Overview
Escherichia coli is a Gram-negative, facultative anaerobic, rod-shaped bacterium and the most abundant facultative anaerobe of the normal gut flora, while also being one of the most intensively studied prokaryotic model organisms in biotechnology. Most strains are harmless commensals, but certain serotypes such as E. coli O157:H7 cause severe food-borne illness.
Klebsiella species are closely related Enterobacteriaceae found as commensals of the mouth, upper respiratory tract and intestine, and are important causes of pneumonia, urinary tract infection, and nosocomial infection. This lesson covers the morphology, culture, biochemical identification, and clinical significance of both genera.
Learning Objectives
After this lesson you will be able to…- Describe the morphology of Escherichia coli and Klebsiella.
- Describe the cultural characteristics of Escherichia coli and Klebsiella.
- Explain the pathogenesis of E. coli and Klebsiella infections.
- Discuss the virulence determinants of pathogenic E. coli.
Clinical Story
Why This MattersA 6-year-old develops bloody diarrhoea and decreased urine output three days after eating an undercooked burger at a family gathering. The paediatrician suspects haemolytic uraemic syndrome. The lab technologist isolates E. coli from stool on sorbitol-MacConkey agar, noting the colourless (sorbitol-negative) colonies characteristic of the dangerous O157:H7 serotype, and immediately alerts the clinical team.
Core Concepts
E. coli is a Gram-negative, facultative anaerobic, non-sporulating rod, about 2.0 µm long and 0.25–1.0 µm in diameter. Motile strains possess peritrichous flagella. It is the most abundant facultative anaerobe of the terminal small and large intestine of mammals, and its presence in the environment usually reflects faecal contamination.
E. coli grows readily on solid or liquid media; different clones produce distinguishable colonies. On MacConkey agar, colonies are pink (lactose fermenting), circular, complete margin, slightly raised, opaque, soft. On Eosin-Methylene Blue (EMB) agar, colonies show a characteristic metallic green sheen. Optimal growth occurs at 37°C, though some strains tolerate up to 49°C. Biochemically, E. coli is MR positive, VP negative, indole positive, citrate negative, urease negative, and produces acid and gas on TSI.
The most dangerous serotype, E. coli O157:H7, produces Shiga toxin (a Shiga toxin-producing E. coli, or STEC) and can cause bloody diarrhoea progressing to haemolytic uraemic syndrome (HUS) — destruction of red blood cells and acute kidney injury requiring dialysis. E. coli is the commonest cause of urinary tract infection and cystitis, and is implicated in intra-abdominal infections, bacteraemia, and rarely neonatal meningitis.
Specimens include urine and stool. Isolation media include nutrient agar, MacConkey's agar, and eosin-methylene blue agar; biochemical media include glucose phosphate broth, motility agar, TSI slant, tryptone water, Simmon's citrate agar, Christensen's urea medium, nitrate broth and nutrient gelatin medium. E. coli is typically sluggishly motile on direct testing and Gram-negative bacilli on staining.
Klebsiella pneumoniae is found as a commensal in the mouth and upper respiratory tract and in moist environments including the intestinal tract, plants, water and soil. On MacConkey and EMB agar, colonies are large (3–4 mm), round, pink, mucoid — the mucoid appearance reflecting the prominent polysaccharide capsule. Klebsiella aerogenes is non-motile, while K. pneumoniae biochemistry differs slightly (K. aerogenes is urease negative and VP positive; K. cloacae/pneumoniae variants differ in MR/VP and urease reactions).
Klebsiella causes pneumonia, urinary tract infections, septicaemia and other pyogenic infections, and occasionally diarrhoea. K. pneumoniae is generally associated with lower respiratory tract and middle ear infections, while K. aerogenes is generally associated with wounds and urinary tract infections. Biochemically Klebsiella is oxidase negative and urease positive, a key distinguishing feature from E. coli (urease negative).
Laboratory Principle
Differentiation within the Enterobacteriaceae, including between E. coli and Klebsiella, relies on the IMViC panel (Indole, Methyl Red, Voges-Proskauer, Citrate) together with motility and urease testing. E. coli's classical IMViC pattern is ++−− (indole and MR positive; VP and citrate negative) with active motility via peritrichous flagella, whereas Klebsiella typically shows the reverse pattern (indole and MR negative; VP and citrate positive) and is non-motile — this biochemical fingerprint, combined with colonial morphology on differential media like MacConkey agar, allows rapid presumptive genus-level identification in the routine diagnostic laboratory.
Equipment Required
Reagents & Materials
| Reagent / Material | Concentration / Grade | Purpose | Storage |
|---|---|---|---|
| MacConkey Agar | Bile salts, lactose, neutral red | Differential/selective medium — lactose fermentation | 2–8°C |
| Eosin-Methylene Blue (EMB) Agar | Selective/differential | Detects metallic sheen (E. coli) vs mucoid pink (Klebsiella) | 2–8°C |
| Triple Sugar Iron (TSI) Agar | Glucose, lactose, sucrose, iron | Fermentation pattern and H₂S detection | 2–8°C |
| Simmon's Citrate Agar | Citrate as sole carbon source | Citrate utilisation test | 2–8°C |
| Christensen's Urea Medium | Urea, phenol red indicator | Urease test — differentiates Klebsiella (+) from E. coli (−) | 2–8°C |
| Kovac's Reagent | p-dimethylaminobenzaldehyde | Indole test | 2–8°C, protect from light |
Step-by-Step Procedure
Collect midstream urine for suspected UTI or fresh stool for suspected enteric infection, in sterile containers.
Confirm Gram-negative bacilli morphology from the specimen or primary culture.
Inoculate nutrient agar, MacConkey's agar, and EMB agar; incubate at 35–37°C for 24 hours.
Note lactose fermentation (pink on MacConkey), metallic sheen (EMB, suggests E. coli), or large mucoid colonies (suggests Klebsiella).
Test motility (E. coli motile, Klebsiella non-motile) and complete the Indole, Methyl Red, Voges-Proskauer, Citrate panel plus urease testing.
E. coli: indole+, MR+, VP−, citrate−, urease−, motile. Klebsiella: indole−, MR variable, VP+, citrate+, urease+, non-motile.
Flow Diagram
Quality Control
Run ATCC reference strains of E. coli (e.g. ATCC 25922) and Klebsiella pneumoniae (e.g. ATCC 700603) with each new batch of MacConkey, EMB, TSI, citrate and urea media to confirm expected reactions before reporting patient results.
Enrol in EQAS programmes for Enterobacteriaceae identification and antimicrobial susceptibility testing, particularly important given the clinical significance of distinguishing Shiga-toxin-producing E. coli and extended-spectrum beta-lactamase (ESBL)-producing Klebsiella.
Reference Values
Key Diagnostic Parameters⚠️ Reference ranges may vary between laboratories. Always apply your laboratory's established reference intervals.
Clinical Interpretation
| Finding | Possible Significance | Action / Follow-up |
|---|---|---|
| Pink, non-mucoid colonies with metallic green sheen on EMB, motile, indole+ | Escherichia coli | Report as E. coli; correlate with UTI/enteric symptoms; consider O157:H7 testing if bloody diarrhoea |
| Large, mucoid, pink colonies on MacConkey, non-motile, urease+ | Klebsiella species | Report as Klebsiella; test for ESBL/carbapenemase given nosocomial risk |
| Sorbitol-negative colonies on sorbitol-MacConkey agar from bloody stool | Suggestive of E. coli O157:H7 (STEC) | Urgent Shiga toxin testing; monitor renal function for HUS |
| E. coli isolated from midstream urine, >10⁵ CFU/mL | Significant urinary tract infection | Treat per antimicrobial susceptibility results |
Common Errors & How to Avoid Them
Cause: Both can appear pink/lactose-fermenting on MacConkey agar, causing confusion without further testing.
Prevention: Always confirm with motility testing and the full IMViC/urease panel — Klebsiella is non-motile, urease-positive and much more mucoid than E. coli.
Cause: Standard MacConkey agar does not differentiate O157:H7 from other E. coli strains.
Prevention: Use sorbitol-MacConkey agar for suspected haemorrhagic colitis — O157:H7 characteristically fails to ferment sorbitol, appearing colourless, unlike most other E. coli.
Cause: Routine susceptibility panels may not flag extended-spectrum beta-lactamase production without specific confirmatory testing.
Prevention: Perform ESBL screening/confirmatory tests on Klebsiella isolates from hospitalised patients, especially with reduced susceptibility to third-generation cephalosporins.
Laboratory Tips from the Bench
The mucoid, tenacious 'string test' when touching a Klebsiella colony with a loop is a quick bedside clue to its heavy capsule, even before biochemical confirmation.
When investigating suspected haemolytic uraemic syndrome, always request sorbitol-MacConkey agar specifically — standard MacConkey will not distinguish O157:H7 from harmless E. coli.
Remember Klebsiella's biochemical signature as the opposite of E. coli: where E. coli is '+ + − −' (Indole, MR, VP, Citrate), Klebsiella is largely '− − + +' — a mirror image.
Important Notes
Haemolytic uraemic syndrome following E. coli O157:H7 infection can progress rapidly to acute kidney injury; antibiotic treatment of STEC infection is generally avoided as it may increase toxin release and worsen HUS risk — supportive care is the mainstay.
Non-pathogenic E. coli strains such as Nissle 1917 are used therapeutically as probiotics — do not confuse these beneficial strains with pathogenic serotypes when interpreting stool culture results in a research or product-testing context.
Interactive Quiz
Test Your KnowledgeFlashcards
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Clinical Case Study
Apply Your Knowledge3 days after eating an undercooked burger at a family gathering, develops bloody diarrhoea, abdominal cramping, and progressively decreasing urine output with pallor.
The sorbitol-negative colonies, positive Shiga toxin test, anaemia and elevated creatinine together confirm Shiga toxin-producing E. coli (STEC, likely O157:H7) infection that has progressed to haemolytic uraemic syndrome, requiring urgent nephrology involvement.
- →Sorbitol-MacConkey agar is essential for screening suspected O157:H7 in bloody diarrhoea cases.
- →Antibiotics are generally avoided in STEC infection due to risk of increased toxin release.
- →HUS requires close monitoring of renal function, haemoglobin and platelet count.
Frequently Asked Questions
Antibiotic-induced bacterial cell death can trigger increased release of Shiga toxin, potentially worsening the risk or severity of haemolytic uraemic syndrome, so supportive care is generally preferred instead.
Motility testing (E. coli motile, Klebsiella non-motile) combined with colony mucoidness (Klebsiella markedly more mucoid due to its capsule) and urease testing (Klebsiella positive, E. coli negative) gives a fast, reliable distinction.
Its polysaccharide capsule provides resistance to phagocytosis and desiccation, and increasing rates of extended-spectrum beta-lactamase (ESBL) and carbapenemase production make it a significant cause of difficult-to-treat nosocomial infections.
Quick Revision
10-Minute ReviewKey Takeaways
- E. coli and Klebsiella are both Enterobacteriaceae but differ sharply in motility, capsule, and key biochemical tests.
- E. coli O157:H7 is a serious food-borne pathogen capable of causing haemolytic uraemic syndrome.
- Klebsiella's prominent capsule underlies both its mucoid colony appearance and its virulence.
- The IMViC panel plus motility and urease testing reliably differentiates these two genera in routine practice.
- Rising antimicrobial resistance (ESBL, carbapenemase) makes Klebsiella a growing nosocomial threat.
Competency Checklist
Track Your MasteryReferences
- National Institute of Open Schooling. Microbiology — Lesson 21: Escherichia coli and Klebsiella.
- Forbes BA, Sahm DF, Weissfeld AS. Bailey & Scott's Diagnostic Microbiology.
- Centers for Disease Control and Prevention. E. coli (Escherichia coli) fact sheets.