Clinical Bacteriology: Order Enterobacterales Isolation and Biochemical Identification

Comprehensive vocabulary flashcards reviewing the classification, culture media, biochemical identification tests, differentiating characteristics, and clinically significant species of the order Enterobacterales.

Order Enterobacterales

A major taxonomic order reorganized since 2016 from the former single family Enterobacteriaceae using whole-genome/ANI analysis, comprising Gram-negative, facultatively anaerobic bacilli and coccobacilli.

Oxidase Test Reaction in Enterobacterales

Negative for all typical members (the defining biochemical trait of the order), with the notable exception of Plesiomonas shigelloides, which is oxidase-positive.

Plesiomonas shigelloides

An opportunistic member of Enterobacterales (formerly classified under Vibrionaceae) that is unique for being oxidase-positive and susceptible to compound O/129, possessing monotrichous or 2 to 5 lophotrichous flagella.

SKY Mnemonic for Non-Motile Enterobacterales

Identifies the genera and species that are non-motile at $37^\circ\text{C}$: Shigella, Klebsiella, and Yersinia pestis.

Temperature-Dependent Motility in Yersinia

Yersinia species (except Y. pestis) are motile at room temperature ($22\text{ to }25^\circ\text{C}$) but non-motile at $35\text{ to }37^\circ\text{C}$. Yersinia pestis is non-motile at both temperatures.

Somatic (O) Antigen

A heat-stable lipopolysaccharide component of the cell wall that serves as an endotoxin and defines 67 serogroups in Salmonella via the Kauffmann-White scheme.

Flagellar (H) Antigen

A heat-labile protein antigen located on flagella (flagellin) used for serotyping; it may exhibit phase variation (Phase 1 and Phase 2) in Salmonella.

Capsular (K) Antigen

A heat-labile polysaccharide capsular antigen associated with bacterial virulence.

Vi Antigen

A specific heat-labile capsular virulence antigen found in Salmonella enterica serotype Typhi used to detect chronic carriers via serum anti-Vi antibodies.

Blood Agar Plate (BAP)

A primary, non-selective, enriched medium consisting of tryptic soy or Columbia agar base with $5\%$ sheep or horse blood, used as a baseline recovery plate to assess hemolysis and colony morphology.

MacConkey (MAC) Agar

A selective and differential medium containing bile salts and crystal violet to inhibit Gram-positives, neutral red indicator, and lactose to differentiate pink-red lactose fermenters from colorless non-lactose fermenters.

Sorbitol MacConkey (SMAC) Agar

A selective/differential screening agar replacing lactose with sorbitol to identify presumptive Shiga toxin-producing Escherichia coli O157:H7, which produces colorless colonies due to its inability to ferment sorbitol.

Eosin Methylene Blue (EMB) Agar

A selective/differential medium containing eosin Y and methylene blue dyes that differentiate strong lactose fermenters such as Escherichia coli (which produce a characteristic green metallic sheen) from slow fermenters and non-fermenters.

Hektoen Enteric (HE) Agar Differential Colony Morphology

A highly selective and differential stool culture medium containing bile salts, lactose, sucrose, salicin, bromthymol blue, acid fuchsin, ferric ammonium citrate, and sodium thiosulfate; lactose fermenters appear orange-salmon/yellow (Arrow A), non-fermenters appear green/translucent, and Salmonella appears green with black centers (Arrow B).

Xylose Lysine Deoxycholate (XLD) Agar Differential Capabilities

A selective and differential stool agar differentiating lactose-fermenting coliforms as yellow (Arrow A), Shigella spp. as red/pink colonies without black centers (Arrow B), and Salmonella spp. as red colonies with black centers (Arrow C) due to xylose exhaustion, lysine decarboxylation (reversion to alkaline), and $H_2S$ production.

Salmonella-Shigella (SS) Agar

A highly selective medium containing bile salts, brilliant green, lactose, neutral red, and sodium thiosulfate/ferric citrate that favors Salmonella (producing colorless colonies with black centers) while suppressing coliforms and some Shigella species.

Brilliant Green Agar (BGA)

A highly selective medium containing brilliant green dye and phenol red indicator, used to recover Salmonella (producing pink-white to red colonies surrounded by red medium) while inhibiting most coliforms, Shigella, and Salmonella Typhi/Paratyphi.

Cefsulodin-Irgasan-Novobiocin (CIN) Agar

A selective and differential medium containing antimicrobial agents, mannitol, and neutral red, yielding characteristic 'bull's-eye' colonies (deep red/maroon center with a colorless translucent border) for Yersinia enterocolitica after incubation at $25^\circ\text{C}$.

CHROMagar (Chromogenic Media)

Selective and differential media incorporating enzyme-specific chromogenic substrates, yielding pink to dark red colonies for E. coli ($\beta\text{-glucuronidase}^+$), blue to metallic-blue colonies for the KEC group, and colorless/brown colonies for Proteae.

Selenite F Broth

An enrichment broth containing sodium selenite that selectively suppresses coliforms while allowing Salmonella to multiply from stool specimens before subculturing onto selective agar after $12\text{ to }18\text{ h}$.

Hugh-Leifson Oxidation-Fermentation (O-F) Test

A semisolid, low-peptone, high-carbohydrate ($1\%$ glucose) test using bromthymol blue; fermentative organisms (Enterobacterales) turn both open and oil-overlaid tubes yellow, oxidative organisms turn only the open tube yellow, and non-saccharolytic organisms turn neither tube yellow.

Triple Sugar Iron (TSI) Agar

A screening medium containing $0.1\%$ glucose, $1\%$ lactose, $1\%$ sucrose, peptone, ferrous sulfate ($H_2S$ indicator), and phenol red to assess carbohydrate fermentation patterns, gas formation, and $H_2S$ production.

TSI Slant Reversion Mechanism

The oxidative degradation of peptones in the presence of atmospheric oxygen produces alkaline amines that turn the slant red (alkaline, K) once low glucose ($0.1\%$) is exhausted, while the butt remains yellow (acidic, A) under anaerobic conditions.

Kligler Iron Agar (KIA)

A screening medium identical in principle to TSI but containing only two sugars ($0.1\%$ glucose and $1\%$ lactose) and lacking sucrose.

Lysine Iron Agar (LIA)

A slant/butt medium containing lysine, a small amount of glucose, bromcresol purple, and $H_2S$ indicators to detect lysine decarboxylation (purple butt, K), lysine deamination (red/burgundy slant, R), and $H_2S$ production (black precipitate).

Lysine Deamination (LDA)

An oxidative enzymatic reaction producing a characteristic red/burgundy slant (R/A) on Lysine Iron Agar, unique to members of the tribe Proteeae (Proteus, Providencia, and Morganella).

SIM Medium

A semisolid medium ($0.4\%$ agar) testing three parameters: $H_2S$ production (blackening), indole formation from tryptophan (red ring upon Kovac's reagent addition), and bacterial motility (diffuse cloudiness spreading from the stab line).

Christensen's Urea Agar

A test medium detecting urease enzyme activity that hydrolyzes urea into ammonia, raising the pH and shifting phenol red from yellow-orange to pink-magenta (rapid positive within $2\text{ to }6\text{ h}$ for Proteus and Morganella).

Simmons Citrate Agar

A defined medium with sodium citrate as the sole carbon source and ammonium dihydrogen phosphate as the sole nitrogen source; utilization yields alkaline products that shift bromthymol blue from green to Prussian blue alongside visible growth.

Methyl Red (MR) Test

A biochemical test detecting sustained mixed-acid glucose fermentation by the development of a stable red color when methyl red indicator is added ($\text{pH} \le 4.4$).

Voges-Proskauer (VP) Test

A biochemical test detecting acetoin (acetylmethylcarbinol), a precursor of 2,3-butanediol, by adding $\alpha\text{-naphthol}$ and $\text{KOH}$ to produce a red-pink complex within $15\text{ to }30\text{ minutes}$.

Decarboxylase Base Media

A Moeller broth containing glucose, peptone, bromcresol purple, and pyridoxal cofactor overlaid with sterile mineral oil; initial glucose acidifies the tube to yellow, followed by amino acid decarboxylation to alkaline amines (e.g., cadaverine or putrescine) that return the test tube to purple.

Phenylalanine Deaminase (PAD) Agar

An agar slant containing phenylalanine used to detect oxidative deamination to phenylpyruvic acid, confirmed by the formation of an intense green color within $1\text{ to }2\text{ minutes}$ after adding $10\%$ ferric chloride (positive in Proteus, Providencia, and Morganella).

ONPG Test (o-Nitrophenyl-β-D-galactopyranoside)

A rapid test detecting $\beta\text{-galactosidase}$ activity in late or slow lactose fermenters lacking lactose permease, yielding a yellow compound (o-nitrophenol) upon enzymatic cleavage.

Nitrate Reduction Test

A broth test detecting reduction of nitrate to nitrite via sulfanilic acid and $\alpha\text{-naphthylamine}$ (red = positive); if clear, zinc dust is added, where persistent clear confirms complete reduction beyond nitrite to $N_2$ (positive) and red indicates unreduced nitrate (true negative).

IMViC Reaction Profile of Escherichia coli

The classic four-test biochemical series (Indole, Methyl Red, Voges-Proskauer, Citrate) producing a $++--$ pattern for Escherichia coli.

Uropathogenic E. coli (UPEC)

The leading bacterial cause of both lower and upper urinary tract infections, equipped with common pili, aerobactin siderophores, and cytolysins, whose upper renal involvement is characterized by white blood cell (WBC) casts.

Enterotoxigenic E. coli (ETEC)

The primary cause of traveler's diarrhea, acting in the proximal small intestine by elaborating heat-labile (LT) and heat-stable (ST) enterotoxins to produce profuse, watery diarrhea without blood.

Enteropathogenic E. coli (EPEC)

The etiologic agent of infantile diarrhea that adheres to the intestinal brush border causing attachment-effacement lesions and loss of microvilli, leading to watery stool containing mucus but no blood.

Enteroinvasive E. coli (EIEC)

An enteric pathogen that invades and destroys intestinal epithelial cells using invasin, resulting in inflammatory dysentery (stool containing blood, mucus, and pus) and a positive Sereny test.

Enterohemorrhagic E. coli (EHEC / STEC)

A Shiga toxin-producing pathotype (classically serotype O157:H7) acquired from ruminants and undercooked ground beef, characterized by hemorrhagic colitis and an inability to ferment sorbitol on SMAC.

Hemolytic Uremic Syndrome (HUS) Triad

The clinical complication associated with Shiga toxins (Stx1 and Stx2) from EHEC characterized by acute kidney injury, thrombocytopenia, and microangiopathic hemolytic anemia.

Enteroaggregative E. coli (EAEC)

An enteroadherent pathotype (notably O104:H4) that adheres to HEp-2 cells in a characteristic 'stacked-brick' pattern and causes persistent watery diarrhea.

MUG Assay

A fluorescence assay testing for $\beta\text{-glucuronidase}$ activity using 4-methylumbelliferyl-$\beta\text{-D-glucuronide}$; typical E. coli fluoresces blue under long-wave UV ($\text{MUG}^+$), whereas E. coli O157:H7 is typically negative.

Klebsiella pneumoniae subsp. pneumoniae

Also known as Friedländer's bacillus, a non-motile, heavily encapsulated Gram-negative rod causing severe community-acquired and nosocomial pneumonia with thick 'currant-jelly' sputum, showing a $--++$ IMViC profile, positive string test ($>5\text{ mm}$), and positive Neufeld-Quellung test.

Currant-Jelly Sputum

Thick, gelatinous, blood-tinged sputum pathognomonic for severe pneumonia caused by the prominent polysaccharide capsule of Klebsiella pneumoniae.

Klebsiella oxytoca

A Klebsiella species differentiated biochemically from K. pneumoniae by being indole-positive, and clinically associated with antibiotic-associated hemorrhagic colitis.

Klebsiella pneumoniae subsp. ozaenae

A Klebsiella subspecies that causes atrophic rhinitis (chronic inflammation and mucosal atrophy) and frequently carries plasmid-mediated extended-spectrum $\beta\text{-lactamases}$ (ESBLs).

Klebsiella pneumoniae subsp. rhinoscleromatis

A biochemically inert Klebsiella subspecies causing rhinoscleroma, a chronic destructive infection of the nasal cavity and upper respiratory tract leading to marked tissue swelling and deformity.

Klebsiella granulomatis

The causative agent of granuloma inguinale (donovanosis), a sexually transmitted infection presenting as painless, beefy-red ulcers, diagnosed by finding Donovan bodies with bipolar 'safety-pin' staining inside mononuclear cells on Wright or Giemsa stains.

Cronobacter sakazakii

A yellow-pigmented (on BHIA at $25^\circ\text{C}$), late lactose-fermenting bacterium associated with powdered infant formula that causes neonatal meningitis, sepsis, and brain abscesses.

Pantoea agglomerans

A yellow-pigmented, triple decarboxylase-negative (LDC-, ODC-, ADC-) opportunistic pathogen associated with nosocomial septicemia from contaminated intravenous (IV) fluids.

Serratia marcescens

A hospital-acquired pathogen notorious for nursery and ICU bacteremia outbreaks, characterized as DNase-positive, gelatinase-positive, lipase-positive, and producing a bright red pigment called prodigiosin on nutrient agar.

Prodigiosin

The characteristic non-diffusible red pigment synthesized on nutrient agar by Serratia marcescens.

Serratia odorifera

A clinical isolate of Serratia known for its pungent, distinct odor resembling rotten potatoes.

Hafnia alvei

A late lactose-fermenting, ONPG-positive, LDC-positive, ODC-positive enteric bacillus that is differentiated from Serratia by being negative for DNase, lipase, and gelatinase.

Proteus mirabilis

An indole-negative, ornithine decarboxylase-positive member of the tribe Proteeae that exhibits swarming on blood agar, produces a burnt-chocolate odor, and drives struvite/staghorn stone formation by rapid urease activity.

Proteus vulgaris

An indole-positive, ornithine decarboxylase-negative Proteus species that ferments sucrose and glucose, yielding an A/A reaction with $H_2S$ on TSI.

Struvite Calculi (Staghorn Calculi) Pathophysiology

Infection stones composed of magnesium ammonium phosphate (MAP) formed when Proteus urease hydrolyzes urea to ammonia, resulting in alkaline urinary pH and mineral precipitation.

Weil-Felix Test

A classic cross-reacting agglutination assay for rickettsial diseases utilizing antigens from non-motile Proteus strains: OX19 and OX2 from Proteus vulgaris, and OXK from Proteus mirabilis (Kingsbury strain).

Providencia rettgeri vs. Providencia stuartii Differentiation

P. rettgeri is urease-positive, adonitol-positive, and trehalose-negative; P. stuartii is urease-negative, adonitol-negative, and trehalose-positive.

Morganella morganii

A motile, non-swarming Gram-negative rod belonging to the tribe Proteeae that is PAD-positive, urease-positive, ornithine decarboxylase-positive, and produces an R/A reaction on LIA.

Edwardsiella tarda

An opportunistic pathogen causing wound infections and bacteremia that resembles Salmonella culturally by producing $H_2S$ and being LDC-positive, but is differentiated by being indole-positive.

Citrobacter koseri

An indole-positive, $H_2S\text{-negative}$ Citrobacter species associated with devastating neonatal nursery outbreaks of meningitis and brain abscesses.

Citrobacter freundii

An indole-negative, $H_2S\text{-positive}$, slow urease-producing enteric bacterium isolated from diarrheal stools, showing late lactose fermentation and an A/A or K/A reaction on TSI.

Salmonella enterica serotype Typhi

The typhoidal etiologic agent of enteric (typhoid) fever that produces slight $H_2S$, no gas, possesses the Vi capsular antigen, exhibits an IMViC pattern of $-+--$, and colonizes the gallbladder in chronic carriers (e.g., 'Typhoid Mary').

Widal Test

An agglutination tube/slide assay detecting anti-O antibodies (indicating active/acute infection) and anti-H antibodies (indicating past exposure or vaccination) in patient sera starting at significant titers of $1:160$ or $1:320$.

Non-Motile Salmonella Exceptions

Salmonella enterica serovar Gallinarum and Salmonella enterica serovar Pullorum.

Shigella Subgroups and Historical Bacilli Names

Group A = Shigella dysenteriae (Shiga's bacillus); Group B = Shigella flexneri (Strong bacillus); Group C = Shigella boydii (Newcastle Manchester bacillus); Group D = Shigella sonnei (Duval's bacillus).

Shigella sonnei

The Group D Shigella species distinguished by being the only subgroup member that is ornithine decarboxylase-positive (ODC+), as well as a late lactose fermenter and ONPG-positive.

Shigella dysenteriae Biochemical Uniqueness

The only Shigella species that fails to ferment mannitol (non-mannitol fermenter).

Yersinia pestis Diagnostic Staining and Broth Growth Pattern

Plague bacillus demonstrating bipolar ('safety-pin') closed-form staining on Wayson or methylene blue stain and a characteristic stalactite-shaped growth extending downward in undisturbed broth culture.

Clinical Plague Transmission Pathways

Infection initiated by rodent flea bites leads to bubonic plague with swollen lymph nodes (buboes), which can seed the bloodstream to cause septicemic plague or progress to primary/secondary pneumonic plague spread via infectious respiratory droplets.

Buboes

Painfully enlarged, inflamed regional lymph nodes (classically inguinal/groin or axillary) pathognomonic for bubonic plague, within which Y. pestis organisms are trapped and cannot spread person-to-person directly.

Pneumonic Plague

The deadliest form of plague with a short incubation period ($1\text{ to }3\text{ days}$) transmitted by respiratory droplet inhalation or aerosol release, making Yersinia pestis a Class A bioterrorism agent.

Yersinia enterocolitica

A psychrotrophic enteric pathogen transmitted by raw/undercooked pork that causes pseudoappendicitis (mesenteric lymphadenitis), fatal sepsis from contaminated packed red blood cells, and forms 'bull's-eye' colonies on CIN agar.

Yersinia pseudotuberculosis

An animal pathogen causing caseous swellings (pseudotubercles) in guinea pigs and rodents, and acute mesenteric lymphadenitis in humans, characterized as motile at $18\text{ to }25^\circ\text{C}$, non-motile at $35^\circ\text{C}$, and urease-positive.