GRAM-NEGATIVE BACILLI Escherichia coli

Enterobacteriaceae • Most significant species in the genus

Escherichia
General Characteristics: • AKA: Colon bacillus
• Gram-negative, non-spore forming, • Important human pathogen associated with
facultatively anaerobic bacilli many disease
• Microscopy: can appear as coccobacilli or as • May inhibit female genital tract & leading cause
straight rods of nosocomial
• All are motile at 35 deg. C. with peritrichous urinary tract infection
flagella except for • Microbiota of large intestine
Klebsiella, Shigella, & Yersenia • Primary indicator of fecal contamination in
• All are non-encapsulated except Klebsiella & water purification
Enterobacter • Motile and possess adhesive fimbriae & sex pili
• All ferments glucose & reduce nitrate to nitrite • Possess O, H, & K antigens
• All are catalase positive & oxidase negative
except P. shigelloides Escherichia coli Properties
• BAP/CAP: large, moist, gray colonies, non- • MAC- flat, dry, pink colonies
hemolytic; Klebsiella & • BAP- some strains are nonhemolytic, others
Enterobacter- large & very mucoid; E. coli- beta beta hemolytic
hemolytic • EMB- greenish metallic sheen
• Ferments glucose, lactose, trehalose, & xylose
Serologic characteristics (Antigens) • Production of indole from tryptophan
• O antigen • Cannot use citrate as a sole carbon source
➢ Somatic antigen • Virulence factor: endotoxin, common pili, K1
➢ Heat stable antigen located on cell wall antigen, intimin
• H antigen
➢ Flagellar antigen Escherichia coli Biochemical Test
➢ Heat labile antigen found on the surface • TSI= A/A (+) Gas, (-) H2S
of flagella • IMViC= + + - -
➢ Structure responsible for motility • PAD (-)
• K antigen • Urease (-)
➢ Heat-labile polysaccharide found only in
encapsulated species
• Vi antigen
➢ Capsular antigen of S. enterica subsp.
Enterica serotype Typhi

Opportunistic Pathogens
• Escherichia
• Klebsiella
• Enterobacter
• Serratia
• Hafnia
• Proteus
• Morganella
• Providencia
• Edwardsiella
• Citrobacter
Escherichia coli strains (Gastrointestinal)

Other species
• E. hermanii
- Clinical significance not yet established
- Yellow-pigmented organism
- CSF, wounds, blood
- Raw milk, beef (same with EHEC
Serotype O157:H7)
• E. vulneris
- More than half of strains are yellow-
pigmented colonies
- Isolated from infected wounds
 Klebsiella species
• Usually found in GIT or humans & animals or Other Klebsiella species
free-living in soil, water, & plants • Klebsiella pneumoniae subsp. Ozaenae
• Associated with opportunistic & hospital- - Causative agent of chronic atrophic
acquired infections-pneumonia, wound rhinitis/ “foul-smelling” atrophic rhinitis
infections, & UTI •Klebsiella pneumoniae subsp.Rhinoscleromatis
• (+) Citrate utilization - Causes granuloma of the nose &
• (-) H2S production oropharynx
• (-) MR, (+) VP • Klebsiella oxytoca & Klebsiella ornithinolytica
• (-) Indole expect for K. oxytoca & K. - Indole positive
ornithinolytica
• Non-motile Enterobacter species
• Encapsulated • Motile
• Hydrolyze urea slowly • Resembles Klebsiella on MAC
• MAC-pink colonies • Common isolate: E. aerogenes & E. cloacae
• (+) citrate utilization
Klebsiella pneumoniae subsp. pneumoniae • (+) KCN
• AKA: Friedlander’s bacillus • (+) ornithine decarboxylase
• Most common isolated species • (+) Lysine decarboxylase except E. aerogenes &
• Have large polysaccharide capsule- virulence E. cloacae
factor and • (+) sorbitol fermentation
causes mucoid & moist colonies in MAC • (+) urease
• Causative agent of community-acquired • (+) malonate test
pneumonia • IMViC - - + +
• “currant jelly like sputum” • TSI A/A (+) gas (-) H2S
• Frequent cause of lower respiratory tract
infections among
immunocompromised patients
• (+) string test
• IMViC= - - + +
• TSI= A/A, (+) gas, (-) H2S
 Serratia species the pH & encourages formation of renal stones)
• Associated with outbreaks in health care • Human pathogens: P. mirabilis, P. vulgaris
settings • P. mirabilis exhibits swarming on nonselective
- S. marcescens, S. rubidaea= urinary/ media (burnt-chocolate/ burnt-gun powder
respiratory tract & in bacteremic odor)
outbreaks in nurseries & cardiac • MAC- NLF; colorless & clear
surgery/ burn units
- Contamination of antiseptic solution Biochemical P. mirabilis P. vulgaris
used for joint injections Test
• Known for resistance to a wide range of Indole - +
antibiotics PAD + +
• MAC- NLF; clear colorless but may be slow or LIA R/A R/A
late lactose IMVic -+vv ++-v
fermenters TSIA K/A (+) gas K/A (+/-) gas
• (+) ONPG, DNase, gelatinase (+)H2S (+) H2S
• IMViC - - + +
• TSI K/A (+) gas (-) H2S
• S. marcescens, S. rubidaea, S. plymuthica Morganella species
produce a • M. morganii only species
characteristics pink to red pigment, prodigiosin, • Same biochemical reactions with P. vulgaris
when cultured at except for citrate
room temp utilization
• S. odorifera have a dirty, musty odor • MAC- NLF; colorless, clear
resembling that of rotten • PAD (+)
potatoes • LIA: R/A
• TSI K/A (+) gas (-) H2S
Hafnia species • (+) urease, ornithine decarboxylase
• H. alvei only species • IMViC + + - -
- H. alvei biotype 1 grows in beer wort of
breweries (has not been isolated Providencia species
clinically) • MAC- NLF; clear & colorless
• Linked to gastroenteritis and occasionally • PAD (+)
isolated from stool • IMViC + + - +
cultures • LIA: R/A
• IMViC - - +(v) + • TSI: K/A (-) gas (+) H2S
• TSI K/A (+) gas (-) H2S
Consist of 5 species:
Proteus species P. alcalifaciens, P. stuartii, P. rettgeri, P.
• Part of Proteeae (PPM) which can deaminate rustigianii, P. heimbachae
phenylalanine • P. stuartii- outbreaks in burn units; isolated in
• Isolated from urine, wounds, ear, & bacteremic urine cultures; resistant to antibiotics
infections • P. rettgeri- pathogen of urinary tract & has
• Can infect proximal kidney tubules & can cause caused outbreaks in health care settings
acute glomerulonephritis (patients w/ urinary • P. alcalifaciens- commonly found in feces of
tract defect or children w/ diarrhea (role not proven)
catheterization)
• Rapid urease producers (split urea in urine,
which raises
 Edwardsiella species • Do not grow in medium containing potassium
• MAC- NLF; clear & colorless cyanide
• Urease (-) • MAC- NLF; clear & colorless
• IMViC + + - - • SSA- colorless with black center
• TSI: K/A (+) gas (+) H2S • Virulence factor: fimbriae, ability to traverse
• Species: E. tarda intestinal mucosa
• Antigenic structures
Citrobacter species - Somatic O & Flagellar H- serologic
• Resembles E. coli on MAC but biochemical test grouping
of Salmonella species - Vi antigen (Salmonella serotype Typhi)-
• All species grow on Simmons citrate medium/ antiphagocytic
citrate (+) • LDC (+) except Salmonella serotype Paratyphi A
• Urease (+)- slowly hydrolyze urease • Urease (-)
• Normal flora in GI tract & associated with • IMViC - + - +
hospital acquired infections (UTI) • TSI: K/A (+) gas (+) H2S
• IMViC - + - +
• TSI: A/A or K/A (+) gas (+) H2S Salmonella species- Clinical Infection
• Acute gastroenteritis
C. freundii - Most common form of food poisoning
• Isolated in diarrheal stool cultures - Source pf infection attributed mainly to
(extraintestinal pathogen- not established) poultry, milk, eggs, & egg products
• UTI, pneumonia, intraabdominal abscesses in - Cooking utensils used in preparing
hospital setting contaminated foods is the mode of
• Endocarditis in drug abusers dissemination
• H2S (+) - Infective dose: 106
• Fails to ferment lactose • Enteric fever (Typhoid fever)
• Colony morphology of C. freundii on primary - Ingestion of food contaminated with the
selective media can be mistaken for Salmonella organism originating from infected
when isolated on stool cultures persons/carriers
- Urea (+); Lysine decarboxylase (-) for C. - Causative agent: Salmonella enterica
freundii subsp. enterica serotype Typhi
- Urea (-); Lysine decarboxylase (-) for - Outbreaks from improper disposal of
Salmonella species sewage, poor sanitation, & lack of
modern potable water system
- Common in tropical & subtropical area
Primary Intestinal Pathogens - Onset of symptoms depends on quantity
• Salmonella of inoculum
• Shigella - During 2nd-3rd week of infection,
• Yersinia organism invades the gallbladder &
Peyer’s patches of the bowl
Salmonella species - Rose spots (blanching, rose-colored
• Gram-negative, facultatively anaerobic bacilli papules around umbilical region
that morphologically appears)
resembles other enteric bacteria • Bacteremia
• Do not ferment lactose - Caused by nontyphoidal Salmonella
• Negative for indole, VP, PAD, & urease - Prolonged fever and intermittent
• Produce H2S except S. Paratyphi A bacteremia
 - Serotype: Typhimurium, Paratyphi, & Shigellosis/ Bacillary dysentery
Choleraesius • Commonly caused by S. dysenteriae type 1
- Observed in (1) young children with • Acute inflammatory colitis & bloody diarrhea
fever & gastroenteritis with brief with WBCs & mucus
episodes of bacteremia, and (2) adults • Only requires a low infective dose to produce a
with transient bacteremia during disease (<200 bacilli)
episodes of gastroenteritis or develop • Young children: rectal prolapse occur due to
symptoms of septicemia without excessive straining
gastroenteritis
Yersinia species
Shigella species Only 3 species are considered human pathogens
• Closely related to Escherichia ➢ Y. pestis- plague
• Not a normal flora in GI tract ➢ Y. pseudotuberculosis- mesenteric
• All Shigella species can cause Bacillary lymphadenitis in human
dysentery ➢ Y. enterocolitica- infection that mimics
• Nonmotile appendicitis
• Does not produce gas from glucose except S. • Other members are found in water, soil, and
flexneri lower animals
• Urease (-)
• H2S (-) Yersinia pestis
• Lysine decarboxylase (-) • Causative agent of ancient disease- plague
• All species possess O antigens (disease primarily of rodents)
• All species are mannitol fermenters except S. • AKA: Plague bacillus
dysenteriae • Class A bioterrorism agent
• MAC-NLF; clear, colorless, fragile • Transmitted by bites of fleas
• SSA- colorless without black centers • 3 forms: bubonic, septicemic, pneumonic form
• IMViC: v + - - - Symptoms: Buboes (swollen lymph
• TSI: K/A (-) gas (-) H2S nodes)
• Humans are the only known reservoir • Gram-negative, short, plump bacillus with a
• Transmission is through direct P2P contact via bipolar staining pattern
fecal –oral route, flies, finger, & food or water • MAC- NLF; colorless, clear
contaminated by infected person • BAP: Pinpoint colonies after 24 hours
• Broth: stalactite-shaped pattern
S. sonnei • IMViC: - + - -
• Decarboxylate ornithine • TSI: K/A (-) gas, (-) H2S
• Slow LF- delayed positive fermentation with
formation of pink colonies on Yersinia enterocolitica
MAC only after 48 hours • Most isolated species of Yersinia
• ONPG (+) • Found in wide variety of animals, including
domestic swine, cats, & dogs (infection can be
S. dysenteriae acquired from contact with pets)
• Most virulent of the species • Infections occur after ingestion of
• Virulence factor: shiga toxin contaminated food- pork & vacuum-packed deli
meat, beef, lamb, chicken, & possibly milk &
water
• Causative agent of enterocolitis or waterborne
gastroenteritis
• Gram-negative, coccobacilli, with a bipolar Other Enterics
staining pattern • Plesiomonas
• Optimal growth temperature: 25-30 degrees • Cronobacter
Celsius • Pantoea
• SIM- Motility seen at 25 degrees Celsius
• Selective media: CIN medium
• MAC- NLF; colorless, clear Plesiomonas shigelloides
• IMViC: v + - - • Straight bacilli, which can occur singly, in pairs,
• TSI: K/A (-) gas, (-) H2S in short chain or filamentous
• Motile (+) polar flagellum either peritrichous or
monotrichous
• Causes Secretory diarrhea
• Only oxidase positive member
• Associated with PLHIV with inflammatory
bowel disease
• Antigenic structure: O & H antigens
• MOT: ingestion of undercooked seafood
(oysters & shrimp) & contaminated water
• MAC- NLF; clear & colorless
• BAP- shiny, opaque, smooth, & non-hemolytic
• Inositol-brilliant green-bile salt agar- white or
green to pink color for other enterics
• IMViC: + + - -
• TSI: K/A (-) gas, (-) H2S
• Decarboxylase (+)

Yersinia pseudotuberculosis Cronobacter sakazakii

• Like Y. pestis, pathogen is primarily of rodents- • Formerly known as Enterobacter sakazakii
guinea pigs • Contaminant of powdered infant formula
• Birds are natural reservoir which may cause neonatal meningitis
• Causes psuedotubercules (caseous swellings) • Isolated from individuals with brain abscesses,
in animals & respiratory and wound infections
• Human infections are rare, only associated with • MAC- pink color (LF)
close contact with infected animals or ingestion • BHIA- yellow pigmentation
of contaminated food/ drink • IMViC: - - + +
• Spreads to mesenteric lymph nodes • TSI: A/A (+) gas, (-) H2S
(generalized infection which is self-limiting)
• SIM- Motility seen at 18-25 degrees Celsius Pantoea species
• MAC- NLF; colorless, clear • Encompasses several species, including
• Urease & rhamnose fermentation (+) Pantoea agglomerans (formerly known as
• TSI: K/A (-) gas, (-) H2S Enterobacter agglomerans), Pantoea dispersa,
Pantoea ananatis, etc.
• Nosocomial outbreaks of septicemia due to
contamination of IV fluids
• Triple decarboxylase negative
• MAC- NLF; clear & colorless
• IMViC: - v + v
• TSI: K/A (-) gas, (-) H2S
Other Non-enteric Gastrointestinal Pathogens • Vibrio cholerae O1- causative agent of cholera
• Vibrio • Rice watery stools (contains numerous flecks of
• Aeromonas mucus)
• Campylobacter • Motility: rapid darting/ shooting stars
• Helicobacter • Virulence factor: Cholera toxin (Choleragen/
CTX)
Vibrio species • String test (+)
• Family Vibrionaceae • Oxidase, Indole, lysine, ornithine
• Found in wide variety of aquatic environment decarboxylase (+)
• 4 clinically encountered species: V. cholerae, V. • TSI: A/A (-) gas (-) H2S
parahaemolyticus, V. vulnificus, V. alginolyticus • LIA: K/K (+) lysine decarboxylation
• Asporogenous, gram-negative rods possessing • Epidemic V. cholerae O1 bio groups:
polar, sheathed - Classical - VP (-), does not agglutinate
flagella when grown in broth but can produce chicken RBC, susceptible to polymyxin B
peritrichous, - El Tor- VP (+) , hemolyzes RBC, resistant
unsheathed flagella when in solid media polymyxin B, able to agglutinate chicken
• Catalase & oxidase (+) RBC
• Reduces nitrate to nitrite except for V.
metschnikovii (negative)
• Susceptibility test: 150 micrograms vibriostatic
O/129 disk in MHA
• MAC- pathogenic are NLF (except V. vulnificus)
• BAP/CAP- medium to large colonies that
appear smooth,
opaque, & iridescent with a greenish hue
• BAP- alpha or beta hemolytic
• TCBS- selective media
➢ Sucrose fermenters: V. cholerae, V.
alginolyticus, V. Metschnikovii Vibrio parahaemolyticus
➢ Non-sucrose fermenters: V. mimicus, V. • Second most common species implicated for
vulnificus, V. parahaemolyticus, V. gastroenteritis
damsela • AKA: Summer Diarrhea
• Alkaline peptone water (pH 8.5)- enrichment • Self-limiting; watery diarrhea, moderate
media (V. cholerae) cramps or vomiting
• Transport medium: Cary-Blair media • Leading cause: V. parahaemolyticus serotype
O3:K6
• Virulence factor: Kanagawa phenomenon
• Heat-stable hemolysin able to lyse human RBCs
in high salt medium
• Selective medium: Wagatsuma agar

Vibrio vulnificus
• Second most serious type of Vibrio- associated
infections
- Primary septicemia & wound infections
• Lactose- positive vibrio
• MOA: eating contaminated raw oysters & fish
Vibrio cholerae - Cholera Vibrio alginolyticus
• Not commonly isolated and least pathogenic - Chronic diarrhea usually lasting more
• Strict halophile that requires a medium with 1- than 10 days
10% NaCl - Cholera-like disease, including rice
• Infections: eye, ear, & wound infections watery stools
- Nebulous syndrome commonly referred
Presumptive Identification to as Traveler’s diarrhea
• Vibrio species- susceptibility to vibriostatic • Septicemia, Meningitis, & wound infection
agent O/129 and (+) (involving leeches), keratitis associated with
string test distinguishes them from Aeromonas contact lens water
• Inability to ferment inositol (except for V. • Causative agent of red-leg disease in
cincinnatiensis & V. metschnikovii) separtes amphibians
them from Plesiomonas • MAC- LF, colonies are pink
• Oxidase reaction (except for V. metschnikovii) • BAP- large, round, raised, white opaque (with
separates them from Enterobacteriaceae extremely strong odor & pigmentation from
(excluding P. shigelloides) translucent & white to buff-colored); hemolysis
• Carbohydrate fermentation metabolism is varied (A. hydrophila, A. veronii biovar sobria,
separates them from oxidative Psuedomonas A.jandaei are beta hemolytic)
• CIN- bulls-eye appearance
Aeromonas species • Indole (+) - A. hydrophila, A. veronii, A. caviae
• Ubiquitous, oxidase-positive, glucose- • TSI: A/A (-) gas (-) H2S- A. caviae
fermenting, gram-negative rods • TSI: A/A (-) gas (+) H2S- A. hydrophila, A. veronii
• Catalase (+) • Positive oxidase distinguishes Aeromonas spp.
• Motile with single polar flagellum and are from Enterobacteriaceae (except P. shigelloides)
facultatively anaerobes • Clinically relevant Aeromonas spp. are indole
• Classified into 2 groups positive
➢ Mesophilic (Optimal growth @ 37 deg. • Distinguishing factor of Aeromonas spp. from
C) Vibrio spp. is string test & vibriostatic O/129
- A. hydrophila complex, A. veronii • Biochemical strip: API 20E
complex, motile strains of A. salmonicida
➢ Psychrophilic (Optimal growth @ 22 Campylobacter species
deg. C) • Faintly staining, gram-negative, small, curved,
- A. salmonicida or S-shaped rods
• Can typically grow at 4-42 deg. C • Old cultures: coccobacilli (Enteric
• Most common isolate: A. caviae campylobacters appear long spirals/ sea-gull
• Common isolate in GI infection: A. caviae shaped)
• Common isolate in HUS: A. hydrophila, A. • Colonies are gray, flat, glistening, & irregular
veronii with tailing effect along the streak line (runny
• Vibriostatic O/129: Resistant spreading growth)
• Inositol fermentation: Negative • Grow in microaerophilic environment except C.
• String test: Negative rectus & C. curvus which are strictly anaerobes
• Level & pattern of virulence is more like the • Causes abortion in domestic animals
multifactorial patterns of various E. coli • Antecedent cause of Guillain-Barre syndrome
subgroups associated with enteric disease • Enteric: C. jejuni, C. coli, C. lari
• Diarrheal presentation • Blood: C. fetus subsp. fetus
- Acute, secretory diarrhea often
accompanied by vomiting
- Acute, dysenteric form of diarrhea (like
shigellosis), w/ blood & mucus
 • Tissue specimen must be kept at 4 deg. C &
processed within 2 hours
of collection
• Urine used for ammonia testing
• Gram stain: 0.1% fuchsin counterstain
(morphology)
- Biopsy stains: Warthin-Starry stain, Silver
stain, Giemsa stain
• Culture: CAP, MTM, Skirrow’s agar, Brucella
• Specimen of choice: Feces, rectal swab, blood agar w/ 5% sheep blood
• Selective media: Campy-BAP for C. jejuni & • Transport media: Stuart Medium (gastric tissue
other enteric campy specimens), cysteine brucella broth w/ 20%
- Brucella agar base, 10% sheep RBC, glycerol, isotonic saline w/ 4% glucose
vancomycin, trimethoprim, polymyxin B,
amphotericin B, cephalothin Helicobacter pylori
• Other media: Butzler medium & Skirrow’s • Major cause of Type B gastritis, peptic ulcer,
medium, Medium V (modification of Butzler gastric cancer
medium with cefoperazone, rifampin, colistin, & • Primary habitat: human gastric mucosa
amphotericin B which inhibits normal colon • Binds with Lewis antigen and monosaccharide
flora), Campy CVA sialic acid
• Transport media: Cary-Blair Medium • Biochemical test: Strong urease producer
• Diagnostic test: Urea breath test
Campylobacter jejuni • Susceptible to metronidazole
• Slow growing, fastidious, saccharolytic
organism Mnemonics!
• Darting motility (monotrichous) and unable to • Non-motile: SKY
grow in media with • Nonencapsulated: KE
high salt concentration • VP (+): ESK
• Most common cause of bacterial • NLF: Edward Clearly Provides Proof Young
gastroenteritis Morgan Slowly Shipped Salmon
• Septic arthritis in PLHIV • H2S (+): Soulless Edward Produces Chemicals
• Optimum temperature for growth: 42 deg. C • Indole (+): Citizen-Killing Pink Vampires Morgan
• MOA: eating contaminated chicken & turkey & Edward Provide Kleptos with oxytocin & Eclairs
• PAD (+): PPM
Helicobacter species
• Campylobacter- like species are Helicobacter &
Wolinella
• Motile by monopolar or multi-bipolar flagella
• Microaerophilic & strong urease activity
• Resembles Campylobacter species in gram
staining
• CAP- gray & translucent
• Oxidase, catalase, urease (+)
• Route of transmission: oral-oral/ fecal-oral
• Specimen: Gastric biopsy tissue, urine, feces,
dental plaque