CLBACT - Order Enterobacterales

Order Enterobacterales

• Primarily agents of gastroenteritis and UTI.
• Facultative anaerobic gram-negative bacilli.
• Glucose fermenters.
• Catalase positive and oxidase negative.
• Nitrate reducers.

Enterobacterales families

Five most significant: Enterobacteriaceae, Hafniaceae, Erwiniaceae, Morganellaceae, Yersiniaceae.

Catalase rule

Catalase Positive EXCEPT Shigella dysenteriae.

Oxidase test

Detects cytochrome oxidase (indophenol oxidase). The enzyme oxidizes a colorless reagent to become a colored product.

Kovac's Oxidase Reagent

Most common oxidase reagent; used in the Spot Method.

ESKY

The non-motile Enterobacterales: Escherichia albertii, Shigella, Klebsiella other than K. aerogenes, and E. asburiae — plus all pathogenic Yersinia at 37 deg C.

Plesiomonas shigelloides

• Outlier in Enterobacterales — the only oxidase positive member.
• Tri-decarboxylase positive.
• Emerging pathogen: watery diarrhea, colitis-like dysentery, subacute/chronic disease.

Primary intestinal pathogens

Escherichia and Shigella — agents of dysentery; can cause outbreaks.

H2S producers

Salmonella and Citrobacter — enteric/typhoid fever and various nosocomial infections.

H2S detection

Detected in specialized agars (HEA, XLD, SSA) and in Sulfide-Indole-Motility medium by the production of a black color.

Voges-Proskauer positive genera

Enterobacter and Klebsiella — mainly pneumonia / UTI and various nosocomial infections.

Family Morganellaceae

Proteus, Providencia, Morganella. Urease producers and deaminase producers.

Family Hafniaceae

Edwardsiella tarda and Hafnia alvei. E. tarda resembles E. coli (indole and MR positive) but is H2S positive.

Family Erwiniaceae

Pantoea agglomerans — wound infections, contaminated intravenous fluids.

Family Yersiniaceae

Yersinia and Serratia. Generally environmental isolates, not in the normal microbiota of man.

O antigen

The lipopolysaccharide of the gram-negative cell wall.

K antigen

The capsular antigen, as in K. pneumoniae. Masks the O antigen in serotyping.

Vi antigen

Capsular antigen found in specific Salmonella enterica serotypes — Typhi and Paratyphi C.

H antigen

The antigen of the flagella. Most Enterobacterales possess flagella.

lacY

Encodes the permease that transports lactose into the bacterial cell.

lacZ

Encodes the enzyme that hydrolyzes lactose into glucose and galactose.

Rapid lactose fermenters

18-24 hrs: Escherichia, Klebsiella, Enterobacter, Cronobacter spp., Pantoea spp.

Late lactose fermenters

ONPG positive: Citrobacter (within 24 hrs), Shigella sonnei, Salmonella bongori, Serratia, Hafnia, Yersinia.

MacConkey Agar

• Selective-differential.
• Selective for gram-negative.
• Differentiates gram-negative according to lactose fermentation.
• Inhibitors of gram-positive bacteria include bile salts.

Eosin-Methylene Blue Agar

• Selective and differential on lactose and sucrose fermentation.
• Red to blue-black colonies.
• Primarily used in coliform counts.
• Indicators and inhibitors: Eosin Y and Methylene Blue.

Greenish metallic sheen

Growth on EMB indicative of Escherichia coli.

CLED agar

Cystine-Lactose-Electrolyte-Deficient agar. For isolation, enumeration and identification of urinary pathogens on the basis of lactose fermentation.

L-Cystine

Component of CLED that supports growth of dwarf coliforms.

CLED indicator

Bromthymol blue.

CLED electrolytes

CLED lacks electrolytes in order to inhibit swarming.

Calibrated loop

Calibrated Loop = 0.001 mL; 1 colony = 1,000 CFU; > 10,000 CFU is significant.

Triple Sugar Iron Agar

• Butt: GLU fermentation; gas production cracks the agar.
• Slant: LAC and SUC fermentation.
• H2S indicators: ferrous sulfate and sodium thiosulfate.
• Inoculate by stabbing the butt, then streaking the slant.

TSI K/K

Red slant, red butt. No gas, no H2S — no fermentation of the three sugars.

TSI K/A

Red slant, yellow butt. No gas, no H2S — glucose only.

TSI A/A

Yellow slant, yellow butt with gas — glucose plus lactose and/or sucrose.

TSI A/A H2S+

Yellow slant, yellow butt, with gas and H2S.

TSI K/A H2S+

Red slant, yellow butt, with gas and H2S.

SIM medium

• Peptone is the source of tryptophan for indole production.
• Peptonized iron and sodium thiosulfate are indicators of H2S production.
• Kovac's Reagent is added only after incubation and after reading motility and H2S.

Motility in SIM

Non-motile: sharp edge at the stab line. Motile: diffuse edge, or turbid throughout the tube.

IMViC

Indole, Methyl Red, Voges-Proskauer, Citrate.

Indole test

Detects the enzyme that converts tryptophan to indole.

Methyl Red test

Detects the ability of an organism to use the mixed acid fermentation pathway.

Voges-Proskauer test

Detects the ability to use the 2,3-butanediol fermentation pathway.

Citrate utilization test

Detects citrate permease, which allows organisms to convert citrate to pyruvate, allowing it to be their carbon source.

Mixed acid pathway

End products: lactic, acetic, succinic and formic acids plus ethanol. Marker: high concentration of stable hydrogen ions.

2,3-butanediol pathway

End products: acetoin (precursor), then 2,3-butanediol and ethanol. Marker: the acetoin (acetylmethylcarbinol) intermediate.

Barritt's reagents

Barritt's A (5% alpha-naphthol) and Barritt's B (40% KOH) — for Voges-Proskauer.

KES

Klebsiella, Enterobacter, Serratia — the VP positive representative taxa.

MRVP broth

• Glucose phosphate broth used for MR and VP.
• A single broth is inoculated, then partitioned into two after incubation.
• Contains dextrose, buffered peptone and dipotassium phosphate.

Simmon's Citrate

Detects citrate permease. Indicator: bromthymol blue. Also contains sodium citrate and magnesium sulfate.

Lysine decarboxylase

Decarboxylates lysine into cadaverine.

Ornithine decarboxylase

Decarboxylates ornithine into putrescine.

Arginine dihydrolase

Decarboxylates arginine into agmatine, and agmatinase hydrolyzes it into putrescine. The dihydrolase converts arginine to citrulline, then ornithine, producing putrescine.

LOA

The decarboxylase triplet, in order: lysine, ornithine, arginine. Prepare 3 tubes; mineral oil is layered on top of the media. Indicator: bromcresol purple.

Christensen's Urea Agar

Urease splits urea into ammonia; ammonia causes pH to alkalinize. Indicator: phenol red. Positive result: pink-to-red.

PPM

The rapid urea hydrolyzers: Providencia rettgeri, Proteus spp., M. morganii.

Phenylalanine Deaminase Test

Phenylalanine agar slant; phenylalanine becomes phenylpyruvic acid, which with FeCl3 gives a green solution. Reagent: 10% FeCl3 added directly to the slant after incubation.

Lysine Iron Agar

• Contains peptones, 0.1% glucose, bromcresol purple, lysine, ferric ammonium citrate, sodium thiosulfate.
• Default result is K/A.
• Blackening of butt means H2S, except in R/A reactions.

LIA R/A

Lysine deamination. The unstable ketoacid reacts with ferric ammonium citrate to produce a red/burgundy color in the presence of O2.

LIA K/K

Lysine decarboxylation.

Stool culture media

Sheep's Blood Agar; MacConkey / Eosin Methylene Blue; Hektoen-Enteric / Xylose-Lysine Deoxycholate / Salmonella-Shigella; Triple Sugar Iron; Lysine Iron.

Hektoen-Enteric Agar

• Sugars: lactose, sucrose, salicin.
• Inhibitors: bile salts, citrate.
• Indicators: bromthymol blue and acid fuchsin.
• Fermenter yellow-orange (more salmon with salicin); non-fermenter green to blue-green.

Salmonella-Shigella Agar

• Sugar: lactose.
• Inhibitors: bile salts, citrate, brilliant green.
• Fermenter pink; non-fermenter colorless.
• H2S positive gives a black center in colonies.

Xylose-Lysine-Deoxycholate Agar

• Sugars: xylose, lactose, sucrose.
• Inhibitor: sodium deoxycholate.
• Fermenter yellow; non-fermenter red.
• Contains lysine to check LDC activity; positive LDC gives red surrounding.

XLD yellow

Acidic pH; fermented xylose and lactose and/or sucrose.

XLD red

Non-fermenter of xylose, lactose/sucrose. Or a possible fermenter of xylose in which lysine decarboxylation alkalinized the medium, reverting it to red.

XLD black center

The organism is an H2S producer.

Enteric H2S system

Source: sodium thiosulfate. Detector: ferric ammonium citrate or ferric ammonium chloride. Positive: black center in colonies.

Family Enterobacteriaceae

• Largest family under Order Enterobacterales.
• Most are commensals.
• Salmonella and Shigella are strictly human pathogens.
• Includes the outlier oxidase-positive Plesiomonas.

Motility rule

Motile EXCEPT Escherichia albertii, Shigella, Klebsiella other than K. aerogenes, E. asburiae.

Indole rule

Indole negative EXCEPT Escherichia, Citrobacter and Klebsiella oxytoca. C. freundii is indole negative.

Methyl Red rule

Methyl Red positive EXCEPT Klebsiella other than K. ozaenae, Enterobacter, and Cronobacter.

Citrate rule

Citrate positive EXCEPT Escherichia, Shigella, S. Typhi and S. Paratyphi A.

H2S rule

H2S negative EXCEPT Salmonella and Citrobacter. S. Paratyphi A and S. Choleraesuis are H2S negative.

Urea rule

Urea negative EXCEPT Citrobacter koseri, Klebsiella, Enterobacter. K. rhinoscleromatis and K. aerogenes are urea negative.

Escherichia and Shigella

Shared features: MR positive, VP negative, citrate negative. Key differences: motility, lactose fermentation and indole.

Escherichia coli

• Part of the normal flora of the intestines.
• Motile gram-negative bacilli; lactose fermenter.
• Pink colonies on MAC; blue-black colonies on EMB with greenish metallic sheen.
• MOT: oral-fecal route.

E. coli biochemicals

Indole +; Methyl Red +; Voges Proskauer -; Citrate -; LDC +; ODC +; ADH -; Acetate +; TSI A/A with no H2S.

Diarrheagenic E. coli

STEC (Shiga-toxin producing), ETEC (enterotoxigenic), EPEC (enteropathogenic), EIEC (enteroinvasive), EAEC (enteroaggregative), DAEC and AIEC.

Extraintestinal E. coli

ExPEC: UPEC (uropathogenic) and MNEC (meningitis/sepsis-associated).

STEC

Shiga-toxin producing E. coli, aka verocytotoxin-producing (VTEC). Encodes Shiga toxins (stx) 1 and/or 2 which mimic the Shigella dysenteriae cytotoxin.

EHEC

Enterohemorrhagic E. coli, a subset of STEC. Causes hemorrhagic colitis and Hemolytic Uremic Syndrome.

Hemolytic Uremic Syndrome

Bloody diarrhea, microangiopathic hemolytic anemia, thrombocytopenia, acute renal failure.

E. coli O157:H7

• Serotype of STEC / EHEC.
• Very low infectious dose; highly transmissible and causes outbreaks.
• Mainly foodborne, from beef and dairy products.
• Detected through antigen testing.
• Non-fermenter of sorbitol.

Sorbitol MacConkey

The medium used to isolate E. coli O157:H7, which is a non-fermenter of sorbitol.

MUG Test

E. coli and other Enterobacteriaceae produce beta-d-glucuronidase, which hydrolyzes beta-d-glucopyranoside-uronic derivatives. STEC strains are the exemption.

MUG principle

4-methylumbelliferyl-beta-d-glucuronide is impregnated into a disk to be hydrolyzed by bacterial enzyme, yielding the 4-methylumbelliferyl moiety, which fluoresces under long wavelength ultraviolet light.

ETEC

Produces a heat-labile enterotoxin (LT) and a heat-stable enterotoxin (ST). An important cause of Traveler's Diarrhea — watery but non-bloody, with high prevalence during warm seasons in tropical areas.

EPEC

• Non-invasive, non-toxin producing, high infectious dose.
• Induces architectural changes in intestinal epithelial cells and microvilli.
• Locus of Enterocyte Effacement pathogenicity island, encoding Tir and Intimin.

Attaching-and-effacing lesions

The A/E lesions produced by EPEC in the intestinal epithelial cells and microvilli.

EIEC

• High genetic and phenotypic similarity to Shigella; contains the pINV plasmid.
• Infections mimic the dysentery caused by Shigella spp.
• Produces enterotoxins set2 and sigA that potentiate fluid loss.

EAEC

• Adheres to epithelial cells focally in a stacking fashion.
• Enhanced biofilm formation and adherence via the aggR gene.
• Acquires virulence factors through conjugation or phage transduction.
• Persistent watery non-bloody diarrhea.

DAEC

Diffuse-adhesive E. coli — diffused adherence pattern covering a large surface.

AIEC

Adherent invasive E. coli — found to have a correlation with Crohn's disease.

UPEC

• Most implicated cause of UTI in children and women.
• Encodes pyelonephritis associated pili (papA and papG).
• Many strains carry Extended-Spectrum Beta-Lactamases.

E. coli ST131

Strain of interest in UPEC. Accounts for 25-70% of ESBL-producing urinary isolates globally. Belongs to serogroup O25.

MNEC

Expresses a capsular antigen, invasins, and cytotoxic necrotizing factor 1, which allows the organism to pass the blood-brain barrier. Implicated in neonatal meningitis; second leading cause of meningitis in immunocompromised hosts.

Escherichia albertii

• Previously known as Shigella boydii serotype 13.
• Primary reservoir: wild birds and racoons.
• Contains cdtB, a cytolethal toxin also found in Clostridioides difficile and STEC.
• May carry stx2. Variable indole production.

Shigella spp.

• Causes Bacillary Dysentery (Shigellosis) — severe diarrhea with blood and mucus.
• Strictly a human pathogen, not found in gut flora.
• Low infective dose: 10-100 organisms.
• Rarely disseminates; may resolve without treatment.

Shigella shedding

The host will still shed viable bacteria in stool for up to 4 weeks.