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
9. Intrinsic resistance
• Resistance to ß-lactams Low affinity of
penicillin binding protein
• Low level resistance to aminoglycosides
Low uptake of these agents
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
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
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
