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)
Presumptive identification of Streptococci & Enterococcci Hemolysis Hippurate BacitracinOrganism 6.5%NaCl Optochin solubility esculin Camp PYR Bile Bile SXT LapGroup A ß S R - - + + - - R -streptococciGroup B ß, none R R + + + - - V R -streptococciGroup C,G & F ß V S - - + - - - R -streptococciGroup D α, ß, R R - V + + + + R -Enterococcci noneOther Group D α, R S - - + - + - R -streptococci noneViridans α, none V S - V + - V - R -streptococciPneumococci α V S - - + - - - S +
Classification (based on phenotypic 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
Intrinsic resistance• Resistance to ß-lactams Low affinity of penicillin binding protein• Low level resistance to aminoglycosides Low uptake of these agents
Acquired resistance• Broad host – range plasmids• Narrow host - range plasmids• Conjugative transposons
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
Vancomycin resistance• Resistance to 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.
Prevalence and antimicrobial resistance pattern of multidrug –resistant enterococci isolated from clinical specimens MM Salem-Bekhit et alIndian Journal Of Medical Microbiology (2012) 30(1): 44-51
Introduction• Evolved from intestinal commensal to 2nd most common nosocomial pathogen.• Common species – Entrococcus fecalis – Enterococcus fecium – E gallinarum – E casseliflavus – E durans – E avium – E hirae
• Rapid increase in 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
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
• 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
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
ß-Lactamase production &inhibition tests• Nitrocefin 5µl• Amoxicillin-clavulanic acid, Ampicillin- sulbactum on disc-agar diffusion method.
Further analysis• DNA isolation• 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
Antibiotic resistance pattern
Resistance patternResistance High level resistanceTetracycline GentamicinErythromycin KanamycinCiprofloxacin AmikacinChloramphenicol Streptomycin
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
PGFE of VRE• 8 isolates tested• 5 isolates had identical profile• Isolates obtained from patients admitted in different wards in same hospital
Discussion• E.fecalis was the 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
• 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.
REFERENCES• Koneman Textbook Of 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