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ORIGINAL ARTICLE
Year : 2019  |  Volume : 149  |  Issue : 2  |  Page : 285-289

Molecular epidemiology & therapeutic options of carbapenem-resistant Gram-negative bacteria


1 Department of Microbiology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, India
2 Department of Microbiology, Ram Manohar Lohia Institute of Medical Sciences, Lucknow, India
3 Department of Pharmacy, Ganesh Shankar Vidyarthi Memorial Medical College, Kanpur, India
4 Department of Microbiology, Assam University, Silchar, India
5 Department of Medicine, Ganesh Shankar Vidyarthi Memorial Medical College, Kanpur, India
6 Department of Microbiology, Ganesh Shankar Vidyarthi Memorial Medical College, Kanpur, India

Date of Submission04-Jan-2018
Date of Web Publication3-Jun-2019

Correspondence Address:
Dr Atul Garg
Department of Microbiology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow 226 014, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmr.IJMR_36_18

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   Abstract 

Background & objectives: The growing incidence and the wide diversity of carbapenemase-producing bacterial strains is a major concern as only a few antimicrobial agents are active on carbapenem-resistant bacteria. This study was designed to study molecular epidemiology of carbapenem-resistant Gram-negative bacterial (GNB) isolates from the community and hospital settings.
Methods: In this study, non-duplicate GNB were isolated from clinical specimens, and phenotypic test such as modified Hodge test, metallo β-lactamase E-strip test, etc. were performed on carbapenem-resistant bacteria. Multiplex PCR was performed to identify the presence of blaIMP, blaVIM, blaKPC, blaOXA48, blaOXA23, blaSPM, blaGIM, blaSIM and blaNDM. Minimum inhibitory concentration (MIC) of colistin, fosfomycin, minocycline, chloramphenicol and tigecycline was also determined.
Results: Of the 3414 GNB studied, carbapenem resistance was 9.20 per cent and maximum resistance (11.2%) was present at tertiary care centre, followed by secondary care (4%) and primary centre (2.1%). Among the carbapenem-resistant bacteria, overall, the most common isolate was Pseudomonas aeruginosa (24%). On multiplex PCR 90.3 per cent carbapenem-resistant isolates were positive for carbapenemase gene. The blaNDM(63%) was the most prevalent gene followed by blaVIM(18.4%). MIC results showed that 88 per cent carbapenem-resistant Enterobacteriaceae were sensitive to fosfomycin, whereas 78 per cent of P. aeruginosa and 85 per cent Acinetobacter spp. were sensitive to colistin.
Interpretation & conclusions: Carbapenem resistance in GNB isolates from the community and hospital settings was found to be on the rise and should be closely monitored. In the absence of new antibiotics in pipeline and limited therapeutic options, prudent use of antibiotics and strict infection control practices should be followed in hospital to limit the emergence and spread of multidrug-resistant bacteria.

Keywords: Antimicrobial resistance - Carba NP test - carbapenemase - colistin - multiplex polymerase chain reaction - NDM


How to cite this article:
Garg A, Garg J, Kumar S, Bhattacharya A, Agarwal S, Upadhyay G C. Molecular epidemiology & therapeutic options of carbapenem-resistant Gram-negative bacteria. Indian J Med Res 2019;149:285-9

How to cite this URL:
Garg A, Garg J, Kumar S, Bhattacharya A, Agarwal S, Upadhyay G C. Molecular epidemiology & therapeutic options of carbapenem-resistant Gram-negative bacteria. Indian J Med Res [serial online] 2019 [cited 2019 Nov 15];149:285-9. Available from: http://www.ijmr.org.in/text.asp?2019/149/2/285/259596

Carbapenemase-producing bacteria have become a major concern. Earlier only nosocomial pathogens such as Pseudomonas aeruginosa and Acinetobacter baumannii had significant carbapenem resistance, however, the emergence of carbapenemases in Enterobacteriaceae is a growing public health problem worldwide because of their high prevalence, wide range of clinical infections, multidrug resistance and rapid dissemination of plasmid-mediated resistance genes from Enterobacteriaceae to other organisms. These enzymes confer resistance to the other β-lactam agents as well and are generally co-associated with resistance genes for aminoglycosides, quinolones and have brought us a step closer to the challenge of extremely drug-resistant bacteria [1],[2]. This study was designed to study molecular epidemiology of carbapenem-resistant bacterial isolates from community and hospital settings from north India and further explore therapeutic options for management of infections caused by carbapenem-resistant Gram-negative bacteria (GNB).


   Material & Methods Top


The present study was conducted from August 2014 to July 2016 at the department of Microbiology, Ganesh Shankar Vidyarthi Memorial Medical College (GSVM), Kanpur, India. The clinical specimens were collected from primary Health Centre Kalyanpur, district hospital Kanpur and LLRM Hospital, a tertiary care centre attached with GSVM Medical College, Kanpur. The study was cleared by the Institutional Ethics Committee.

Non-duplicate GNB isolated from various specimens were identified using conventional techniques [3]. Antimicrobial susceptibility was performed by Kirby Bauer disk diffusion method [3] and minimum inhibitory concentration (MIC) breakpoints of carbapenems for the isolates which were resistant by disc diffusion testing was determined by E-test (BioMérieux, France). Further to look for treatment options for these carbapenem-resistant isolates MIC of other antibiotics such as fosfomycin, minocycline, chloramphenicol and tigecycline was also determined using E Strip and colistin MIC was determined using broth microdilution method, results were interpreted as per Clinical and Laboratory Standards Institute (CLSI) guidelines [4].

The isolation of genomic DNA of carbapenem-resistant bacteria was done by QuiAmp mini DNA extraction kit (Qualigens, Germany) and multiplex PCR was performed to identify the presence of following genes blaIMP, blaVIM, blaKPC, blaOXA48, blaOXA23, blaSPM, blaGIM, blaSIM and blaNDM using the primers and protocol described earlier [5]. New Delhi metallo-β-lactamase (NDM) positive amplicons were sequenced and previously published sequences of NDM isolates retrieved from the National Center for Biotechnology (http://www.ncbi.nlm.nih.gov) were used as the reference sequence for result interpretation. Phenotypic tests such as modified Hodge test (MHT)[1], metallo-β-lactamase (MBL), E-strip test [1], Neo-Sensitabs Test (Rosco Diagnostica, Denmark) and Rapidec Carba NP Test (BioMérieux, France)[6] were performed on carbapenem-resistant PCR-positive bacterial isolates.


   Results & Discussion Top


A total of 8973 samples were processed and 3414 GNB were isolated; of which 312 (9.20%) isolates were carbapenem-resistant. Maximum resistance (11.2%) was present at tertiary care centre, followed by secondary care (4.0%) and primary centre (2.1%). Amongst the carbapenem-resistant bacteria; overall, the most common isolate was Pseudomones aeruginosa (24%) followed by Acinetobacter spp. (22%) and  Escherichia More Details coli (16%) [Table 1]. In a community-based study from south India Sekar et al[7] also documented three per cent carbapenem resistance in members of Enterobacteriaceae, however in the treatment guidelines document released by the Indian Council of Medical Research [8], surveillance data were collected and compiled from four tertiary care centres in India, and a high meropenem resistance of 42, 47 and 62 per cent was reported among members of Enterobacteriaceae, P. aeruginosa and A. baumannii, respectively.
Table 1: Aetiology of carbapenem resistant bacteria isolated from different healthcare level

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On multiplex PCR 282 of 312 (90.3%) isolates were positive f or carbapenemase gene. The blaNDM178 (63%) was the most prevalent gene followed by blaVIM(18.4%). The blaKPC, blaGIM and blaSIM were not isolated in this study; blaNDM and blaOXA48 were co-observed in 20 per cent isolates [Table 2]. Sequencing was performed on 178 blaNDM positive isolates and 133 (75%) isolates were carrying blaNDM-1 and the rest were harbouring blaNDM-5 genes. The findings were in concurrence to previously published reports from India [9],[10]. Some NDM-positive isolates were earlier screened for the coexistence of ESBL genes, 16s methyltransferase genes determining aminoglycosides resistance and quinolones resistance determinants and it was found that NDM positive isolates were co-harbouring several other resistance determinants [11]. In contrast to western literature [1], blaKPC was not isolated in this study.
Table 2: Molecular epidemiology of carbapenem resistant Gram-negative bacteria

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Phenotypic carbapenemase detection test was performed on 261 PCR confirmed isolates.

Rapidec Carba NP test, Neo-Sensitabs and MHT and showed a sensitivity of 90, 73 and 20 per cent, respectively. MIC of the isolates resistant to carbapenem was determined for other antibiotics such as chloramphenicol, colistin, fosfomycin, minocycline and tigecycline [Table 3].
Table 3: Antimicrobial susceptibility pattern of carbapenem resistant Gram-negative bacteria (GNB)

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Carbapenemases are generally encoded by a genetic element found on different plasmids that may jump from bacteria to bacteria easily causing the rapid emergence of multidrug-resistant bacteria [1]. Thus for carbapenem-resistant isolates MIC was also determined for chloramphenicol, colistin, fosfomycin, minocycline and tigecycline. Fosfomycin which was previously used mainly as oral treatment for uncomplicated urinary tract infections, currently attracts clinicians' interest worldwide. Particularly, the reported activity against pathogens with advanced resistance suggests that this antibiotic may provide a useful option for the treatment of patients with these difficult to treat infections [12]. In our study 88 per cent CRE were sensitive to fosfomycin.

Colistin and polymyxin B have recently regained significant interest as a consequence of the increasing incidence of infections due to carbapenem-resistant bacteria and are reconsidered as last-resort antibiotics [13]. Results of this study demonstrated that 78 per cent of P. aeruginosa and 85 per cent Acinetobacter spp. were sensitive to colistin. Indian data on colistin resistance from ICMR document [8] reported colistin resistance of 10 per cent in P. aeruginosa and 22 per cent in A. baumannii complex. Another study from north India reported colistin resistance in carbapenem resistance A. baumannii as 16 per cent [14]. The use of polymyxins has been challenged by the emergence of the plasmid-borne mobile colistin resistance gene (mcr-1)[15]. Since MCR-1 is capable of horizontal transfer between different strains of a bacterial species and after its discovery in November 2015 in E. coli (strain SHP45) from a pig in China, it has been found in E. coli,  Salmonella More Details enterica, Klebsiella pneumonia, Enterobacter aerogenes and Enterobacter cloacae[15].

Results of our study show that 70 per cent of Acinetobacter spp. and 50 per cent carbapenem-resistant enterobacteriaceae (CRE) were sensitive to minocycline. The study results were in concurrence to other Indian and western literature [16],[17]. Tigecycline is a structural analogue of minocycline that was designed to avoid tetracycline resistance mediated by ribosomal protection and drug efflux [18]. It is indicated for the treatment of complicated skin infections, intra-abdominal infections and community-acquired bacterial pneumonia [19]. This study results showed 36 per cent tigecycline resistance in CRE in concurrence with other Indian studies [20],[21].

In conclusion, carbapenem resistance in the GNB from the community and hospital settings is on rise and should be closely monitored. In the absence of new antibiotics in pipeline and limited therapeutic options, it is important to prudently use antibiotics and strict infection control practices should be followed in the hospital to limit the emergence and spread of multidrug-resistant bacteria.

Financial support & sponsorship: None.

Conflicts of Interest: None.



 
   References Top

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da Silva LM, Nunes Salgado HR. Tigecycline: a review of properties, applications, and analytical methods. Ther Drug Monit 2010; 32 : 282-8.  Back to cited text no. 18
    
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    Tables

  [Table 1], [Table 2], [Table 3]


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