Identifying Enterobacteriaceae to genus and species level from biochemical tests is a foundational clinical microbiology skill. Understanding why each test gives the result it does, rather than just memorising which species gives which pattern, builds the deeper knowledge that helps you reason through unusual presentations, atypical results, and the growing challenge of novel variants that may not match perfectly with published biochemical tables.
This page covers the family characteristics, the key differential tests, the major genera and species with their distinguishing features, clinical significance of each, and the antibiotic resistance patterns most associated with each organism.
Family-Level Characteristics
All Enterobacteriaceae share these features: gram-negative rods (bacilli), facultatively anaerobic (grow in both aerobic and anaerobic conditions), ferment glucose (not oxidise: they use fermentation pathways generating acid and sometimes gas), reduce nitrates to nitrites, are oxidase-negative (lacking the cytochrome c oxidase of Pseudomonas and other non-fermenters), and are catalase-positive.
The oxidase-negative result is the most useful single test: it places an organism within the Enterobacteriaceae and excludes non-fermenters (Pseudomonas, Acinetobacter, Stenotrophomonas), which are oxidase-positive or present other distinctive features.
On MacConkey agar (a selective and differential medium containing crystal violet and bile salts to inhibit gram-positives, with lactose and a neutral red pH indicator), Enterobacteriaceae are subdivided by lactose fermentation: lactose fermenters turn the medium pink-red (acid production from lactose lowers pH and the indicator turns pink), lactose non-fermenters produce colourless or pale colonies.
Lactose fermenters: Escherichia coli (strongly lactose fermenting, distinctive metallic sheen on EMB agar), Klebsiella (large mucoid pink colonies), Enterobacter (pink colonies, smaller than Klebsiella), Serratia marcescens (some strains lactose-positive, characteristically produce a red pigment on blood agar or nutrient agar at room temperature).
Lactose non-fermenters: Salmonella, Shigella (pale colonies on MacConkey), Proteus (produces characteristic swarming on blood agar, inhibited on MacConkey and CLED).
Escherichia coli: The Reference Organism
E. coli is the most common gram-negative isolate in clinical microbiology. Its biochemical fingerprint: oxidase-negative, catalase-positive, facultative anaerobe, strongly ferments lactose and glucose with acid and gas production, indole-positive (tryptophanase cleaves indole from tryptophan), methyl red positive (mixed acid fermentation), Voges-Proskauer negative, citrate-negative (IMViC: ++--), hydrogen sulphide negative, urease-negative, non-motile or motile (most strains motile with peritrichous flagella).
On EMB (Eosin Methylene Blue) agar, E. coli produces the characteristic metallic green sheen: a combination of E. coli's vigorous lactose and sucrose fermentation producing acid that precipitates the dye, creating the sheen. This feature is almost specific for E. coli among common clinical isolates.
Pathogenic E. coli strains: STEC (O157:H7 and other serotypes producing Shiga toxins), ETEC (enterotoxigenic, produces heat-stable and heat-labile toxins causing traveller's diarrhoea), EPEC (enteropathogenic, attaches to and effaces intestinal microvilli, common cause of infant diarrhoea in developing countries), EIEC (enteroinvasive, invades intestinal epithelium, produces dysentery similar to Shigella), UPEC (uropathogenic, the dominant cause of UTI with P-fimbriae and Type 1 fimbriae).
Klebsiella Species: The Mucoid Encapsulated Rods
Klebsiella pneumoniae is a common cause of community and hospital-acquired pneumonia, UTI, and bloodstream infection, particularly in immunocompromised patients, alcoholics, and those with structural lung disease. On MacConkey agar, Klebsiella produces large, mucoid, pink colonies: the mucoid appearance reflects the abundant polysaccharide capsule (the primary virulence factor providing anti-phagocytic protection).
Biochemical profile: non-motile (important differential from Enterobacter), urease-positive, citrate-positive, VP-positive, MR-negative (butanediol fermentation), indole-negative (contrast with E. coli). IMViC pattern: --++.
The "string test": when a colony of K. pneumoniae is touched with a loop and the loop is slowly withdrawn, the viscous extracellular polysaccharide stretches into a string of more than 5 mm. This simple test identifies hypermucoviscous K. pneumoniae strains associated with liver abscess (particularly in Asian patients), endophthalmitis, and metastatic abscess formation.
Klebsiella oxytoca: indole-positive (distinguishing it from K. pneumoniae), otherwise similar biochemical profile. Increasingly recognised as a cause of antibiotic-associated haemorrhagic colitis through production of cytotoxins.
Proteus, Morganella, and Providencia: The Urease Group
Proteus mirabilis is the most important member of the Proteus-Morganella-Providencia tribe clinically. Key features: strongly urease-positive (produces urease so rapidly it often turns urea broth pink within 15 to 60 minutes, compared to hours for other urease-positive organisms), hydrogen sulphide-positive, indole-negative, swarming motility on blood agar (concentric rings from spreading swarmer cells), phenylpyruvic acid (PPA)-positive.
The clinical importance of urease in P. mirabilis: urease hydrolysis of urea produces ammonia, raising urinary pH. Alkaline urine promotes precipitation of struvite (magnesium ammonium phosphate) crystals, forming infection stones (struvite calculi). Struvite calculi harbour bacteria within their crystal matrix, protected from antibiotics, causing recurrent UTI that cannot be eradicated without surgical removal of the stone.
Proteus vulgaris: indole-positive (differentiating from P. mirabilis), also urease-positive, also PPA-positive.
Morganella morganii: indole-positive, urease-positive, ornithine decarboxylase-positive, H2S-negative. Intrinsically resistant to many beta-lactams through chromosomal AmpC. Frequently isolated in polymicrobial wound infections and complicated UTI.
Salmonella and Shigella: The Enteric Pathogens
Salmonella and Shigella are the two major intestinal pathogen genera within Enterobacteriaceae, both appearing as pale, non-lactose fermenting colonies on MacConkey agar.
Salmonella: glucose fermented with H2S production (except Salmonella Typhi, which is H2S-variable and typically produces small amounts), lactose-negative, indole-negative, urease-negative, motile (peritrichous flagella). Serological classification by Kauffmann-White scheme using O (somatic) and H (flagellar) antigens identifies over 2,500 serovars. S. Typhi and S. Paratyphi are the agents of enteric fever (typhoid): they are taken up by M cells in Peyer's patches, invade macrophages, and disseminate systemically, causing prolonged fever, rose spots, and splenomegaly. Non-typhoidal Salmonella typically causes self-limiting gastroenteritis.
Shigella: the only Enterobacterium that does not produce gas from glucose fermentation (acid only, no gas, distinguishing it from all other Enterobacteriaceae except S. Typhi). Non-motile. Indole-negative (most species). No H2S. Does not use citrate. Causes bacillary dysentery by invasion of colonic epithelial cells. The infective dose is extremely low (10 to 100 organisms), allowing person-to-person transmission via the faecal-oral route and making it a major cause of outbreaks in institutional settings.
Enterobacter and Serratia: Hospital-Associated and AmpC-Mediated
Enterobacter cloacae complex and Serratia marcescens are significant hospital-acquired pathogens, particularly in ICU patients, immunocompromised patients, and neonates.
Enterobacter cloacae: motile (distinguishing from Klebsiella), citrate-positive, VP-positive, MR-negative, ornithine decarboxylase-positive (distinguishing from Klebsiella). Intrinsic inducible AmpC beta-lactamase (chromosomally encoded). Exposure to cephalosporins can select for constitutively overexpressing AmpC mutants (stably derepressed), causing clinical failure even against initially susceptible isolates.
Serratia marcescens: characteristic red pigment (prodigiosin) at room temperature on blood agar and nutrient agar (not produced at 37 degrees Celsius). DNase-positive (differentiating from Enterobacter). Lipase-positive. Gelatinase-positive. Also carries inducible AmpC. Associated with respiratory infections in ICU patients and outbreaks linked to contaminated multiuse medication vials.
Frequently Asked Questions
What are Enterobacteriaceae?
Enterobacteriaceae (or Enterobacterales in current taxonomy) are a family of gram-negative facultative anaerobic rods that are oxidase-negative, catalase-positive, and ferment glucose. They include E. coli, Klebsiella, Salmonella, Shigella, Proteus, Enterobacter, Serratia, Citrobacter, Morganella, and many other clinically important genera.
What is the significance of oxidase-negative result in gram-negative rods?
A gram-negative rod that is oxidase-negative is within the Enterobacteriaceae. An oxidase-positive gram-negative rod is most likely a non-fermenter (Pseudomonas, Acinetobacter) or another oxidase-positive species. The oxidase test is the critical first branching point in gram-negative rod identification.
What is MacConkey agar?
MacConkey agar is a selective and differential medium for gram-negative bacteria. It contains crystal violet and bile salts (inhibiting gram-positive organisms) and lactose with a neutral red pH indicator (differentiating lactose fermenters from non-fermenters). Lactose fermenters produce pink-red colonies; non-fermenters produce pale or colourless colonies.
What is the IMViC pattern?
IMViC is a set of four biochemical tests: Indole, Methyl Red, Voges-Proskauer, and Citrate. The pattern of positive (+) and negative (-) results identifies key Enterobacteriaceae: E. coli is ++-- (indole positive, MR positive, VP negative, citrate negative), Klebsiella is --++ (indole negative, MR negative, VP positive, citrate positive).
Why does Proteus mirabilis cause kidney stones?
P. mirabilis produces urease, hydrolysing urea to ammonia and carbon dioxide. Ammonia alkalinises urine, promoting precipitation of struvite (magnesium ammonium phosphate) crystals. These infection stones harbour bacteria protected from antibiotics. Eradicating P. mirabilis UTI in patients with struvite stones is impossible without surgical stone removal.
What is AmpC beta-lactamase?
AmpC beta-lactamases are chromosomally encoded beta-lactamases in organisms such as Enterobacter, Serratia, Citrobacter, and Pseudomonas (ESCPM organisms). They can be induced by beta-lactam exposure. Constitutively overexpressing AmpC mutants arise during treatment with cephalosporins, causing clinical failure. ESCPM organisms should not be treated with cephalosporins for serious infections even if initially susceptible.
What is the Kauffmann-White scheme?
The Kauffmann-White scheme is the serological classification system for Salmonella, identifying serovars by their O (somatic, lipopolysaccharide) and H (flagellar) antigens. Over 2,500 Salmonella serovars are defined by specific O and H antigen combinations. Serotyping is important for epidemiological tracking of outbreaks.
Why is Shigella different from other Enterobacteriaceae?
Shigella is the only Enterobacterium that does not produce gas from glucose fermentation (produces acid only). It is also non-motile, non-H2S producing, and non-citrate using. These features reflect its highly adapted host-restricted pathogenic lifestyle: Shigella infects only primates, requires a very low infective dose (10 to 100 organisms), and causes disease through direct invasion of colonic epithelial cells.
What is the string test for Klebsiella?
The string test is performed by touching a Klebsiella colony with a loop and slowly withdrawing it. Hypermucoviscous K. pneumoniae strains, associated with liver abscess and metastatic infection, produce a string of viscous polysaccharide material exceeding 5 mm in length. This test identifies strains with the hypermucoviscous phenotype (often carrying the rmpA gene regulating polysaccharide capsule production).
What is the clinical significance of ESBL-producing Klebsiella?
ESBL-producing Klebsiella pneumoniae is resistant to all penicillins and all cephalosporins. Treatment of invasive infections requires carbapenems or, depending on susceptibility and clinical severity, alternatives such as ceftazidime-avibactam, piperacillin-tazobactam (if MIC is low), or fosfomycin (for UTI). Hospital outbreaks with ESBL-Kp are common and require enhanced contact precautions.