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Enterococci
Enterococci are Gram-positive cocci that are natural inhabitants of the gastrointestinal tract. They have become important nosocomial pathogens due to their intrinsic and acquired antibiotic resistance. This study found that Enterococcus faecalis was the most common species isolated from clinical specimens in two Saudi hospitals. Many isolates showed resistance to tetracycline, ciprofloxacin, and chloramphenicol. Vancomycin resistance was observed in 3.9% of isolates, with the VanA phenotype being most common. Pulsed-field gel electrophoresis identified identical clones of E. faecalis isolated from different hospital wards, suggesting intra-hospital transmission. The high resistance rates indicate a need for improved infection control and antibiotic steward
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Overview of Enterococci; Gram-positive, GIT inhabitants, and unique growth characteristics.
Presumptive identification techniques based on hemolysis and phenotypic classification of Enterococcus species.
Growth characteristics of various Enterococcus species under different conditions.
Virulence factors such as cytolysin and extracellular proteins that contribute to pathogenicity.
Clinical relevance of Enterococci in various infections including UTIs and endocarditis.
Intrinsic and acquired antibiotic resistance mechanisms affecting Enterococci.
Detailed mechanisms leading to vancomycin resistance in Enterococci and involved gene clusters.
Study introduction, prevalence, and increase in Vancomycin Resistant Enterococci (VRE) cases.
Study methodology including sample distribution and antimicrobial susceptibility testing.
Results of antibiotic susceptibility tests and patterns of resistance observed.
Findings related to VRE isolates, their resistance profiles, and discussion on clinical significance.
Summary of findings, implications for infection control, and the need for ongoing surveillance.
Cited references that inform the presentation and the study's foundation.
Thank you note to the audience.
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Enterococci
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- 2. Enterococcus • Gram positive cocci, non motile, non sporing • Catalase Negative • Previously classified as group D streptococci • Natural inhabitants of GIT • Distinct features – Ability to grow at 10°C and 45°C – Ability to grow in 6.5%NaCl – Ability to grow at 9.6pH – Ability to hydrolyze esculin in 40% bile – Ability to process pyrrolidonyl arylamidase (PYR)
- 3. Presumptive identification of Streptococci & Enterococcci Hemolysis Hippurate Bacitracin Organism 6.5%NaCl Optochin solubility esculin Camp PYR Bile Bile SXT Lap Group A ß S R - - + + - - R - streptococci Group B ß, none R R + + + - - V R - streptococci Group C,G & F ß V S - - + - - - R - streptococci Group D α, ß, R R - V + + + + R - Enterococcci none Other Group D α, R S - - + - + - R - streptococci none Viridans α, none V S - V + - V - R - streptococci Pneumococci α V S - - + - - - S +
- 4. Classification (based onphenotypic characters in clinical isolates) Group 1 Group 3 Group 5 E.avium E.dispar E.columbae E.gilvus E.durans E.canis E.malodartus E.hirae E.moraviensis E.pallens E.ratti E. pseudoavium E.villorum E.raffinosus E.sacchrolyticus Group 2 Group 4 E.fecalis E.asini E.fecium E.cecorum E.casseliflavus E.sulfures E.gallinarum E.phoeniculicola E.mundtii Enterococcus sp E.hemoperoxidus Enterococcus sp Koneman textbook of diagnostic microbiology
- 5. MANNITOL Species PYRUVATE RAFFINOSE SORBOSE ARABINOS GLUCOSE SUCROSE 6.5%Nacl SORBITOL MGP 10°C 45°C ADH PYR LAP HIP E E.fecalis + + + + + + + + + - - + - + + - E.fecium + + + + + + - + + - + V V + - - E.casselifl + NA + + + + - + + - + V + + V + avus E.gallinar + + + + + + + + + - + - + + - + um E.durans + NA + + + + V + - - - - - - - - E.hirae + + + + + + - + - - - - + + - - E.avium + NA + + + - V + + + + + - + + V LAP-leucine aminopeptidase, PYR- pyrrolidonyl arylamidase, ADH- arginine dihydrolase, HIP – hippurate, MGP- methyl α-D-glucopyranoside
- 6. Virulence Factors • Cytolysin/hemolysin • Aggregation substance • Extracellular surface protein • Extracellular superoxide • Lipoteichoic acid • Coccolysin
- 7. Clinical relevance • Complicated UTI • Bacteremia • Endocarditis • Intra abdominal & pelvic infection • Wound and soft tissue infection • Neonatal sepsis • Meningitis (rarely)
- 8. Antibiotic resistance Intrinsic resistance Acquired resistance • Penicillinase resistant • Chloramphenicol penicillins • Erythromycin • Cephalosporins • Tetracyclins • Low level aminoglycosides • High level clindamycin • Low level clindamycin • High level aminoglycosides • Fluroquinolones • Penicillins • Vancomycin
- 9. Intrinsic resistance • Resistanceto ß-lactams Low affinity of penicillin binding protein • Low level resistance to aminoglycosides Low uptake of these agents
- 10. Acquired resistance • Broadhost – range plasmids • Narrow host - range plasmids • Conjugative transposons
- 11. Acquired resistance (Cont’d) •Chloramphenicol mediated by chloramphenicol acetyl transferase, prevents binding to 50S ribosome • Erythromycin resistance occurs as a part of Macrolide- Lincosamide-streptogramin B resistance phenotype. – Transferred by resistance deretminant ermB carried on Tn917. – Leads to methylation of adenosine residue in 23S rRNA. – Also confers high level resistance to clindamycin • Tetracycline resistance transferred by plasmid pAMα1 – Promotes active efflux of tetracycline from cells – Protects ribosomes from inhibition by tetracycline • Aminoglycosides – Cell membrane bound inactivating enzymes – Decreased affinity of ribosomal proteins to aminoglycoside (mutation) – Decreased uptake of drug
- 12. Vancomycin resistance • Resistanceto glycopeptides is due to alteration of peptidoglycan precursor D-Ala –D- Ala to D-Ala – D-Lactate / D-Ala-D-Ser • Genes involved are VanS/VanR • VanS gene activates D-Lac/D-Ser peptidoglycan precursor • D-Lac has 1000 times less affinity to vancomycin • D-Ser exhibits 7fold less affinity to vancomycin • Six gene clusters have been identified – VanA, VanB, VanC, VanD, VanE & VanG • VanA is acquired by Transposon Tn1546 • vanB is acquired by Transposon Tn1547/Tn5382 • Genetic material transferred via conjugation involving a pheromone induced system.
- 13. Prevalence and antimicrobialresistance pattern of multidrug –resistant enterococci isolated from clinical specimens MM Salem-Bekhit et al Indian Journal Of Medical Microbiology (2012) 30(1): 44-51
- 14. Introduction • Evolved fromintestinal commensal to 2nd most common nosocomial pathogen. • Common species – Entrococcus fecalis – Enterococcus fecium – E gallinarum – E casseliflavus – E durans – E avium – E hirae
- 15. • Rapid increasein colonization and infection with Vancomycin Resistant Enterococci(VRE) • Resistance intrinsic/ plasmid mediated. • Resistance likely due to widespread use of Vancomycin and Cephalosporins • Vancomycin resistance – Van A High level resistance – Van B – Van B2 – Van D – Van C Intrinsic low level resistance • VRE most common in E.fecium • Transfer resistance via plasmids to MRSA
- 16. Materials & Methods Distribution of samples • Period of study January 2009 – March 2010 • Place of study • King Khaled University Hospital (140samples) Urine 32 • King Saud Medical City Hospital (100 Samples) Blood 30 • Ethical Burns committee approval 17 • Approval from both hospital ethical committee & from Saudi ministry Throat swabs of Health 12 • Isolation Bed sore 11 – Urine, sterile body fluids & wounds Devices 14 • Trypticase soy agar with 5% sheep blood – Stool samples Stool samples 47 • Inoculated into enterococcal37 Pus broth , incubated overnight at 35°C • Subcultured to BHIA with 6µg vancomycin/ml & BHIA without Body fluids Vancomycin 16 Wound discharge 24
- 17. • Presumptive identification – Growth characteristics on blood agar – Gram staining morphology, – Catalase reaction, – Ability to grow in 6.5%Nacl, – Bile esculin hydrolysis & – Biochemicals using API Strep system. • Enterococcal samples stored in 16%glycerol at -70°C
- 18. Antimicrobial Susceptibility Testing Drug Concentration Ampicillin 10µg Vancomycin 30µg Teicoplanin 30µg Erythromycin 15µg Ciprofloxacin 5µg Chloramphenicol 30µg Amikacin 200µg Streptomycin 300µg Gentamycin 10µg Kanamycin 200µg Tetracycline 30µg Linezolid 30µg Quality control strain E.fecalis ATCC 51299
- 19. MIC • E-test Vancomycin , Teicoplanin • Agar dilution method Gentamycin, Kanamycin, Streptomycin, Amikacin & Linezolid. • Vancomycin resistance any enterococcal isolate with MIC to vancomycin if atleast 16µg/ml
- 20. ß-Lactamase production &inhibition tests • Nitrocefin 5µl • Amoxicillin-clavulanic acid, Ampicillin- sulbactum on disc-agar diffusion method.
- 21. Further analysis • DNAisolation • Detection of vancomycin determinants – Denaturation at 94°C for 3 min – Anneling at 60°C for 45seconds – Extension at 72°C for 1min – Final extension at 72°C for 2min – Amplicons analysed by electrophoresis on 1% agarose gel containing ethidium bromide • Restriction fragment length analysis by pulse field gel electrophoresis
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- 24. Resistance pattern Resistance High level resistance Tetracycline Gentamicin Erythromycin Kanamycin Ciprofloxacin Amikacin Chloramphenicol Streptomycin
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- 26. VRE • Van A – 8 isolates – 5 E.faecium and 3 E.fecalis – Resistant to vancomycin & Teicoplanin • Van B – 1 isolate – Intermediate resistance to Vancomycin and sensitive to Teicoplanin • Van C – 4 isolates – 3 E.gallinarum and 1 E.casseliflavus – Intermediate resistance to Vancomycin and sensitive to Teicoplanin
- 27. PGFE of VRE •8 isolates tested • 5 isolates had identical profile • Isolates obtained from patients admitted in different wards in same hospital
- 28. Discussion • E.fecalis wasthe predominant pathogen • Many isolates were resistant to Tetracycline, Ciprofloxacin & Chloramphenicol • Resistance to Erythromycin was lower compared to other countries • Nosocomial VRE reported is 3.9% (0.3% in 1989 – 11% in 1996) • Isolates of VRE in this study were from immunocompromised patients or with h/o Nosocomial infection • Vancomycin resistant phenotype Van A was predominant and resulted in high level resistance • Van B phenotype showed moderate vancomycin resistance • Being the mainstay of treatment, ampicillin resistance causes concern
- 29. • E.gallinarum ,a rare enterococcal species in human infection was isolated • The clone of E.fecalis obtained from two hospital were identical. This may be due to intra hospital dissemination. • Aminoglycoside resistance along with vancomycin resistance indicates a need for regular surveillance studies, infection control measures and antibiotic policy • As vancomycin resistance can spread via plasmids to other species like S.aureus there is an increased chance of seeing more vancomycin resistant cases in future.
- 30. REFERENCES • Koneman TextbookOf Diagnostic Microbiology • Kater fisher et al . The ecology, epidemiology and virulence of enterococcus Microbiology(2009), 155,1749-1757 • PM Giridhara upadhyaya et al. Review of virulence factors of enterococcus: an emerging nosocomial pathogen IJMM (2009) 27(4): 301-5 • Barbara E.Murray. The life and times of enterococcus Clinical microbiology reviews jan 1990. p 46-65
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