PPM (Proteus, Providencia,
  = Purple; (-) = yellow Morganella)
o Butt: Decarboxylation
  = Red; (-) = Purple
IMViC Test
o Indole Test: o Methyl Red Test
 Tryptophan  Indole  Detects mixed acid from glucose fermentation
 Kovac’s / Ehrlich’s Reagent: PDAB (p- (LAFS)
dimethylaminobenzaldehyde)  Methyl Red: Indicator
 : red ring   = red; Control: E. coli
 (-) yellow; Control: E. cloacae

o Citrate Utilization Test o Voges-Proskauer

 Simmon’s Citrate medium  Detects butylene glycol from glucose
  Blue; Control: Pseudomonas fermentation
aeruginosa  Reagent: Acetoin or acetylmethylcarbinol
 (-) Green; Control: E.coli   Red; (-) Yellow
Note: same base medium: MRVP, or Clark & Lubs
Dextrose broth

Organism I M V C
E. coli + + - -
TSI:
Klebsiella: Urease  - - + +
A/A gas  H2S neg
Enterobacter: Urease neg - - + +
TSI: Salmonella - + - +
K/A Shigella - + - -
Proteus mirabilis - + - V
Urease , LIA R/A
Proteus vulgaris + + - V
o Urease test  Sulfide Indole Motility Medium (SIM)
 Urea is broken down into CO2, H2O, and o Sulfide:  = Black color in butt
Ammonia in the presence of Urease o Indole:  = Pink ring formation after
 Indicator: Phenol Red addition of Kovac’s reagent
  = Pink (-) = Yellow o Motility:  = movement away from stab
 Rapid Urease:
 PPM (Proteus, Providencia, Morganella)
 Slow/Late Urease:
 CKEYS (Citrobacter, Klebsiella,
Enterobacter, Yersinia, Serratia)

o Deaminase Reactions  Gelatin Hydrolysis

 Lysine, Tryptophan, Phenylalanine   Gelatin Liquefaction
 Reagent: 10% Ferric Chloride  Serratia
  Phenylalanine = Green
  Tryptophan = Brown
Enterobacteriaceae  All are motile with peritrichous flagella except:
 Glucose Fermenters, Facultatively anaerobic Shigella, Klebsiella, Yersinia (motile at RT)
 Oxidase Negative, Catalase , Nitrate Reduction  Antigens:
 o Somatic “O” – heat stable
  Opportunistic/Commensals of GIT, o Flagellar “H” – heat labile
 Except: Salmonella, Shigella, Yersinia o Capsular “K” – heat labile
Escherichia coli E. coli biotypes
o Causes: UTI, bacteremia/sepsis, neonatal Traveler’s diarrhea;
Enterotoxigenic
meningitis, pneumonia due to cholera-like toxin
(ETEC)
o Normal flora of GIT of humans Montezuma’s Revenge
o Marker of fecal contamination in water Enteropathogenic Infantile diarrhea
(Coliform) (EPEC)
o Related to Shigella (O antigen) Dysentery; shigella-like diarrhea (no
Enteroinvasive
o Culture: WBCs)
(EIEC)
 MAC: Flat, Dry, pink colonies Non-motile
 BAP: gray-mucoid, -hemolytic Hemolytic-Uremic Syndrome (HUS)
 EMB: “Green-metallic sheen” Enterohemorrhagic  Verotoxin (Shiga-like toxin)
(EHEC) E.coli O157:H7:
Sorbitol Mac (-), MUG (-)
Enterobacter Enteroaggregative Acute & Chronic Diarrhea,
o May resemble Klebsiella on MAC (EAEC) “Stacked-brick” appearance
Uropathogenic UTI
E.coli
Klebsiella pneumoniae
o Aka Friedlander’s Bacillus o Causes: Pneumonia (Currant Jelly-like sputum)
o Capsulated (Mucoid colonies) o UTI
o LOA: Lysine , Urease  Other Klebsiella:
 K. oxytoca: Indole 
 K. rhinoscleromatis: Inert

Proteus Group (PPM) o Providencia:

o Rapid Urease  - assoc. with Renal stones  Nosocomial outbreaks involving burn units
o Phenyl Deaminase ,  Antimicrobial resistant
o Lysine Deamination  (R/A) Urease
o Proteus: H2S  Providencia rettgeri 
 Associated with “burnt gun powder” Providencia stuartii negative
odor
 Swarming on agar; o Morganella
 “Diene’s phenomenon”  Morganella morganii: Citrate (-), Urease 
Indole ODC
Proteus vulgaris  Neg
Proteus mirabilis negative 
Salmonella o S. typhi = typhoid fever
o The most serious pathogen for humans  Assoc. with “Rose spots”
o Associated with chicken, egg, poultry products  1st week: Blood
o Culture:  2nd week: Urine
 MAC: Clear, Colorless colonies  3rd week: Stool
 SSA: Colorless with black center (Fisheye)  Confirmatory: Bone Marrow Culture
  Selenite F broth  Carrier status: Gallbladder
o Gas , except S. typhi
o Antigens: Vi, O, H Salmonella Shigella
S. typhi Typhoid bacillus Motility  Neg
S. typhosa Eberth’s bacillus H2S  Neg
S. enteritidis Gardner’s bacillus Resembles Citrobacter E.coli
Shigella O Antigen Remarks
o Related to Escherichia S. dysenteriae Mannitol (-);
o NOT a normal GI flora A
(Shiga bacillus) most virulent
o Biochemically inert, Nonmotile S. flexneri “Gay bowel”
B
o Associated with dysentery (Strong’s bacillus) syndrome
 Low dose needed (<200) S. boydii
C
o Culture: (Boyd’s bacillus)
 MAC: Clear, colorless colonies S. sonnei ONPG 
D
 SSA: Colorless colonies, no black center (Duval’s bacillus)

Yersinia pestis Yersinia enterocolitica

 NOT normal flora  Motile at 22 C but not at 35 C
 Plague bacillus – Black Death  Gastroenteritis
 Two forms: Bubonic (1st), Pulmonary (2nd)  Cold enrichment at 4 C
 Safety pin appearance – Bipolar bodies  Bulls eye colony on Cefsulodin Irgasan Novobiocin (CIN)
(Wayson stain)  Blood bag contaminant
 Stalactite appearance on broth
 Non motile

Plesiomonas shigelloides Serratia marsescens

 Oxidase  o DNAse , Lipase , Gelatinase 
 Resembles Shigella (K/A, gas (-), H2S (-)) o Produces red pigment (prodigiosin) at RT
 Found in fresh water, seafood o Nosocomial infections, UTI
 Inositol fermenter

 Comma shaped, motile

Vibrionaceae  Halophilic, except V. cholerae
 Oxidase , Catalase , Indole , Nitrate   Alkalophilic

Vibrio cholera  Biotypes: Classical, El Tor

 Cause of Cholera (rice watery stools)  Culture:
 Rapid, shooting-star motility o Yellow halo on TCBS (Sucrose Fermenter)
 Non halophilic o Enrichment: APW
 String Test  (0.5% Na desoxycholate)
Vibrio parahemolyticus Vibrio vulnificus
 Halophilic  Lactose 
 Nonsucrose fermenter
 “Summer Diarrhea”; assoc. with Gastroenteritis
(seafood)
 Kanagama  (-h on Wagatsuma)

Campylobacter Helicobacter
 Curved, darting motility Only bacteria associated with Cancer
 Seagull wings  Peptic ulcer
 Species: C. jejuni, C. coli  Strong urease producer
 Microaerophilic, capnophilic  Urea Breath test 
 Media: Campy’s Blood Agar, Skirrow’s Medium,  Gold standard: Biopsy, staining
Bolton Medium  Stain: Warthin Starry
 K/K, H2S negative
Nonfermentative Organisms  Not normal flora

Pseudomonas aeruginosa  Causes many opportunistic infections:

o Oxidase positive, Oxidizes Glucose  Cystic Fibrosis, Pneumonia, Meningitis, Burns,
o Catalase , Nitrate  Wounds,
o Pyocyanin, Pyoverdin – “green metallic sheen”  Swimmer’s Ear
o Grape like / tortilla-like odor (2-  Resistant to: Iodine, Disinfectants
aminoacetophenone)
 o Acetamide, Cetrimide, 42 C

Burkholderia mallei Burkholderia pseudomallei (Whitmore bacillus)

 Glander’s Disease  Vietnamese time bomb
 Oxidase , Non motile  Melioidosis
Acinetobacter
 Acinetobacter baumanii
 Acinetobacter lwoffi
Haemophilus X Factor: Hemin
V Factor: NAD
Haemophilus influenza Haemophilus aegypticus
 Pfeiffer’s Bacillus  Koch-Week bacillus
 Normal flora  Pink eye conjunctivitis
 Encapsulated
 Mousy odor on plated media Haemophilus ducreyi
 Satellitism  Soft chancre
 6 serotypes; B: associated with meningitis  School of fish

X-Factor V-Factor D-ALA (Porphyrin)

H. influenzae + + -
H. parainfluenzae - + +
H. haemolyticus + + -
H. parahaemolyticus - + +
H. aegypticus + + -
H. aphrophilus - - +
H. paraphrophilus - + +
H. ducreyi + - -

HACEK Group:  Haemophilus parainfluenzae

 Subacute Bacterial Endocarditis  Aggregatibacter
 Cardiobacterium
 Eikenella
 Kingella
Brucella  Brucella abortus
 Bang’s bacillus  Brucella melitensis
 Nonmotile; nonhemolytic
 Media: Castañeda’s Bottle
 Capnophilic
 Growth is inhibited by dyes
Bordetella pertussis Legionella
 Strict aerobe  Legionnaire’s Disease
 Whooping cough  Media: BCYE
 Nonmotile except: B. bronchiseptica  Specimen: Sputum
 Media: Bordet Gengou  L. pneumophila: Pontiac Fever
 Regan Lowe (preferred)  L. micdadei: Pittsburgh Pneumonia
 Mercury like droplet colonies  L. bozemanii: WIga’s agent
Francisella Pasteurella
 Tularemia  P. multocida
 Rabbit Fever  Shipping fever
Streptobacillus monilliformis Gardnerella
 Rat bite fever / Haver hill fever  Bacterial vaginosis
 Clue cells
 Media: HBT
Chromobacterium violaceum Eikenella corrodens
 Violacein – violet pigment  Human bite
 Bleach-like odor; Pitting on agar