Enterobacteriaceae: How to Identify and Tell Them Apart

A stool sample lands on your bench. You streak it on MacConkey agar and wait. The next morning, some colonies are bright pink, others are colorless. That single color difference is the first fork in a decision tree: the pink colonies are usually ordinary gut bacteria, but a colorless colony is the one that could be Salmonella or Shigella, the organisms you cannot afford to miss.

The Enterobacteriaceae are the most common bacteria you will grow in a clinical laboratory. They share enough biology to be grouped as one family, but they cause diseases as different as a simple bladder infection and fatal septicemia. This article is about the shared rules that hold the family together, and the few tests that tell its members apart. For the detailed information of each organism you can check the hyperlink for their own article.

The Enterobacteriaceae are a large family of Gram-negative rods that share a set of common properties. Many of the bacteria you meet most often in a clinical laboratory belong here, including Escherichia coli, Klebsiella, Salmonella, Shigella, Enterobacter, Proteus, and Yersinia.

In 2016 the old single family was reorganized into a larger order called Enterobacterales, and several genera were moved into separate families within that order. In everyday clinical and laboratory use, and in most exams, the term "Enterobacteriaceae" is still used broadly for this whole group of Gram-negative enteric rods. This article uses the term in that common, practical sense. If you see a source that gives a different genus count or places an organism in a different family, this reclassification is usually the reason.

Figure: LF and NLF colonies in MacConkey Agar

Why the Enterobacteriaceae matter

Three facts explain why this one family takes up so much of a diagnostic laboratory's time.

First, they are everywhere. Many live in the human gut as normal flora, so they are the usual cause of infections that start from the bowel: urinary tract infections, wound infections after abdominal surgery, and bloodstream infection when gut bacteria escape into the blood.

Second, a few members are true intestinal pathogens rather than normal flora. Salmonella, Shigella, and certain strains of E. coli cause diarrheal disease and enteric fever. Telling a harmless gut E. coli from Salmonella on the same plate is a daily task, and the whole identification workflow below exists to make that call reliably.

Third, this family carries much of the antibiotic resistance that now limits treatment. Extended-spectrum beta-lactamases and carbapenem resistance appear here more than in almost any other group. That is covered in the resistance section near the end.

Common Characteristics

Every member of this family shares the same core set of properties. These are the features that let a laboratory recognize an isolate as an enteric Gram-negative rod before any genus-level testing.

• Gram-negative, non-spore-forming rods.
• Facultatively anaerobic: they grow with or without oxygen.
• Ferment glucose, and produce acid as a result.
• Oxidase-negative. This single test is the fastest way to separate them from oxidase-positive Gram-negative rods such as Pseudomonas and Vibrio. If a Gram-negative rod is oxidase-positive, it is not in this family.
• Reduce nitrate to nitrite (with a few exceptions).
• Catalase-positive (with few exceptions).

The oxidase result is the one to hold onto. It is the first gate: a Gram-negative rod that is oxidase-negative and grows on MacConkey agar is very likely an enteric organism, and the biochemical tests below then sort it to a genus.

Figure: Antigens of the Enterobacteriaceae family

Surface antigens

Three surface antigens are used to type members of this family, and a fourth is specific to Salmonella. These antigens are the basis of serotyping schemes such as the one used for Salmonella and for E. coli.

• O antigen: part of the lipopolysaccharide in the outer membrane. Heat-stable.
• H antigen: the flagellar protein. Present only in motile organisms, so a non-motile organism such as Shigella or Klebsiella has no H antigen. Heat-labile.
• K antigen: the capsular polysaccharide, when a capsule is present.
• Vi antigen: a special capsular antigen of Salmonella Typhi. Its name comes from "virulence." It can mask the O antigen in fresh isolates.

Medically important genera

These are the genera you will actually encounter. The table groups them by the kind of disease they cause, which is more useful at the bench than an alphabetical list.

The lactose fermentation split: your first sorting step

On MacConkey agar, the single most useful early clue is whether an organism ferments lactose. Fermenters lower the pH and turn pink. Non-fermenters stay pale or colorless. This one reading splits the family into two working groups before you run a single biochemical test.

Strong lactose fermenters (pink on MacConkey): CEEK

- Citrobacter (often a slow or late fermenter, see below)

• Escherichia
• Enterobacter
• Klebsiella

Non-lactose fermenters (pale on MacConkey):ShYPS are the pathogens to watch

• Shigella
• Yersinia
• Proteus
• Salmonella

There is a clinical reason this split is worth memorizing. The two organisms you least want to miss in a stool sample, Salmonella and Shigella, are both non-lactose fermenters. So a colorless colony on MacConkey from a diarrheal stool is the one that earns further work, while a pink colony is more often ordinary gut flora. The color is not a diagnosis, but it tells you which colony to chase.

One honest caution, because sources disagree and students get caught by it: Citrobacter and some E. coli strains can be slow or weak lactose fermenters and may look pale at first read. "Lactose fermenter" is a useful sorting rule, not an absolute identity. Confirm with the biochemical tests below.

Primary Isolation Media

1. Blood Agar (BA)
2. MacConkey (MAC) agar
3. Cystine-lactose-electrolyte deficient (CLED) agar
4. Desoxycholate citrate agar (DCA)
5. Xylose-lysine-desoxycholate agar (XLD)
6. Brilliant Green Agar (BGA)
7. Cefixime-tellurite-sorbitol-MacConkey (CT-SMAC) agar
8. Thiosulphate-citrate-bile salt (TCBS) agar
9. Cefsulodin-Irgasan-novobiocin (CIN) agar
10. Chromogenic media

Colonial Appearance

How the family is sorted into genera

Once an isolate is confirmed as an oxidase-negative enteric Gram-negative rod, a small panel of biochemical tests separates it into a genus. Each test asks one question about the organism's metabolism. No single test identifies an organism; the pattern across the panel does.

• Indole test: can the organism split tryptophan and release indole? Full method and reading: Indole Test.
• Methyl red (MR) test: does it perform mixed-acid fermentation, dropping the pH sharply? Read in detail about Methyl red (MR) test
• Voges-Proskauer (VP) test: does it ferment glucose to neutral acetoin instead? MR and VP are usually opposite results and are run as a pair. Read in detail about Voges-Proskauer (VP) test
• Citrate utilization test: can it use citrate as its only carbon source? Read in detail about Citrate utilization test
• Urease test: can it split urea? A strong rapid positive points to Proteus. Read in detail about Urease test
• Triple sugar iron (TSI) agar: reads sugar fermentation, gas, and H₂S production together in one tube. This is the workhorse first-look test for this family. Read in detail about Triple sugar iron (TSI) agar
• Motility test: is the organism motile? Separates motile genera from non-motile Klebsiella and Shigella. Read in detail about Motility test

A common memory aid for the core four is IMViC (Indole, Methyl red, Voges-Proskauer, Citrate). The classic teaching contrast is E. coli, which is IMViC + + − −, versus Klebsiella / Enterobacter, which is − − + +. That single contrast anchors the whole panel: the gut commensal splits tryptophan and acidifies, the environmental organisms do not and instead use citrate.

Summary of biochemical reactions of Enterobacteriaceae

This table is a family-level snapshot for comparison. For how to read each result and the full reaction logic for any one organism, see that organism's own article.

Antimicrobial resistance in this family

This family carries a large share of the resistance that now limits treatment of Gram-negative infections. Three mechanisms matter most, and each has its own dedicated article. Here is the family-level overview and how to recognize which organism raises which concern.

Inducible AmpC beta-lactamase. Some genera carry a chromosomal AmpC enzyme that can be switched on and then destroys many penicillins and cephalosporins. The organism can test susceptible on day one and resistant a few days into treatment. The classic AmpC producers are remembered by the mnemonic HYPERM or the related SPACE group:

• Hafnia
• Yersinia enterocolitica
• Providencia
• Enterobacter species
• Serratia marcescens
• Morganella morganii

and, importantly, Citrobacter freundii and Klebsiella aerogenes (the organism formerly named Enterobacter aerogenes). The practical rule is that for a serious infection with one of these organisms, many clinicians avoid third-generation cephalosporins even if the isolate looks susceptible, because AmpC can emerge on treatment. The full mechanism is in the beta-lactamase / AmpC article.

Extended-spectrum beta-lactamases (ESBLs). These plasmid-carried enzymes break down most cephalosporins and are common in E. coli and Klebsiella. Detail and detection are in the ESBL article.

Carbapenem-resistant Enterobacteriaceae (CRE). When an organism becomes resistant even to carbapenems, treatment options collapse. Resistance arises either by producing a carbapenemase enzyme (of which Klebsiella pneumoniae carbapenemase, KPC, is the most common) or by combining an AmpC or ESBL with loss of the outer-membrane porins the drug uses to enter. CRE are a major global concern. Detection and the full picture are in the carbapenem resistance / CRE article.

How to remember

The oxidase gate. Before any other test, one question sorts a Gram-negative rod into or out of this family: is it oxidase-negative? Picture a gate that only opens for oxidase-negative rods. Pseudomonas and Vibrio are turned away at the gate; the enteric rods walk through. Ask yourself: a Gram-negative rod grows on MacConkey and is oxidase-positive. Is it in this family? No.

CEEK stays pink, the pathogens stay pale. The strong lactose fermenters are Citrobacter, Escherichia, Enterobacter, Klebsiella (CEEK), and they turn MacConkey pink. The stool pathogens you must not miss, Salmonella and Shigella, stay pale. So in a diarrheal stool, the pale colony is the suspect, the pink one is usually a bystander.

IMViC tells the commensal from the environment. E. coli is + + − − : it splits tryptophan and pours out acid, the behavior of a gut organism living on your food. Klebsiella and Enterobacter are − − + + : they stay quiet on those and instead eat citrate, the behavior of organisms that also live in soil and water. The mnemonic is not four random letters; it is a gut organism versus an environmental one.

Key exam facts in one table

References

1. Tille, P. M. (2022). Bailey & Scott's Diagnostic Microbiology (15th ed.). Elsevier.
2. Procop, G. W., et al. (2017). Koneman's Color Atlas and Textbook of Diagnostic Microbiology (7th ed.). Wolters Kluwer.
3. Madigan, M. T., Bender, K. S., Buckley, D. H., Sattley, W. M., & Stahl, D. A. (2021). Brock Biology of Microorganisms (16th ed.). Pearson.
4. Adeolu, M., Alnajar, S., Naushad, S., & Gupta, R. S. (2016). Genome-based phylogeny of the order Enterobacterales. International Journal of Systematic and Evolutionary Microbiology, 66(12), 5575–5599.