|Year : 2013 | Volume
| Issue : 3 | Page : 250-256
Incidence of blaNDM-1 gene in Escherichia coli isolates at a tertiary care referral hospital in Northeast India
A Bora1, GU Ahmed1, NK Hazarika2, KN Prasad3, SK Shukla3, V Randhawa4, JB Sarma5
1 Department of Biotechnology, Gauhati University, Guwahati, Assam, India
2 Department of Microbiology, Gauhati Medical College and Hospital, Guwahati, Assam, India
3 Department of Microbiology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
4 Biotechnology division, Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh, India
5 Northumbria Healthcare NHS Foundation Trust, Rake Lane, NE 28 8NH, United Kingdom
|Date of Submission||19-Jun-2012|
|Date of Acceptance||04-May-2013|
|Date of Web Publication||25-Jul-2013|
G U Ahmed
Department of Biotechnology, Gauhati University, Guwahati, Assam
Source of Support: None, Conflict of Interest: None
Purpose: Increasing reports on New Delhi metallo-β-lactamase-1 (NDM-1) producing Escherichia coli constitute a serious threat to global health since it is found to be highly resistant to most of the currently available antibiotics including carbapenems. This study has been performed to find out the incidence blaNDM-1 in E. coli isolates recovered from the various clinical samples at a tertiary care referral hospital in Northeast India. Materials and Methods: A total of 270 non-duplicated E. coli isolates were recovered from the various clinical samples at a tertiary care referral hospital in Northeast India. All isolates with reduced susceptibility to meropenem or ertapenem (diameter of zones of inhibition, ≤21 mm) were further phenotypically confirmed for carbapenemase production by modified Hodge test. All screened isolates were also subjected to the polymerase chain reaction detection of blaNDM-1 gene and additional bla genes coding for transmission electron microscopy, SHV, CTX-M, and AmpC. Results: Out of 270 E. coli isolates, 14 were screened for carbapenemase production on the basis of their reduced susceptibility to meropenem or ertapenem. All screened isolates were found to be positive for blaNDM-1 . Each of the blaNDM-1 possessing isolate was also positive for two or more additional bla genes, such as blaTEM , blaCTX-M and blaAmpC . Phylogenetic analysis showed very less variation in blaNDM-1 gene with respect to blaNDM-1 possessing E. coli isolates from other parts of India and abroad. Conclusions: Our findings highlight the incidence of blaNDM-1 in E. coli isolates with a reduced susceptibility to meropenem or ertapenem.
Keywords: Antibiotic resistance, carbapenemases, Escherichia coli, extended-spectrum β-lactamases, New Delhi metallo-beta-lactamase-1
|How to cite this article:|
Bora A, Ahmed G U, Hazarika N K, Prasad K N, Shukla S K, Randhawa V, Sarma J B. Incidence of blaNDM-1 gene in Escherichia coli isolates at a tertiary care referral hospital in Northeast India. Indian J Med Microbiol 2013;31:250-6
|How to cite this URL:|
Bora A, Ahmed G U, Hazarika N K, Prasad K N, Shukla S K, Randhawa V, Sarma J B. Incidence of blaNDM-1 gene in Escherichia coli isolates at a tertiary care referral hospital in Northeast India. Indian J Med Microbiol [serial online] 2013 [cited 2020 May 25];31:250-6. Available from: http://www.ijmm.org/text.asp?2013/31/3/250/115628
| ~ Introduction|| |
The successful use of 3 rd generation cephalosporins in medicine was considered as a landmark in antimicrobial chemotherapy. Unfortunately, the increased use of these cephalosporins has led to the emergence of resistance in enterobacterial species, possessing extended-spectrum β-lactamases (ESBLs).  The emergence of ESBLs and cephamycins resistance, particularly in Escherichia coli and Klebsiella pneumoniae, enforced clinician to often use the carbapenems as drugs of last resort to treat serious infections caused by these bacteria.  As a consequence of selective pressure exerted by the carbapenems, carbapenem-hydrolyzing β-lactamases (carbapenemases) has emerged. Carbapenemases found in Enterobacteriaceae can be metallo-β-lactamases, expanded-spectrum oxacillinases or clavulanic-acid inhibited β lactamases. 
New Delhi metallo-β-lactamase-1 (NDM-1) is a novel type of metallo-β-lactamase, which is named after the city of origin following a common practice of naming for transferable metallo-β-lactamases (e.g. Verona integron- encoded metallo-β-lactamase 1 named after Verona, Italy).  NDM-1 was first reported in 2009 in K. pneumoniae and E. coli, both recovered from a Swedish patient of Indian origin, who was previously admitted to a hospital in New Delhi, India.  NDM-1 attains significant global attention as the gene encoding NDM-1, known as blaNDM-1 is located on transmissible plasmid, which may also include a number of other antibiotic resistance genes resulting into extensive drug resistant phenotypes (the so-called 'superbugs').  In general, the term "superbugs" is a colloquial reference to a bacterium that carries resistant genes to many antibiotics. 
In August 2010, Kumarasamy et al.,  reported the emergence of NDM-1 producing Enterobacteriaceae (mostly, E. coli and K. pneumoniae) in India, Pakistan and the United Kingdom. Since, then several cases of NDM-1 producing E. coli are being reported from several other parts of the world.  Most of these cases were reported from patients who had a history of visiting India, Pakistan or Bangladesh with or without receiving medical care. In India, NDM-1 producing E. coli has been reported from different parts of the country. ,,,, However, the exact prevalence of NDM-1 is still not known due to lack of adequate epidemiological data. Hence, the present study was undertaken to evaluate the incidence of blaNDM-1 gene in E. coli isolates at a tertiary care referral hospital in Northeast India.
| ~ Materials and Methods|| |
A total of 270 non-duplicated E. coli isolates were recovered from the various clinical samples such as urine (n = 214), pus (n = 15), sputum (n = 24) and blood (n = 17) at a tertiary care referral hospital in Northeast India. The total number of E. coli urinary isolates was much higher than that of other sample sources as E. coli is the predominant cause of both community and nosocomial urinary tract infections. Samples were obtained from both outpatients and inpatients between August 2009 and July 2010. Standard microbiological techniques were used for isolation and identification of the isolates.  Prior to testing, all isolates were stored in 15% glycerol-supplemented Luria-Bertani medium at − 80C. The study was carried out with the consent from the Institutional Ethics Committee.
Antimicrobial susceptibility testing
The susceptibilities to antimicrobial agents were determined by Kirby-Bauer disc diffusion method with β-lactam and non-β-lactam antibiotic discs and results were interpreted as per Clinical and Laboratory Standards Institute (CLSI) guidelines.  All antibiotic discs and media were procured from Hi-Media, Mumbai, India, except doripenem, which was procured from BD Diagnostics, Franklin Lakes, NJ, USA. E. coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 strains were used for quality control. Minimum inhibitory concentration (MIC) values for imipenem, meropenem, ertapenem, doripenem and tigecycline add colistin were determined by using Etest strips (bioMerieux, France) following the manufacturer's protocol.
Screening and phenotypic detection of carbapenemase production
By disc diffusion, all isolates with reduced susceptibility to meropenem or ertapenem (diameter of zones of inhibition, ≤21 mm) were screened for production of carbapenemase according to CLSI guidelines.  Phenotypic detection for carbapenemase production was performed by modified Hodge test (MHT), using ertapenem disc (10 μg) as described by CLSI. 
Molecular detection of β-lactamase genes
Plasmid DNA was extracted from all screening positive isolates by using Sure Spin Plasmid Mini Kit (Genetix Biotech Asia Pvt. Ltd., India) following the manufacturer's protocol. The extracted plasmid DNA of each isolate was subjected to the polymerase chain reaction (PCR) detection of blaNDM-1 gene with target specific primer set, NDM-Fm (5′-GGTTTGGCGATCTGGTTTTC-3′,) and NDM-Rm (5′-CGGAATGGCTCATCACGATC-3′).  These isolates were also examined for the presence of additional plasmid-mediated β-lactamases genes such as blaTEM , bla SHV , blaCTX-M and blaAmpC by PCR amplification described elsewhere. ,,, The PCR products were analysed by electrophoresis with 1.5% agarose gels in 1x tris-acetate-EDTA buffer at 100 V for 30 min. After staining with ethidium bromide (0.5 μg/ml), the gels were visualised under a gel documentation system (Bio-Rad, USA).
Purified PCR products of blaNDM-1 gene for three randomly selected representative isolates (EC-27, EC-35 and EC-160) were further sequenced with the same set of primers as used for amplification by 3730XL DNA analyser (Applied Biosystems, CA, USA) based on Sanger's sequencing method.
E. coli isolates positive for blaNDM-1 by PCR and confirmed by gene sequencing were used as a positive control in this study.
The blaNDM-1 nucleotide sequences of three E. coli isolates were compared with those of other blaNDM-1 nucleotide sequences of E. coli obtained from the National Centre for Biotechnology Information GenBank database. To determine the nearest phylogenetic neighbours, each partial sequence of the nucleotide sequence was subjected to the nucleotide sequence homology searches using basic local alignment search tool homology search tool.  BLASTN was used for the searches with default parameters. The hits were selected keeping the 'E value' (minimum) and 'query coverage' (maximum) into consideration. Alignment and comparative nucleotide sequence analysis were carried out by using embedded version of ClustalW multiple sequence alignment in MEGA 5 software at default conditions of 15 gap opening penalty and 6.66 gap extension penalty iteration. ,
For phylogenetic analysis, the neighbour-joining method was used to construct phylogenetic trees for the nucleotide sequences, using the program MEGA 5.  The robustness of the phylogenetic hypothesis was tested by Felsenstein's bootstrap test,  a means of assessing the reliability in a particular phylogeny, which is evaluated by using Efrons bootstrap resampling technique.  In the present study, all bootstrap analyses of nucleotides involved 1000 replications of the data.
Nucleotide sequence accession numbers
The nucleotide sequence of the blaNDM-1 genes from three E. coli (EC-27, EC-32 and EC-160) isolates have been registered in GenBank under accession numbers JN575035, JN575036 and JN575037, respectively. The accession numbers (in parentheses) of the other blaNDM-1 nucleotide sequences used in the phylogenetic tree development are as follows: E. coli strain A2861 (JN255860), E. coli strain NDM-1 Dok01 (AB604953), E. coli strain MH01 (HQ917683), E. coli strain B10533 (HQ259057), E. coli strain and EC-19 (AB571289).
| ~ Results|| |
During the study period, out of 270 E. coli isolates, a total of 14 isolates were screened for carbapenemase production on the basis of their reduced susceptibility to meropenem or ertapenem. Out of these 14 E. coli isolates, 10 were from urine, one was from pus and three were from blood. None of the sputum isolates was found to be positive in screening for carbapenemase production. Screen positive isolates were also positive for MHT, although two isolates gave weakly positive reactions. The age of patients with NDM-1 positive isolates ranged from 3 days to 72 years and the male to female ratio was 1.33:1.
All the screening positive 14 isolates were found to be positive for plasmid mediated blaNDM-1 gene by PCR. Furthermore, these blaNDM-1 possessing isolates were found to be also positive for two or more additional plasmid mediated bla genes [Table 1]. These isolates were PCR negative for blaSHV gene. Co-existence of both blaTEM and blaCTX-M with blaNDM-1 was established in 57.14% (8/14) isolates as well as both blaCTX-M and blaAmpC in 21.43% (3/14) isolates. Co-existence of all three additional bla genes (blaTEM, blaCTX-M and blaAmpC ) with blaNDM-1 was also established in 21.43% (3/14) isolates.
|Table 1: Different characteristics of NDM-1 positive Escherichia coli isolatesa|
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By the disc diffusion susceptibility testing, 85.71% (12/14) of the NDM-1 producers were resistant to amikacin and 92.85% (13/14) were resistant to ciprofloxacin. All 14 isolates were resistant to gentamicin, co-trimoxazole, nalidixic acid, ampicillin, cefpodoxime, cefotaxime, ceftazidime, ceftriazone, cefepime, cefoxitin and piperacillin/tazobactam. None the isolates were found to be susceptible to imipenem, meropenem, ertapenem or doripenem; however, all the isolates were susceptible to tigecycline and colistin by disc diffusion method. The MIC values for different carbapenems varied widely among the NDM-1 positive isolates [Table 2]. The range of MIC values for imipenem, meropenem, ertapenem and doripenem were 2-8 μg/ml, 3-16 μg/ml, 8->32 μg/ml and 2-6 μg/ml respectively. Among the NDM-1 positive isolates, highest values observed for imipenem, meropenem, ertapenem, and doripenem were 8, 16, >32, and 6 μg/ml, respectively. MIC value less than one was found for both tigecycline (range, 0.125-0.75 μg/ml) and colistin (range: 0.125-0.5 μg/ml).
Homologous comparison of the three E. coli sequences showed 99% identity to those present in the GenBank database and none were exactly identical to the sequences obtained from the database. The phylogenetic relationships among three E. coli from Northeast India and other sequences obtained from GenBank were investigated. [Figure 1] shows the phylogenetic tree of 5 closely related nucleotide sequences of blaNDM-1 genes of E. coli isolates. As indicated in [Figure 1], the phylogenetic tree based on the E. coli gene sequence homologies was divided into two branches. The majority of E. coli isolates formed an independent lineage distinct from other E. coli isolates. The strain A2861, strain B10533 and strain EC-19 from India together with the strain MH01from Canada and strain NDM-1 Dok01 from Japan form one clade. The EC-160 isolate falls in a second clade of the first branch. There were 99.56-99.61% identities between isolate EC-160 and the rest of the strains in the first branch. The progressive multiple sequence alignment identity among the sequences in this lineage was between 94% and 100%. EC-27 and EC-32 isolates belonged to another branch and formed third clade of the phylogenetic tree.
|Figure 1: Phylogenetic tree (topology only) based on the nucleotide sequences showing the relationship among the blaNDM-1 genes belonging to different Escherichia coli isolates. Accession numbers are presented in parentheses. The tree was constructed using p-distance and the method was neighbour-joining. Bootstra P values (1000 replicates) are shown at the nodes. Scale bar represents the number of inferred nucleotide substitution per site|
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| ~ Discussion|| |
The increasing reports on NDM-1 producing Enterobacteriaceae have addressed a potential threat to global health. The present study gives an initial insight on the incidence of blaNDM-1 gene in the clinical isolates of E. coli in the Northeast India. Among the 270 E. coli isolates collected from various clinical samples during a period of 1 year, we detected blaNDM-1 in 14 isolates (5.18%). A pervious single-day point-prevalence study from this region also showed the incidence of blaNDM-1 in E. coli isolates.  Deshpande et al., reported the incidence of NDM-1 in 9 E. coli isolates among 24 carbapenem resistant Enterobacteriaceae in a tertiary care centre in Mumbai.  A very recent study carried out in the intensive care unit (ICU) and wards of Sir Ganga Ram Hospital, Delhi showed 8.1% overall prevalence of NDM-1 in E. coli isolates.  However, true prevalence of NDM-1 in the community cannot be deduced as the samples were collected from only one tertiary centre.
Out of 14 blaNDM-1 positive E. coli isolates, 10 isolates were recovered from inpatients (prevalence, 71.42%) and 4 isolates were from non-hospitalized individuals (prevalence, 28.58%). Although, NDM-1 positive isolates were most frequently isolated from ICU in Indian hospitals, , we observed higher incidence of NDM-1producing E. coli in non ICU patients (64.28%, 9/14) than that in ICU patients (35.72%, 5/14). Among the NDM-1producing ICU E. coli isolates, one isolate was recovered from neonatal intensive care unit. A point prevalence survey carried out in an Indian rural hospital described the presence of blaNDM-1 possessing E. coli in a sick new-born care unit. 
E. coli isolates possessing blaNDM-1 gene were found to be resistant to several classes of β-lactam and non β-lactam antibiotics by disc diffusion susceptibility testing. All the E. coli isolates producing NDM-1 were non-susceptible to imipenem, meropenem, ertapenem and doripenem. Resistance to imipenem, meropenem, ertapenem and doripenem was observed in 72.43% (10/14), 92.86% (13/14), 100% (14/14) and 78.57% (11/14), respectively. Out of 14 NDM-1 producing isolates, only two isolates were sensitive to amikacin and one isolate was sensitive to ciprofloxacin. All the 14 isolates showed a high level of resistance to penicillin, third and fourth generation cephalosporins, cephamycin, and aztreonam as well as to β-lactam-β-lactamase inhibitor combination tested in the study. This finding is similar with other Indian reports ],[ except one in which several NDM-1 producing isolates of Enterobacteriaceae were susceptible to various carbapenems as well as to piperacillin/tazobactam by disc diffusion testing. 
The variations in the MIC value for different carbapenems depends on the type and expression of carbapenemase enzyme, the bacterial species and the presence of other resistance mechanisms such as ESBLs and AmpC β-lactamases, reduced permeability and/or efflux pumps.  Surprisingly, low MIC value for both imipenem and doripenem was observed in the majority of NDM-1 positive isolates [Table 2]. The lowest MIC value for both imipenem and doripenem was 2 μg/ml. MIC value for meropenem were found to be higher than imipenem and doripenem for all NDM-producing isolates, except one isolate that showed same MIC value (4 μg/ml) for both imipenem and meropenem. Among the NDM-1 producing isolates, higher MIC value was observed constantly for ertapenem in comparison to other carbapenems tested in the study. A previous study reported that higher resistance for ertapenem than for imipenem or meropenem may result from the combinations of a beta-lactamase (CTX-M or AmpC enzyme) in addition to decreased permeability or increased efflux.  Therefore, the comparative high resistance towards ertapenem observed in this study might be due the presence of blaCTX-M and/or blaAmpC in association with blaNDM-1 in the NDM-1 positive isolates. Tigecycline and colistin were found to be most active agents in all the NDM-1 producing E. coli isolates but both agents have limitations in clinical implementation. Usually, NDM-1 producing bacteria remain susceptible to tigecycline and colistin,  but the incidence of tigecycline and colistin resistance in carbapenemase producing Enterobacteriaceae has a great impact on available therapeutic options. 
CLSI recommends MHT to detect carbapenemase production in Enterobacteriaceae isolates; though, the sensitivity and specificity of the test for detecting low-level metallo-β-lactamase production are not known.  In our study, out of the 14 E. coli possessing blaNDM-1 , two isolates (14.3%) gave weakly positive results in MHT. All remaining isolates (84.7%) were clearly positive in MHT. These results signify that MHT is highly sensitive in the detection of carbapenemase production in NDM-1 producing E. coli isolates. Similar results for MHT were also observed among NDM-1 producing Enterobacteriaceae isolates in a recent study from Pakistan.  In contrast, a previous study reported weakly positive MHT results in 11 of 15 NDM-1-producing Enterobacteriaceae isolates, including E. coli.  Very recently, false positive results for MHT in carbapenemase negative E. coli and K. pneumoniae isolates have also been documented. 
Since all the NDM-1 possessing isolates exhibited high-level of resistance to a different generation cephalosporins and aztreonam, PCR detection for some of the important types of ESBL genes as well as AmpC gene was performed. As expected, each of the blaNDM-1 positive isolate harbored two or more additional bla genes. Of these, blaCTX-M was the most common and found in all isolates, whereas blaTEM was found in 78.57% (11/14) isolates. Only 21.43% (3/14) of NDM-1 producing isolates was positive for plasmid-mediated blaAmpC . However, sequencing of these additional bla genes could have given additional information. Earlier studies from India  and abroad,  also reported the co-existence of different types of ESBL genes (mostly, blaTEM-1 and blaCTX-M-15 ) along with AmpC genes (mostly, blaCMY ) in blaNDM-1 positive E. coli isolates. The presence ESBL and AmpC genes in the blaNDM-1 positive isolates might contribute to the high level of resistance to aztreonam observed in this study.
The phylogenetic analysis revealed that the E. coli isolate EC-160 from Northeast India, has very less variation in blaNDM-1 gene with respect to the blaNDM-1 possessing E. coli isolates from other parts of India as well as Canada and Japan. Whereas, E. coli isolates EC-27 and EC-32 from Northeast India were found to be in the same clade; thus, showing less genetic variation between these two isolates. However, isolates EC-27 and EC-32 appear to be genetically different from EC-160 and other E. coli isolates as shown in the phylogenetic tree [Figure 1]. Although, all the three E. coli isolates (EC-27, EC-32 and EC-160) were recovered from urine samples, the MIC value for carbapenems in EC-160 isolate was higher than in EC-27 and EC-32. Isolate EC-27 and EC-32 showed similar MIC value for all four carbapenems and these MIC value found to be lower than other NDM-1 producing E. coli from India and abroad. ,
The rapid emergence of antibiotic resistance is a major concern for both developed and developing countries. Dissemination of NDM-1 producing organisms may be facilitated by the conditions like overcrowding, over-the-counter availability of antibiotics, low level of hygiene, and weak hospital antibiotic policies.  Transmission of NDM-1 producing bacteria mainly takes place by faecal-oral route and inadequate sewage system further intensifies the problem.  Walsh et al., recently reported the presence of NDM-1 producing bacteria in various genera of Enterobacteriaceae and in some other non-fermenting Gram negative bacteria in drinking water and seepage samples from Delhi.  India is the second-largest populated country in the world (1.3 billion people). Regrettably, it was estimated that approximately 700 million Indian people lack basic sanitation and at least 100 million Indian residents carry NDM-1-positive bacteria as normal gut flora.  The identification of ultraviolet light resistance gene in NDM-1 producing E. coli plasmid might enhance the survivability of the bacterium in the environment.  These reports indicate an alarming risk of rapid dissemination of NDM-1 in Indian environment. Our findings also indicate the high probability of NDM-1 producing E. coli in Northeast India.
| ~ Conclusion|| |
Even though carbapenemase production is rare in E. coli, NDM-1 producing E. coli is now being increasingly reported not only from India, but also from different parts of the world. E. coli is ubiquitous in the environment and lives commensally in the guts of most mammals, including human. The natural tendency of this bacterium for frequent genetic exchange leads to the emergence of new pathogenic strains with a larger concentration of antibiotic resistance genes. Resistance strains of E. coli can be easily acquired by means of contaminated food and water. Besides, presence of NDM-1 gene with other resistance determinants on transmissible plasmid constitutes a significant threat to global health-care for being readily transferable between different clinically relevant bacteria. Therefore, early recognition of NDM-1 producing Enterobacteriaceae, especially E. coli with any reduced carbapenem susceptibility should have primary importance as high mortality rates are associated with patients infected with these bacteria.
| ~ Acknowledgment|| |
The first author (Arijit Bora) acknowledges University Grants Commission (UGC), India for providing UGC-RFSMS fellowship to carry out his research and also Department of Science and Technology (DST), Govt. of India for providing the visiting fellowship for N.E. Research student to carry out the molecular analysis part of this study in Department of Microbiology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, India.
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[Table 1], [Table 2]
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| ||Journal of Antimicrobial Chemotherapy. 2014; |
|[Pubmed] | [DOI]|