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ENTEROCOCCI
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
                            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          +
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
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
Virulence Factors
•   Cytolysin/hemolysin
•   Aggregation substance
•   Extracellular surface protein
•   Extracellular superoxide
•   Lipoteichoic acid
•   Coccolysin
Clinical relevance
•   Complicated UTI
•   Bacteremia
•   Endocarditis
•   Intra abdominal & pelvic infection
•   Wound and soft tissue infection
•   Neonatal sepsis
•   Meningitis (rarely)
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
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 al

Indian 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
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
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
Results
Antibiotic resistance pattern
Resistance pattern
Resistance                 High level resistance
Tetracycline               Gentamicin
Erythromycin               Kanamycin
Ciprofloxacin              Amikacin
Chloramphenicol            Streptomycin
VRE
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
THANK YOU!!

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Enterococci

  • 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 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
  • 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 • Resistance to ß-lactams  Low affinity of penicillin binding protein • Low level resistance to aminoglycosides  Low uptake of these agents
  • 10. Acquired resistance • Broad host – 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 • 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.
  • 13. Prevalence and antimicrobial resistance 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 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
  • 15. • 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
  • 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 • 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
  • 24. Resistance pattern Resistance High level resistance Tetracycline Gentamicin Erythromycin Kanamycin Ciprofloxacin Amikacin Chloramphenicol Streptomycin
  • 25. VRE
  • 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 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
  • 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 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