Prevalence of ESBL and MBL antibiotic resistance genes in Klebsiella pneumoniae in Pretoria Academic Hospital Maningi NE, Ehlers MM, Hoosen AA, Makgothlo E, Omar S and Kock MM Department of Medical Microbiology, Faculty of Health Sciences, University of Pretoria/NHLS INTRODUCTION

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Extended spectrum β-lactamase (ESBL) are group of β lactamases that are plasmid mediated that have the ability to hydrolyse the newer cephalosporins and aztreonam and are inhibited by clavulanic acid

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Extended spectrum β-lactamase producing K. pneumoniae is the cause of most of the multi-drug resistant nosocomial infections

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The ESBL-producing K. pneumoniae can be identified by the presence of the genes blaCTX-M, blaSHV and blaTEM coding for the β-lactamases cef enzymes

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The transmission of ESBLs is due to clonal spread, plasmid dissemination or both mechanisms among or between species

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Metallo β-lactamases has recently emerged as a resistant class of broad-spectrum β-lactamases in K. pneumoniae and it can be identified by the presence of blaVIM gene

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These ESBLs and MBL enzymes can be detected

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This study investigated the prevalence of ESBL and MBL antibiotic resistance genes in K. pneumoniae isolates from Pretoria Academic Hospital

MATERIALS AND METHODS

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This study was a prospective descriptive study in which ninety seven K. pneumoniae isolates were obtained from clinical specimens sent for analysis to the Microbiology laboratory, National Health Laboratory Services, Pretoria Academic Hospital

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The VITEK 2 (bioM érieux, France) was used for phenotypic

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identification of the isolates as ESBL-producing K.

pneumoniae

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The MagnaPure LC Compact (Roche Applied Science, Germany) was used for the automated extraction of DNA according to manufacturer’s instructions

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The multiplex PCR assay amplifying the blaCTX-M, blaSHV,

blaTEM and blaVIM genes was performed using the QIAGEN

Multiplex PCR kit (QIAGEN, USA) following the manufacture’s instructions

REFERENCES

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Aranzazu Valverde, Teresa MC, Lucia GM, Fernando B and Rafael C (2008) Complex molecular epidemiology of extended-spectrum β-lactamases in Klebsiella pneumoniae: a long-term perspective from a single intitution in Madrid. Journal of antimicrobial Chemotherepy 61:64-72 Franceco L, Jean-Denis D, Celine C, Adrea E, Gianluigi L, Gianfranco A, Gian M and Antoniolo (2004) Emergence in K. pneumoniae and E. cloacae clinical isolates of the VIM-4 Mettallo β-Lactamase encoded by a conjugative plasmid. Antimicrobial agents and chemothetapy 48:648-650 Michel A, Pfaller and John S (2006) Overview of the epidemiological profile and laboratory detection of extended spectrum β-lactamases. Clinical ifectiouse diseases 42:153-163

RESULTS AND DISCUSSION

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Multiplex PCR detected the presence of CTX-M, SHV and TEM genes in the K. pneumoniae isolates from the Pretoria Academic Hospital (Figure 1)

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No VIM genes were detected with the multiplex PCR in the K.

pneumoniae isolates included in this study

L 1 2 3 4 5 6 7 8 9 10 11 12 13 1 4 15 C L

747 bp

bla

SHV 445 pb

bla

TEM 593 bp

bla

CTX-M

100 bp DNA ladder

Figure 1: Multiplex PCR simultaneously amplified the bla CTXM and bla TEM (593 bp),

bla

SHV (747 bp) (445 bp). In lanes 2, 5, 6 7, 8, 9 10, 11, 12,13 and 14 ESBL genes were detected while lanes 1, 3, 4, and 15 were negative for all three genes. Negative control was included in lane C and lanes L represent the molecular weight marker (100 bp DNA ladder, Promega, Medison, USA)

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The ESBL genes were detected in different combinations as only one gene, two genes or all three genes in one isolate (Figure 2)

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Most of the ESBL-producing K. pneumoniae isolates harboured three ESBL genes (Figure 2)

SHV, CTX, TEM, 36% SHV, TEM, 3% CTX, TEM, 18% SHV, 16% TEM, 4% N, 13% VIM 0 CTX, SHV 3% CTX-M, 3%

Figure 2: Pie chart representation of Multiplex PCR results of three ESBL genes bla CTX-M ,

bla

SHV and bla TEM detected in bacterial isolates

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Multiplex PCR results obtained in this study of K. pneumoniae isolates revealed that 89% (87/97) were positive for ESBL genes

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This prevalence of ESBL-producing K. pneumoniae genes is alarming compared to the 22% prevalence of these gene in SA 1998-1999 (Michel et al., 2006)

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Carbapenems are the preferred antibiotic for the treatment of serious infections caused by ESBLs, since they are resistant to the newer expanded-spectrum cephalosporins

CONCLUSION

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There is a high prevalence of ESBL-producing K. pneumonia isolates in Pretoria Academic Hospital

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This leads to a need for improved, adequate infection control measures to be instituted and more rational use of antibiotics in Pretoria Academic Hospital