Phenotypic and Genotypic Characterisation of Vancomycin

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Phenotypic and Genotypic Characterisation of Vancomycin Powered By Docstoc
					Phenotypic and Genotypic Characterisation of
   Vancomycin Resistant Enterococci from
              Clinical Samples
               Ira Praharaj,S.Sujatha,S.C.Parija

                     Department of Microbiology,
Jawaharlal Institute of Postgraduate Medical Education and Research
                              Puducherry
                  Introduction

• Enterococci have emerged as important nosocomial
  pathogens

• Increasing trend of resistance to various
  antimicrobial agents including glycopeptides like
  vancomycin

• Limited number of Indian studies dealing with
  vancomycin resistance in enterococci
                   Objectives
• To study the antimicrobial resistance pattern of
  isolates of Enterococcus species from clinical samples
  with reference to commonly used antibiotics

• To determine the prevalence of vancomycin
  resistance among isolates of Enterococcus species

• To determine the genetic basis of vancomycin
  resistance using Polymerase Chain Reaction
Materials & Methods
• Place of study
  – Department of Microbiology,
    Jawaharlal Institute of Postgraduate Medical Education and
    Research(JIPMER),Puducherry


• Period of study
  – 1 year(November 2008 –October 2009)
              Inclusion Criteria
• All isolates of Enterococcus species from the
  diagnostic bacteriology laboratory

   – Isolates from clinical specimens like
     blood,urine,sterile body fluids like CSF,peritoneal
     fluid,pleural fluid and exudates
      Identification of Enterococci
• Gram positive cocci in
  pairs and chains
• catalase negative
• Bile-esculin hydrolysis
• 6.5% NaCl tolerance
• Heat tolerance
• Lancefield Grouping-
  Group D
• PYR test
Antimicrobial Susceptibility Testing
• Kirby-Bauer Disk diffusion testing(CLSI guidelines)
• Mueller Hinton Agar
   –   Ampicillin
   –   Ciprofloxacin
   –   Tetracycline
   –   Vancomycin
   –   Teicoplanin
   –   Linezolid
   –   Nitrofurantoin(only for urine isolates)
         Glycopeptide Resistance
• Vancomycin
   – Disk diffusion testing
   – Vancomycin screening agar(6µg/ml)
   – Minimum Inhibitory Concentration-Agar dilution(CLSI
     guidelines)-vancomycin(HiMedia)
• Teicoplanin
   – Disk diffusion testing
   – Minimum Inhibitory Concentration-Agar dilution
Vancomycin Screen Agar
                Speciation of VRE
• Facklam and Collin’s Characterisation Scheme
• Tests used
   –   Arginine dihydrolysis
   –   Fermentation of Mannitol,Sorbose,Arabinose,Raffinose
   –   Pyruvate utilisation
   –   Growth on Potassium Tellurite agar
   –   Motility
   –   Pigment production
           Speciation




Enterococcus faecalis– Arginine dihydrolysed
                       Sorbose not fermented, Mannitol fermented
                       Pyruvate utilised,Arabinose not fermented
                       Growth on PTA
 Enterococcus gallinarum-arginine dihydrolysed,sorbose not
fermented,mannitol fermented ,Pyruvate utilised,Arabinose
                     fermented,motile
Molecular Basis of Vancomycin Resistance

• DNA extraction-Rapid alkaline lysis method
  [Dutka-Malen et al,JCM,1990)
   – Phenol-Chloroform extraction method for DNA purification


• Primers (Sigma Aldrich,USA)

• Multiplex PCR to detect vancomycin resistance genes
Vancomycin Resistance     Primers                    Amplicon size(PCR product)
  • Primers
gene


vanA                      5’-GGGAAAACGACAATTGC-3’    732 bp
                          5’-GTACAATGCGGCCGTTA-3’


vanB                      5’-ATGGGAAGCCGATAGTC-3’    635bp
                          5’-GATTTCGTTCCTCGACC-3’


vanC1                     5’-GGTATCAAGGAAACCTC-3’    822bp
                          5’-CTTCCGCCATCATAGCT-3’


vanC2/C3                  5’-CTCCTACGATTCTCTTG-3’    439bp
                          5’-CGAGCAAGACCTTTAAG-3’



                        Dutka-Malen et al,JCM,1995
Results & Discussion
• Total number of Enterococcus species isolates in the
  one year study period was 367




• Overall prevalence of Enterococcus species among
  clinical isolates was 0.78%
Distribution of Enterococcal
           Isolates
            35

                        Urine
 78                     Blood
                        CSF
                        Peritoneal Fluid
                 217    Pus/Wound swabs
  19                    Others
       14
  4
           Resistance to Antibiotics
                                    %resistance
80.00%
                  74.38%
                           71.38%
70.00%

60.00%

50.00%   45.23%

40.00%                                 37%

30.00%

20.00%                                            17%

                                                        8.70%   7.60%
10.00%
                                                                        0%
 0.00%
                 Glycopeptide Resistance
       No.       No.     No.                MIC in µg/ml
       Sensiti   Inter   Resi
       ve        medi    sta
                 ate     nt   <2    2   4      8     16    32   64   128   >128


Va     335       1       31   326   5   4      1     0     0    0    4     27
(n=
367)



Tec    338       0       29   333   5   1      0     0     1    5    15    8
(n=
367)
        Glycopeptide Resistance
• 32 isolates were identified as Vancomycin Resistant
  Enterococcus(VRE) after MIC testing
   – 29 of VRE were resistant to Teicoplanin

• 100% concordance between vancomycin screening
  agar and MIC testing

• Overall prevalence of vancomycin resistance among
  Enterococcus isolates was 8.7%
         Distribution of VRE isolates


        Others             2
          Urine                                                            13
  Wound swab                                                 8
         Blood                         4
            CSF                3
Peritoneal fluid       2
                   0   2           4          6          8       10   12    14


                                       No. of isolates
• All VRE isolates were from inpatients,thus reiterating
  the fact that VRE is a nosocomial pathogen




• 37.5% of the patients(12 patients) infected with VRE
  had been admitted in ICUs at some point of time
                   Mortality
• Mortality rate among patients infected with VRE was
  considerable at 31.25%
  (10 out of 32 patients infected with VRE expired)
Vancomycin Resistant Phenotypes
                          Phenotype   Species of   Number
                                      Enterococcus

     1                    VanA        -E.faecalis   27
 2
                                      -E.gallinarum 1
                                      -E.mundtii    1
              VanA
              Phenotype
                          VanB        -E.faecalis   2
              VanB
              Phenotype
              VanC
              Phenotype
                          VanC        -E.gallinarum 1
         29
  Vancomycin Resistance Genotypes
            VanC
  VanB    Phenotype
Phenotype    ,1
   ,2




                  VanA
                Phenotype,
                      29
vanA Resistance Genotype




 5   4   3   2   1   NC   PC    M


                                     1000 bp
                                    700 bp


                                    100 bp

                           732 bp
vanA gene in Enterococcus gallinarum
• An isolate of E.gallinarum from a CSF sample was
  found to have both vanC1 and vanA genes by
  multiplex PCR

• vanA gene is transferable and inducible in contrast to
  the vanC1 gene which is chromosomal

• Acquisition of the vanA gene altered the phenotype
  of the E.gallinarum isolate and conferred high-level
  glycopeptide resistance(VanA phenotype)
         9   8   7   6   5   4   3   2   1   M
                                                 1000bp
822 bp
732 bp                                           822 bp

320 bp                                           173 bp
                                                  100 bp
• No other study from India has yet reported
  the coexistence of vanA and vanC1 genes in a
  single VRE isolate
                    Conclusion
• Study demonstrates that enterococci are emerging
  nosocomial pathogens in our setup,isolated from
  various clinical specimens

• High rate of resistance to antimicrobials like
  fluoroquinolones,tetracycline,ampicillin and
  aminoglycosides

• Vancomycin resistance rate among enterococcal
  isolates was 8.7% which is quite high compared to
  other Indian reports
                      Conclusion
• VanA phenotype of vancomycin resistance commonest in
  concordance with studies elsewhere in the country and the
  world

• vanA genotype commonest in our study

• Other genotypes seen-vanC1 associated with intrinsic
  vancomycin resistance

• First Indian report of coexistence of vanC1 and vanA genes in
  an isolate of E.gallinarum conferring high level glycopeptide
  resistance to the isolate

				
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