Overview
Enterococcus is a genus of lactic acid bacteria comprising catalase-negative, Gram-positive cocci that often occur in pairs or short chains. They are part of the normal enteric flora but have become one of the most important causes of nosocomial infection worldwide because of their remarkable antibiotic resistance.
Two species dominate clinical practice β E. faecalis (90β95% of infections) and E. faecium (5β10%, but increasingly antibiotic-resistant). This lesson covers their identification, differentiation from streptococci, and the growing problem of vancomycin resistance.
Learning Objectives
After this lesson you will be able toβ¦- Describe the general characteristics of Enterococcus.
- Identify Enterococci in the laboratory using biochemical tests.
- Explain the pathogenesis of enterococcal infection.
- Discuss intrinsic and acquired antibiotic resistance in Enterococcus, including vancomycin resistance.
Clinical Story
Why This MattersAn elderly patient in the intensive care unit develops a catheter-associated urinary tract infection that fails to respond to first-line cephalosporins. The microbiology lab isolates a catalase-negative, Gram-positive coccus that grows in 6.5% NaCl and hydrolyses bile-aesculin β raising immediate suspicion of Enterococcus and the need to test for vancomycin resistance before treatment can proceed.
Core Concepts
Enterococci are facultative anaerobes capable of respiration in both oxygen-rich and oxygen-poor environments. Although they do not form spores, they tolerate a remarkably wide range of conditions: temperature 10β45Β°C, pH 4.5β10.0, and high sodium chloride concentrations. They are normal inhabitants of the gastrointestinal flora and were previously classified as Group D streptococci.
Enterococci are Gram-positive cocci occurring singly, in pairs, and short chains; cells may appear coccobacillary from agar growth but more oval and chain-like from thioglycolate broth. They can exhibit beta or gamma haemolysis (occasionally alpha) on sheep blood agar and grow optimally at 35Β°C, over a much wider temperature range (10β45Β°C) than streptococci.
Because the group D antigen is a teichoic acid and not a good serological marker, Enterococci are identified by phenotype: growth in broth with 6.5% NaCl, hydrolysis of aesculin in the presence of 40% bile salts (bile-aesculin medium), hydrolysis of pyrrolidonyl-Ξ²-naphthylamide (PYR positive, except E. cecorum, E. columbae, E. saccharolyticus), and production of leucine aminopeptidase (LAP positive in most strains).
Once confirmed as Enterococcus, species are divided into 5 groups based on acid formation in mannitol, sorbitol and sorbose broths and arginine hydrolysis. Pigmentation testing (yellow pigment) identifies E. casseliflavus, E. mundtii and related species. Pyruvate utilisation differentiates E. faecalis from E. faecium, while the tellurite tolerance test also separates these two clinically important species.
Enterococci are intrinsically resistant to cephalosporins, penicillinase-resistant penicillins, and monobactams, with low-level intrinsic resistance to aminoglycosides and intermediate-to-resistant susceptibility to fluoroquinolones. They are inhibited but not killed by beta-lactams such as ampicillin. Vancomycin, the primary alternative therapy, is increasingly compromised by Vancomycin-Resistant Enterococci (VRE), most common in E. faecium but also seen in E. faecalis.
Enterococci are among the most frequent causes of nosocomial infection, especially in intensive care units, selected for by cephalosporin therapy to which they are intrinsically resistant. They are transmitted primarily via the hands of hospital personnel and occasionally via medical devices. Common infection sites include the urinary tract, wounds, biliary tract, and blood; they may cause neonatal meningitis/bacteraemia and adult endocarditis.
Laboratory Principle
Presumptive identification of Enterococcus exploits its unusual physiological hardiness relative to other streptococci: it can hydrolyse aesculin to aesculetin in the presence of 40% bile (a concentration that inhibits most other Gram-positive cocci), and it can grow in hypertonic 6.5% NaCl broth and at temperatures up to 45Β°C β conditions that streptococci cannot tolerate. Combined with a positive PYR and LAP enzymatic test, this phenotypic fingerprint reliably distinguishes Enterococcus from Streptococcus without relying on the poorly antigenic group D teichoic acid.
Equipment Required
Reagents & Materials
| Reagent / Material | Concentration / Grade | Purpose | Storage |
|---|---|---|---|
| Bile-Aesculin Agar | 40% bile salts, aesculin | Detects aesculin hydrolysis (blackening) | 2β8Β°C |
| 6.5% NaCl Broth | 6.5% sodium chloride | Tests salt tolerance of Enterococcus | Room temperature |
| PYR Reagent/Disc | L-pyrrolidonyl-Ξ²-naphthylamide | PYR hydrolysis test | 2β8Β°C, protect from light |
| LAP Disc | Leucine-Ξ²-naphthylamide | Leucine aminopeptidase test | 2β8Β°C |
| Sheep Blood Agar | 5% sheep blood | Haemolysis pattern & primary culture | 2β8Β°C |
| Hydrogen Peroxide (3%) | Catalase reagent | Confirms catalase-negative reaction | Room temperature, dark bottle |
Step-by-Step Procedure
Prepare a smear from the colony/broth and Gram stain to confirm Gram-positive cocci in pairs and short chains.
Add a colony to 3% hydrogen peroxide on a slide; absence of bubbling confirms catalase-negative status, consistent with Enterococcus.
Streak onto bile-aesculin agar; blackening of the medium within 24β48 hours indicates aesculin hydrolysis in the presence of bile.
Inoculate 6.5% NaCl broth; turbidity after incubation at 35Β°C confirms growth in hypertonic conditions.
Apply the isolate to PYR and LAP discs/reagents; a positive (red/orange) colour reaction within 5 minutes supports Enterococcus identification.
Perform mannitol, sorbitol, sorbose, arginine hydrolysis, pigmentation, pyruvate utilisation, and tellurite tolerance tests to differentiate E. faecalis from E. faecium and other species.
Flow Diagram
Quality Control
Include a known Enterococcus faecalis (ATCC 29212) and a Streptococcus species (bile-aesculin negative, salt-intolerant) as positive and negative controls with every batch of bile-aesculin agar and 6.5% NaCl broth. Run vancomycin susceptibility controls (VRE and vancomycin-sensitive strains) whenever new antibiotic discs are introduced.
Enrol in a national or regional EQAS programme for antimicrobial susceptibility testing, specifically covering vancomycin resistance detection in Enterococcus, given its major infection-control significance in hospital settings.
Reference Values
Key Diagnostic Parametersβ οΈ Reference ranges may vary between laboratories. Always apply your laboratory's established reference intervals.
Clinical Interpretation
| Finding | Possible Significance | Action / Follow-up |
|---|---|---|
| Catalase-negative, bile-aesculin positive, grows in 6.5% NaCl | Confirms genus Enterococcus | Proceed to species identification and susceptibility testing |
| Vancomycin-resistant Enterococcus (VRE) | Serious nosocomial pathogen, especially in E. faecium | Contact/infection control precautions; alternate antibiotics (e.g. linezolid) |
| Enterococcus isolated from blood in patient with heart murmur | Possible infective endocarditis | Blood cultures, echocardiography, combination antibiotic therapy |
| Enterococcus in mixed urine culture, low colony count | Possible contamination/colonisation rather than true infection | Correlate with symptoms and repeat culture if needed |
Common Errors & How to Avoid Them
Cause: Relying on group D antigen agglutination, which is a poor teichoic-acid-based marker, gives unreliable identification.
Prevention: Use phenotypic tests β bile-aesculin, 6.5% NaCl, PYR, LAP β instead of serogrouping for definitive identification.
Cause: Standard susceptibility panels may not flag intrinsic low-level aminoglycoside resistance, leading to inappropriate monotherapy.
Prevention: Perform high-level aminoglycoside resistance screening before considering synergistic combination therapy for serious infections such as endocarditis.
Cause: Vancomycin resistance can be subtle and take longer to express phenotypically on standard susceptibility testing.
Prevention: Follow full recommended incubation times and use validated VRE screening media (e.g. vancomycin-containing selective agar) when infection control is a concern.
Laboratory Tips from the Bench
A quick bedside differentiator: Enterococcus grows readily at 45Β°C and in 6.5% NaCl broth, conditions that inhibit virtually all streptococci β use this simple physiological test when serology is ambiguous.
Always test for high-level aminoglycoside resistance before recommending synergistic penicillin-aminoglycoside combination therapy for enterococcal endocarditis, as intrinsic low-level resistance can mask true synergy failure.
Remember 'BEST' for Enterococcus ID: Bile-aesculin positive, 6.5% NaCl growth, Salt/temperature tolerant, Tests PYR/LAP positive.
Important Notes
Vancomycin-resistant Enterococcus faecium is a WHO priority pathogen. Strict infection control β hand hygiene, contact precautions, and environmental cleaning β is essential to prevent hospital-wide spread.
Even when susceptible in vitro, enterococci are typically only inhibited (bacteriostatic effect), not killed, by beta-lactams alone β hence combination therapy with an aminoglycoside is often needed for bactericidal effect in serious infections.
Interactive Quiz
Test Your KnowledgeFlashcards
Tap to flipClick or tap any card to reveal the answer. Use arrow keys to navigate in single-card mode.
Clinical Case Study
Apply Your KnowledgeDevelops fever (38.6Β°C) and cloudy, foul-smelling urine 5 days after catheter placement. No response to empiric cephalosporin therapy after 48 hours.
The catalase-negative, bile-aesculin positive, salt-tolerant coccus confirms Enterococcus, and vancomycin resistance identifies this as VRE β explaining both the catheter-associated UTI and the failure of standard cephalosporin therapy, to which Enterococcus is intrinsically resistant.
- βEnterococci are intrinsically resistant to cephalosporins β never expect them to work.
- βVRE requires alternative agents such as linezolid or daptomycin.
- βStrict contact precautions are needed to prevent VRE transmission in ICU settings.
Frequently Asked Questions
The group D cell wall antigen is a teichoic acid rather than a protein or polysaccharide, making it a poor immunogen for reliable serological grouping, so phenotypic tests are preferred.
Yes β it commonly causes wound, biliary tract and bloodstream infections, and can cause endocarditis in adults and meningitis/bacteraemia in neonates.
Beta-lactams inhibit cell wall synthesis but enterococci have tolerance mechanisms that prevent full bactericidal killing, so combination therapy with an aminoglycoside is often required for cure in serious infections.
Quick Revision
10-Minute ReviewKey Takeaways
- Enterococcus is a leading cause of hospital-acquired infection due to its inherent hardiness and antibiotic resistance.
- Phenotypic tests (bile-aesculin, salt tolerance, PYR, LAP) are more reliable than serology for identification.
- Cephalosporins should never be used to treat suspected enterococcal infection due to intrinsic resistance.
- Vancomycin-resistant Enterococcus (VRE) requires strict infection control measures.
- Combination therapy is often needed for bactericidal effect in serious enterococcal infections like endocarditis.
Competency Checklist
Track Your MasteryReferences
- National Institute of Open Schooling. Microbiology β Lesson 17: Enterococcus.
- Murray PR, Rosenthal KS, Pfaller MA. Medical Microbiology.
- Clinical and Laboratory Standards Institute (CLSI) guidelines for antimicrobial susceptibility testing.