African Journal of
Bacteriology Research

  • Abbreviation: J. Bacteriol. Res.
  • Language: English
  • ISSN: 2006-9871
  • DOI: 10.5897/JBR
  • Start Year: 2009
  • Published Articles: 123

Full Length Research Paper

Prevalence and characterization of NDM-1 and OXA-48 carbapenemase gene harboring Enterobacteriaceae in a tertiary care hospital, South India

Radha Srinivasan*
  • Radha Srinivasan*
  • Department of Microbiology. JIPMER, Puducherry-605 006, India.
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Kalaiarasan Ellappan
  • Kalaiarasan Ellappan
  • Department of Microbiology. JIPMER, Puducherry-605 006, India.
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Harish Belgode Narasimha
  • Harish Belgode Narasimha
  • Department of Microbiology. JIPMER, Puducherry-605 006, India.
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  •  Received: 17 June 2015
  •  Accepted: 12 August 2015
  •  Published: 30 October 2015

 ABSTRACT

The occurrence of carbapenem resistance among Enterobacteriaceae has reached critical levels worldwide. The present study was carried out to screen for carbapenem resistance among Enterobacteriaceae isolates from blood, surgical wound and tracheal samples in different wards and intensive care units between the period of 2010 through 2012 in a teaching hospital attached to JIPMER, south India. A total number of 425 meropenem-resistant isolates belonging to Enterobacteriaceae were included. Genes encoding β-lactamases (blaNDM-1 and blaOXA-48) and influx of β-lactams through the major porin OmpK36 were characterized. Multiplex PCR amplification assays indicated that out of 425 meropenem- resistant isolates, 75 and 187 isolates harbored blaNDM-1 with and without OmpK36, respectively. One and five Klebsiella pneumoniae isolates harbored blaOXA-48 with presence and absence of OmpK36, respectively. Transferability of the plasmids was tested by conjugation assays and it was found that 110 transconjugants carried blaNDM-1. blaOXA-48 gene was not transferable by conjugation analysis. The study highlights the emergence of blaNDM-1 and blaOXA-48 genes in this center.
 
Key words: Enterobacteriaceae; blaNDM-1; blaOXA-48; OmpK36; conjugation.


 INTRODUCTION

Antibiotic resistance is one of the most important growing global challenges to humans in the environment (Korzeniewska and Harnisz, 2013; Prabaker and Weinstein, 2011; Rhomberg and Jones, 2009; Duijn et al., 2011). Carbapenems are very useful beta-lactams for treatment of infections due to MDR Enterobacteriaceae. Several investigations have been reported on the importance of long term care facilities especially hospitals as reservoirs of multidrug-resistant bacteria (MDRB) and colonization with these bacteria may last for an extended period, and patients in long term care facilities represents a major route for the introduction of MDRB to acute-care facilities (Korzeniewska and Harnisz, 2013; Lewis et al., 2007; Mansouri and Abbasi, 2010; Pitout and Laupland, 2008).

Acquisition of carbapenemase enzymes and loss of porins are the main mechanism of resistance to this group of antibiotic (Cuenca et al., 2003; James et al., 2009). The most prevalent carbapenemases are the molecular class B metallo-β-lactamase (MBL), mainly blaNDM-1 and blaOXA-48 (Castanheira et al., 2011; Kilic et al., 2011). Particularly, in last few years blaNDM-1 (Castanheira et al., 2013) and blaOXA-48 (Pitart et al., 2011) genes are carried on plasmids that enable their transfer between different species of Enterobacteriaceae (Mohamed et al., 2013). Outer membrane protein (OMP) serve as a major gateway for the passage of β-lactams and deficiency of this protein has emerged as a major mechanism in multidrug resistant (MDR) Enterobacteriaceae (Jacoby et al., 2004; Pfeifer et al., 2012). Furthermore, previous investigations have been reported that deficiency in OMP expression resulting in the elevation of MIC in K. pneumoniae than that of non-deficient strains (James et al., 2009; Jacoby et al., 2004; Pfeifer et al., 2012).

In this work, an attempt has been made to study the characteristics of the recent dissemination of blaOXA-48 and blaNDM-1-producing Enterobacteriaceae in OmpK36 deficient strains and to determine its prevalence in strains isolated from patients. 


 MATERIALS AND METHODS

Bacterial isolates and susceptibility tests
 
A total of 425 meropenem-resistant Enterobacteriaceae isolates were collected between 2010 and 2012 from clinical specimens and tested for decreased susceptibility to carbapenems by agar dilution method, according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI 2011). According to CLSI recommendations, the production of carbapenemase among the isolates was phenotypically confirmed by the modified Hodge test and meropenem/ethylene diamine tetra-acetic acid (EDTA) double-disk synergy test as per CLSI guidelines 2011. Escherichia coli ATCC 25922 was used as a negative control.
 
PCR amplification       
 
Total DNA was extracted from all strains by boiling lysis method. A multiplex polymerase chain reaction (PCR) was used to amplify genes encoding blaNDM-1 and blaOXA-48 and OmpK36 (Table 1). A total of 30 cycles were performed with a thermal cycler (Master cycler gradient, eppendorf) consisting initial denaturation step for 15 min at 95°C, denaturation step for 1 min at 95°C, an annealing step for 1 min at 59°C and extension step for 1 min 30 s at 72°C. After the final cycle there was a step of final extension of 10 min at 72°C. The PCR products generated were visualized by ethidium bromide staining after electrophoresis in a gel containing 1.5% agarose.
 
Conjugation experiments
 
Conjugation was carried out at 30°C using sodium azide resistantE. coli J53 as recipient. Transconjugants were selected on MacConkey agar plates supplemented with meropenem (4 mg/L) and sodium azide (100 mg/L) (Jamal et al., 2012). Plasmid was extracted from transconjugants, respectively and analyzed for blaNDM-1 and blaOXA-48 genes. Multiplex PCR was carried with respective blaNDM-1 and blaOXA-48 primers (Table 1).
 
 


 RESULTS

The 425 meropenem-resistant isolates included Klebsiella spp.  229, Enterobacter spp. - 89, E. coli - 85, Citrobacter spp - 14, Providencia spp - 5, Proteus spp - 3. Figure 1 shows the presence of MBL-encoding genes such as blaNDM-1 and blaOXA-48 in both OmpK36-deficient and non-deficient strains, respectively.
 
 
Conjugation experiments were carried out on all 425 clinical isolates using sodium azide-resistant J53 E. coli strain as a recipient. Transconjugants were selected on MacConkey agar plate containing meropenem and sodium azide. 117 (27%) isolates had shown positive on agar plate with sodium azide and meropenem. Plasmid was extracted from all the selected transconjugants by alkaline lysis method, and blaNDM-1 and blaOXA-48 genes were analyzed by multiplex PCR assays (Figure 2). From this study, it was observed that 110 out of 117 transconjugants carrird blaNDM-1 and none carried blaOXA-48.
 


 DISCUSSION

The emergence of carbapenemase-producing Enterobacteriaceae has been reported in several studies worldwide (Castanheira et al., 2011; Mohamed et al., 2013; Duin et al., 2013; Castanheira et al., 2011; Poirel et al., 2011). The present study showed the emergence of blaNDM-1 and blaOXA-48 co-producers among Enterobacteriaceae isolates. It was also observed that there is loss of porin OmpK36 in Enterobacteriaceae isolates contributing to the carbapenem resistance. Multiplex PCR analysis illustrated the presence of MBL-encoding genes such as blaNDM-1 and blaOXA-48 in both the OmpK36 deficient and non-deficient Enterobacteriaceae isolates. Out of 425 isolates, 136 isolates had shown the presence OmpK36.
 
A total number of 187 isolates harboured blaNDM-1 without OmpK36 gene, 5 isolates harboured blaOXA-48 without OmpK36, 75 isolates harboured blaNDM-1 with OmpK36 and one Klebsiella pneumoniae harboured blaOXA-48 with OmpK36. All the 5 isolates carrying blaOXA-48 without OmpK36 was found to be K. pneumoniae. Conjugation results using sodium azide-resistant recipient E. coli J53 with selection of 110 transconjugants on MacConkey agar carried only blaNDM-1 and no transconjugants were found to carry blaOXA-48. Several studies worldwide have reported the prevalence of transconjugants bearing blaNDM-1 plasmids (Balm et al., 2013).
 
The present work highlights the prevalence of blaNDM-1 and blaOXA-48 in the clinical isolates of Enterobacteriaceae isolated in this centre. It is also remarkable to note that blaNDM-1 is found to be the mostprevalent in this centre accounting to nearly (264) 62% of the meropenem-resistant isolates, but the isolates with blaOXA-48 were only 1.4%. Furthermore, conjugation studies corroborated that 57 strains of Klebsiella spp, 18 strains of Enterobacter spp. and 21 strains of E. coli harboured blaNDM-1 plasmids and they were successfully transferred to the recipient strain E. coli J53 by conjugation. The results in this study indicated that there is increasing frequency of transmissible resistance genes such as blaNDM-1 and blaOXA-48 among the meropenem-resistant Enterobacteriaceae strainsisolated form the clinical samples.


 CONCLUSION

blaNDM-1 and blaOXA-48 appear to be an emerging cause of carbapenem resistance in Enterobacteriaceae in India. The present study shows that blaNDM-1 is the most frequent resistance gene identified among the carbapenem-resistant isolates, and for some isolates, resistance to carbapenems is also related to the presence of blaOXA-48. The relationship between the occurrence of the resistance genes (blaNDM-1 and blaOXA-48) and the presence or absence of OmpK36 gene has been worked out in the study. 


 CONFLICT OF INTERESTS

The author(s) did not declare any conflict of interest.


 ACKNOWLEDGEMENT

This study was financially supported by Indian Council of Medical Research (ICMR).



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