Overview
Citrobacter, Edwardsiella, Enterobacter and Serratia are members of the family Enterobacteriaceae — Gram-negative, motile bacilli with peritrichous flagella that are oxidase-negative, catalase-positive, and reduce nitrates to nitrites. They are generally regarded as environmental contaminants or opportunistic commensals, isolated from soil, water, and the faeces of humans and animals.
Although once considered low-virulence organisms, infections caused by this group are increasingly recognised, particularly in hospital settings, and they are often multidrug resistant. This lesson focuses on the distinguishing biochemical characteristics and pathogenic potential of each genus.
Learning Objectives
After this lesson you will be able to…- Describe the characteristics of Citrobacter, Edwardsiella, Enterobacter, and Serratia.
- Describe the pathogenicity of Citrobacter, Edwardsiella, Enterobacter, and Serratia.
- Differentiate between Citrobacter, Edwardsiella, Enterobacter, and Serratia using biochemical reactions.
Clinical Story
Why This MattersA newborn in the neonatal intensive care unit develops fever and lethargy. Blood culture grows a Gram-negative bacillus initially suspected to be Salmonella because it shares the O antigen — until the microbiology team runs a full biochemical panel and correctly identifies Citrobacter koseri, a rare but recognised cause of neonatal meningitis, changing both the diagnosis and the infection-control response.
Core Concepts
Citrobacter belongs to the tribe Citrobacteriaceae. Members are motile, grow well on ordinary media forming smooth, convex, non-pigmented 2–3 mm colonies, and appear pale to pink on MacConkey agar due to (sometimes late) lactose fermentation. Three species exist: C. freundii, C. koseri, and C. amalonaticus. They are indole positive (except C. freundii), MR positive, VP negative, citrate positive, weakly urease positive, and H₂S is produced by C. freundii; mannitol fermentation is always positive and they are always ONPG positive. As normal gut commensals, pathogenicity relates to endotoxins, O and H antigens, capsular antigen and adhesion proteins; they share O antigen with Salmonella (risking misidentification) and can cause UTI, gall bladder and middle ear infection, with C. koseri occasionally causing neonatal meningitis.
Edwardsiella belongs to tribe Edwardsielleae; E. tarda is the only recognised human pathogen. It is non-capsulated, motile, and produces H₂S in KI medium. On MacConkey agar, colonies are pale, becoming pink on further incubation due to late lactose fermentation ('tarda' refers to this slow/weak sugar fermentation — only glucose and maltose are fermented). It is indole positive, utilises citrate, and is urease negative. Its normal habitat is the intestine of cold-blooded animals and fresh water; it is mainly pathogenic to water animals but causes occasional human infection of uncertain significance, isolated from wound, urine, blood and CSF.
Enterobacter belongs to tribe Klebsielleae. Members are motile, capsulated, and form pink, mucoid colonies on MacConkey agar. They are MR negative, VP positive, indole negative, citrate positive, and do not produce H₂S. Of the 12 species, E. aerogenes and E. cloacae are most common clinically; E. aerogenes is urease negative while E. cloacae is urease positive. Both species are widely distributed in water, sewage, soil and vegetables, and are associated with opportunistic infection including UTI, respiratory tract infection and cutaneous wounds; they may occasionally cause meningitis and septicaemia, and are an important cause of hospital-acquired infection.
Serratia belongs to tribe Klebsielleae. Members are motile, Gram-negative coccobacilli that may form capsule. S. liquifaciens, S. rubidaea and S. marcescens are clinically significant, with S. marcescens most frequently encountered. It forms smooth, convex colonies with crenated edges and produces the red pigment prodigiosin (insoluble in water, best formed at 15–20°C though growth is poor at this temperature; soluble in alcohol, ether, acetone, chloroform). Serratia is ONPG positive but fails to ferment lactose, forms pale colonies on MacConkey agar, and is indole, MR and urease negative but citrate and VP positive, with no H₂S production. As a saprophyte from water, soil and food, it causes increasingly frequent nosocomial infection, especially in newborns and immunosuppressed patients, associated with meningitis, endocarditis, septicaemia, peritonitis and respiratory tract infections; multidrug resistance is common.
All four genera are motile and ONPG positive. Key differentiators: Citrobacter and Edwardsiella are MR positive and H₂S positive, whereas Enterobacter and Serratia are MR negative, VP positive, and H₂S negative. Urease distinguishes further — Enterobacter is variable/positive (E. cloacae positive, E. aerogenes negative) while Citrobacter, Edwardsiella and Serratia are largely negative or only Citrobacter weakly positive. Lactose fermentation is variable in Citrobacter, slow/late in Edwardsiella, positive in Enterobacter, and negative in Serratia.
Laboratory Principle
These four genera are distinguished within the Enterobacteriaceae using a standard biochemical panel — indole, methyl red (MR), Voges-Proskauer (VP), citrate utilisation, urease, and hydrogen sulphide (H₂S) production — because their colony appearance on MacConkey agar (pale to pink, lactose-fermenting to variable) alone cannot reliably separate them. The MR/VP pair is particularly informative: MR-positive/VP-negative organisms (Citrobacter, Edwardsiella) use the mixed-acid fermentation pathway, while MR-negative/VP-positive organisms (Enterobacter, Serratia) use the butanediol fermentation pathway, reflecting a fundamental metabolic divergence that underlies their laboratory differentiation.
Equipment Required
Reagents & Materials
| Reagent / Material | Concentration / Grade | Purpose | Storage |
|---|---|---|---|
| MacConkey Agar | Bile salts, lactose | Colony morphology and lactose fermentation screening | 2–8°C |
| Kligler Iron Agar / TSI | Glucose, lactose, iron salts | H₂S production and sugar fermentation pattern | 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 | Urease test | 2–8°C |
| MR-VP Broth | Glucose phosphate broth | Methyl Red and Voges-Proskauer tests | 2–8°C |
| Kovac's Reagent | p-DMAB reagent | Indole test | 2–8°C, protect from light |
Step-by-Step Procedure
Collect urine, blood, wound swab or CSF as clinically indicated; inoculate onto MacConkey agar and incubate at 35–37°C for 18–24 hours.
Note colour (pale to pink), size, texture and pigment (e.g. red prodigiosin pigment suggests Serratia marcescens at 15–20°C).
Confirm Gram-negative bacilli; test motility (all four genera are typically motile).
Test indole, methyl red, Voges-Proskauer, and citrate utilisation to place the isolate into the correct metabolic group.
Test urease production and H₂S formation on TSI/Kligler iron agar to further differentiate genera and species.
Cross-reference the full biochemical profile against the standard characteristics table to reach a confident genus/species-level identification.
Flow Diagram
Quality Control
Maintain reference strains of Citrobacter freundii, Enterobacter cloacae, and Serratia marcescens for use as positive/negative controls with every new batch of MR-VP broth, citrate agar and urea medium, confirming expected reaction patterns before reporting clinical isolates.
Participate in EQAS schemes covering less-common Enterobacteriaceae identification, since misidentification (e.g. Citrobacter mistaken for Salmonella due to shared O antigen) can have significant clinical and public-health reporting consequences.
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 |
|---|---|---|
| MR+, VP−, H₂S+, indole variable, pale/pink colonies on MacConkey | Citrobacter species | Correlate with UTI, gall bladder, or (rarely) neonatal meningitis (C. koseri) |
| H₂S+ in KI medium, indole+, urease−, slow lactose fermenter | Edwardsiella tarda | Correlate with wound, urine, blood, or CSF isolation; consider aquatic exposure history |
| MR−, VP+, indole−, citrate+, mucoid pink colonies | Enterobacter species | Report as Enterobacter; consider nosocomial UTI/respiratory/wound infection |
| MR−, VP+, red pigment at 15–20°C, no H₂S | Serratia marcescens | Report as Serratia; assess for multidrug resistance and nosocomial source |
| Isolate sharing O antigen with Salmonella from faecal sample | Possible Citrobacter misidentified as Salmonella | Confirm with full biochemical panel before reporting as Salmonella |
Common Errors & How to Avoid Them
Cause: Citrobacter shares O (somatic) antigen with Salmonella, which can cause serological cross-reaction and misidentification in faecal samples.
Prevention: Always confirm identification biochemically (urease, H₂S pattern, citrate) rather than relying on O antigen agglutination alone.
Cause: Enterobacter forms pink, mucoid colonies on MacConkey agar similar in appearance to Klebsiella.
Prevention: Differentiate using motility testing — Enterobacter is motile while Klebsiella is non-motile — plus the full biochemical panel.
Cause: Prodigiosin (red pigment) is best formed at 15–20°C; incubating exclusively at 37°C may yield non-pigmented, harder-to-recognise colonies.
Prevention: If Serratia is suspected but colonies are non-pigmented, consider incubating a duplicate plate at room temperature (15–20°C) to allow pigment expression.
Laboratory Tips from the Bench
The red pigment of Serratia marcescens is a helpful visual clue, but remember it is temperature-dependent — a lack of pigment at 37°C does not rule out Serratia; biochemical confirmation is still required.
When a faecal isolate agglutinates with Salmonella O antiserum but the organism is urease-positive or shows other atypical Salmonella reactions, always suspect Citrobacter and run a confirmatory biochemical panel before finalising the report.
Remember the MR/VP metabolic divide: 'Citrobacter and Edwardsiella use the Mixed-acid path (MR+), while Enterobacter and Serratia take the Butanediol path (VP+)' — this single pair of tests splits the four genera into two clean groups.
Important Notes
All four genera — but particularly Enterobacter and Serratia — are increasingly implicated in hospital-acquired infections and are often multidrug resistant, making accurate identification and antimicrobial susceptibility testing clinically essential.
These organisms are widely distributed in water, soil, sewage and on vegetables; their presence in a clinical specimen should always be interpreted in the context of possible environmental contamination versus true infection.
Interactive Quiz
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Clinical Case Study
Apply Your KnowledgeDevelops fever (38.2°C), lethargy and poor feeding on day 5 of NICU stay. Blood culture is drawn as part of a sepsis workup.
The weak, atypical agglutination with Salmonella O antiserum combined with weak urease positivity strongly suggests Citrobacter rather than true Salmonella, since Citrobacter shares the O antigen but shows different biochemical behaviour; C. koseri is a recognised, though uncommon, cause of neonatal sepsis and meningitis.
- →Never rely on O antigen agglutination alone in neonatal or faecal isolates — confirm biochemically.
- →Citrobacter koseri is a rare but important cause of neonatal meningitis.
- →Prompt correct identification changes both antibiotic choice and infection-control response.
Frequently Asked Questions
Although traditionally viewed as low-virulence environmental or commensal organisms, they are increasingly recognised as important opportunistic and nosocomial pathogens, particularly in immunocompromised, neonatal, or hospitalised patients, and are often multidrug resistant.
Both form mucoid pink colonies on MacConkey agar, but Enterobacter is motile while Klebsiella is non-motile — a simple motility test is usually the fastest differentiator.
Prodigiosin pigment production is temperature-dependent and best expressed at 15–20°C; at the standard 37°C incubation temperature used for most clinical cultures, growth is favoured over pigment production, so colonies often appear non-pigmented.
Quick Revision
10-Minute ReviewKey Takeaways
- Citrobacter, Edwardsiella, Enterobacter and Serratia are opportunistic Enterobacteriaceae of growing clinical importance.
- Serological cross-reactivity (e.g. Citrobacter with Salmonella) makes biochemical confirmation essential.
- The MR/VP test pair cleanly divides these four genera into two metabolic groups.
- Serratia's red pigment (prodigiosin) is a useful but temperature-dependent visual clue.
- Multidrug resistance is increasingly common across this group, especially in hospital settings.
Competency Checklist
Track Your MasteryReferences
- National Institute of Open Schooling. Microbiology — Lesson 22: Citrobacter, Edwardsiella, Enterobacter and Serratia.
- Forbes BA, Sahm DF, Weissfeld AS. Bailey & Scott's Diagnostic Microbiology.
- Murray PR, Rosenthal KS, Pfaller MA. Medical Microbiology.