|Year : 2016 | Volume
| Issue : 2 | Page : 173-182
New Delhi metallo-β-lactamase and extended spectrum β-lactamases co-producing isolates are high in community-acquired urinary infections in Assam as detected by a novel multiplex polymerase chain reaction assay
VV Borah1, KK Saikia1, P Chandra2, NK Hazarika3, R Chakravarty4
1 Department of Bioengineering and Technology, Gauhati University, Guwahati, Assam, India
2 Medical Research Center, Hamad Medical Corporation, Doha, Qatar
3 Department of Microbiology, Gauhati Medical College and Hospital, Guwahati, Assam, India
4 Department of Microbiology, Gloria Diagnostics - A Unit of East End Nursing Home and Research Center, Guwahati, Assam, India
|Date of Submission||11-Feb-2015|
|Date of Acceptance||13-Dec-2016|
|Date of Web Publication||14-Apr-2016|
K K Saikia
Department of Bioengineering and Technology, Gauhati University, Guwahati, Assam
Source of Support: None, Conflict of Interest: None
Background: The ability of microorganisms to evade antibiotic pressure is challenging in healthcare as patients have little or no drug treatment options. Detection of the prevalence of antibacterial resistance pattern helps towards improved antibiotic policy and empirical treatment. Objectives: We carried out antibiogram profiling and documented the prevalence and co-prevalence of New Delhi metallo-β-lactamase (NDM) and extended spectrum β-lactamases (ESBL) encoding genes in urinary Escherichia coli and Klebsiella pneumonia isolates. Materials and Methods: Antibiotic susceptibilities were tested for 241 isolates of E. coli and K. pneumoniae from urine samples collected from out- and hospitalised patients. Polymerase chain reaction (PCR) was carried out on isolates tested positive for phenotypic production of metallo-β-lactamase and ESBL. A multiplex PCR assay was designed to detect the genes. Results: Multiplex PCR assay designed had a limit of detection of 10 3 CFU/mL in vitro. NDM detected was significantly higher among K. pneumoniae compared to E. coli (69.2% vs. 18.2%; P = 0.001). Of 17, 14 NDM positive isolates also harboured ESBL genes. The co-production of CTX-M + TEM + NDM (3/9; 33.3% and 5/8; 62.5%) was most common in K. pneumoniae and E. coli, respectively while CTX-M + TEM + SHV + NDM was found in one isolate. Of the 156 phenotypically ESBL producing isolates, CTX-M, TEM and SHV was detected by PCR in 85, 53 and 24 isolates, respectively. Conclusion: NDM and ESBL co-producing isolates were both community (64.7%) and hospital (35.29%) acquired among E. coli. Antibiotic resistance can be effectively evaluated by a cost and time effective molecular method, such as the multiplex PCR used in this study, which complement culture and sensitivity tests.
Keywords: Community-acquired infections, multiplex-polymerase chain reaction, New Delhi metallo-β-lactamase, SHV, TEM
|How to cite this article:|
Borah V V, Saikia K K, Chandra P, Hazarika N K, Chakravarty R. New Delhi metallo-β-lactamase and extended spectrum β-lactamases co-producing isolates are high in community-acquired urinary infections in Assam as detected by a novel multiplex polymerase chain reaction assay. Indian J Med Microbiol 2016;34:173-82
|How to cite this URL:|
Borah V V, Saikia K K, Chandra P, Hazarika N K, Chakravarty R. New Delhi metallo-β-lactamase and extended spectrum β-lactamases co-producing isolates are high in community-acquired urinary infections in Assam as detected by a novel multiplex polymerase chain reaction assay. Indian J Med Microbiol [serial online] 2016 [cited 2019 Jun 20];34:173-82. Available from: http://www.ijmm.org/text.asp?2016/34/2/173/176853
| ~ Introduction|| |
Antibiotic resistant genes confer an evolutionary advantage to bacteria for survival during an antibiotic pressure. However, this trait has currently become an immense concern in health care as it is often associated with the emergence of multidrug-resistant isolates for which very few antibiotic options remain available. , A detection strategy that complements the regular laboratory investigation of culture and sensitivity (minimum inhibitory concentration [MIC] cut-off) will provide an early and rapid detection of the resistance determinants. , This can possibly help in not only in the generation of data of commonly prevalent antibiotic resistant genes in a particular region but also in deciding the treatment options. Of the many prevailing resistance determinants worldwide, bacteria harbouring extended spectrum β-lactamases (ESBL) and the New Delhi metallo-β-lactamase (NDM) genes are most commonly disseminated in Assam identified in both Escherichia More Details coli and Klebsiella pneumoniae. , CTX-M, TEM and SHV are common worldwide while India is considered to be the main reservoir of NDM; , including neighbouring countries like Bangladesh.  The ESBLs derived from TEM-1, TEM-2 or SHV-1 confer a wide range of enzymatic activity against third-generation cephalosporins or aztreonam.  Variants of NDM-1 have evolved new sub-types of this Group-3a enzyme and Lahey.org reports 12 variants of this enzyme since its first detection in 2008.  This group of enzymes not only confer resistance to the entire class of β-lactams (except aztreonam) but make treatment options very limited, if any.  Furthermore, the co-prevalence of other ESBL genes (most commonly CTX-M with variable MIC cut-offs against aztreonam) greatly increases the resistance parameter in bacteria. 
This study was aimed at documenting the prevalence pattern of antibacterial resistance to commonly prescribed antibiotics as per Clinical Laboratory Standard Institute (CLSI) guideline, 2011 and phenotypic and genotypic detection of ESBL and carbapanemase resistance in circulating E. coli and K. pneumaniae isolates in Assam, India. We are also reporting here a novel multiplex polymerase chain reaction (PCR) assay targeted towards the detection of CTX-M, TEM, SHV and NDM. This study reports prevalence of NDM and ESBL co-producers in community as well as hospital settings.
| ~ Materials and Methods|| |
Following ethical clearance by Institutional Ethical Committee of Gauhati University (GU/ACA/Ethics/2012/3993 dated 10/1/12) in between November 2012 to August 2014, a total of 241 non-repetitive, consecutive bacterial isolates received at bacteriology laboratory at Gauhati Medical College and Hospital, a Tertiary Care Centre and East End Nursing Home Hospital and Research Center in Assam, India from both out- and in-patients [Figure 1] were included in the study. Of the 241 patients, age ranged from 0.6 to 79 years, with a mean age of 41.25 ± 20.70 years and it was observed that age was not significantly associated with incidence of both E. coli and K. pneumoniae infection (P > 0.05).
|Figure 1: Percentage distribution of Escherichia coli and Klebsiella pneumonia isolates. (a) percentage distribution of all collected isolates on the basis of different community and hospital setting. (b) Percentage prevalence of bacterial infection on the basis of four age groups. (c) Percentage distribution of bacterial isolates collected on the basis of gender|
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Antibiotic susceptibility and phenotypic detection tests
Antibiotic susceptibility testing
Disk susceptibility tests were performed and interpreted as per the CLSI guidelines 2011.  The susceptibility tests were performed to screen the production of ESBLs and metallo-β-lactamases as well as to assess the antibiotic susceptibility profile of these isolates. The antibiotics used were Penicillins: Ampicillin (10 μg), β-lactam/β-lactamase combination: Pipericillin/tazobactam (100/10 μg), ampicillin/sulbactum (10/10 μg), Cephems: Cefepime (30 μg), cefotaxime (30 μg), ceftazidime (30 μg), cefoperazone (30 μg), cefexime (5 μg), Monobactams: Aztreonam (30 μg), Carbapenems: Imipenem (10 μg), ertapenem (10 μg), doripenem (10 μg), meropenem (10 μg), Aminoglycosides: Kanamycin (30 μg), gentamycin (10 μg), Fluoroquinolones: Gemifloxacin (5 μg), Quinolones: Nalidixic acid (30 μg), Folate pathway inhibitor: Trimethoprim-sulfamethoxazole (1.25/23.75 μg) and Nitrofurans: Nitrofurantoin (300 μg). All antibiotic discs were purchased from HiMedia Pvt. Ltd., Mumbai.
Detection of extended spectrum β-lactamases producers
Isolates resistant or intermediately resistant to aztreonam, cefotaxime and/or ceftaxidime were phenotypically detected for the presence of ESBL by the Double Disk Synergy test using cefotaxime (30 μg) and cefotaxime + clavulanic acid (30/10 μg) and ceftazidime and ceftazidime (30 μg) + clavulanic acid (30/10 μg).  K. pneumoniae ATCC 700603 was used as the ESBL positive control and E. coli ATCC 25922 were used as the negative control.
Detection of carbapenemase producers
Isolates resistant or intermediately resistant to imipenem, ertapenem and/or meropenem were phenotypically detected for the production of carbapenemases by the modified Hodge test using ertapenem (10 μg) as an indicator disc  and by comparing the zone diameter surrounding ertapenem discs supplemented with and without 0.5M EDTA (750 μg); an increase of zone diameter by ≥4 mm suggested the production of metallo-carbapenemase. 
Complete gene sequences for NDM, CTX-M, TEM and SHV were downloaded from NCBI nucleotide database also referring to designations given in Lahey.org/Studies/(for NDM gene). A consensus sequence for each gene created using BioEdit v7.2.5 was used as the template for primer design using Primer-BLAST in NCBI. The accession numbers from which the consensus sequences were derived are enlisted in [Table 1] along with the primers. Oligos were ordered from Imperial Life Sciences (P) Ltd., Haryana.
|Table 1: Primers designed for multiplex detection of extended spectrum beta - lactamases and New Delhi metallo-beta-lactamase genes in this study|
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Standardization of the multiplex polymerase chain reaction assay
Bacterial DNA isolation for all experiments was done by boiling lysis. 2 μL of this crude DNA extract was used as template for all the reactions. The reaction mix contained 2 mM of MgCl 2 , 0.2 mM dNTP mix, 0.4 μM of each primer for TEM and CTX-M and 0.7 μM for SHV and NDM with 1U AmpliTaq; DNA Polymerase (Applied Biosystems, California, USA) in a 1× PCR buffered reaction. Positive controls used for the multiplex PCR were K. pneumoniae ATCC 700603 (SHV-18), J53 pMG298 (CTX-M-15), clinical isolates each containing TEM and NDM detected by uniplex PCR followed by sequence confirmation was used as a representative positive control and autoclaved distilled water was used as negative control. The cycling conditions for amplifying the genes were as follows: Initial denaturation at 95°C for 5 min and 30 cycles at 95°C for 30 s, 55°C for 30 s and 72°C for 40 s, followed by a final extension of 5 min at 72°C. PCR products were analysed in a 1.5% agarose gel. A molecular ladder of 100 bp was run with every gel.
Efficiency of the multiplex polymerase chain reaction assay
Detection limit of the multiplex polymerase chain reaction assay
The ability of the multiplex PCR assay to detect the desired genes in a given DNA sample was assessed by serial dilution of the bacterial cultures. Bacterial cultures with OD of 1.09 at 600 nm corresponded to 10 8 CFU/ml was serially diluted 10-fold to obtain 10 2 CFU/ml. Template DNA was prepared accordingly for all the dilutions followed by PCR.
Sensitivity evaluation of the multiplex polymerase chain reaction assay
To estimate the sensitivity of the primer pairs in the multiplex system, the minimum template concentration necessary to amplify a product was identified for all targets by serially diluting template DNA of a known concentration in the ratio of 1:10 and using them under the following conditions: (1) Only one type of target DNA as template and (2) a mix containing all targets at equal concentrations as template.
Qualitative and quantitative data values were expressed as frequency along with percentage and mean ± SD. Descriptive statistics were used to summarise demographic, genetic and all other clinical isolates characteristics of the participants. A well-structured data capture form in view of research study design and objectives was designed to collect all required data. Quality of data (review of completeness, data verification, validation and accuracy, security and confidentiality of data) was performed and maintained by lead research investigators. The primary outcome variable is the prevalence and co-production of NDM and other ESBL encoding genes in clinical urinary isolates of E. coli and K. pneumoniae was estimated and presented along with 95% confidence interval (CI). Associations between two or more qualitative variables were assessed using Chi-square test and Fisher Exact test as appropriate. Pictorial presentations of the key results were made using appropriate statistical graphs. A two-sided P <0.05 was considered to be statistically significant. All statistical analyses were done using statistical packages SPSS 19.0 (SPSS Inc. Chicago, IL, USA).
| ~ Results|| |
Antibiotic susceptibility and phenotypic detection tests
Antibiotic susceptibility testing
E. coli has shown highest resistance against nalidixic acid (154/188, 81.91%) and ampicillin (155/188, 82.44%). Among the third generation cephalosporins, resistance was highest against cefexime (134/188, 71.27%), followed by ceftazidime (125/188, 66.48%) and cefoperazone (115/188, 61.17%). Cefepime and aztreonam were neutralized by 49.46% (93/188) and 54.25% (102/188) of the E. coli isolates, respectively.
K. pneumoniae, on the other hand, showed 100% resistance against ampicillin followed by ertapenem (35/53, 66%), cefotaxime (33/53, 62.2%), nalidixic acid (31/53, 58.4%) and same resistance percentage against cefexime, cefoperazone and ceftazidime (30/53, 56.6%).
The most effective antibiotics were imipenem (6/188, 3.19% and 5/53, 9.4%), pipericillin/tazobactum (20/188, 10.63% and 10/53, 18.8%), gentamycin (29/188, 15.42% and 11/53, 20.7%) and doripenem (46/188, 24.46% and 5/53, 9.4%) against E. coli and K. pneumoniae respectively.
However, in comparison to the first two carbapenems, meropenem was ineffective against 61.7% (116/188) and 45.2% (24/53) while ertapenem against 54.25% (102/188) and 66% (35/53) of the isolates, respectively. Comparison between the bacteria and between the community and hospital acquired infection is represented in Supplementary Table 1 [Additional file 1] and [Figure 2].
|Figure 2: Distribution of the New Delhi metallo-β-lactamase and extended spectrum β-lactamases producing bacterial isolates. The distribution is based on the detection of New Delhi metallo-β-lactamase and extended spectrum β-lactamases genes and their division into out-patients, hospitalised patients and those from Intensive Care Unit|
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Extended spectrum β-lactamases production confirmatory tests
Double Disk Synergy Test showed 123/188 (65.43%) E. coli and 33/53 (62.26%) K. pneumoniae isolates were ESBL producers [Table 2]. These isolates were resistant to all cephems and monobactams and ertapenem but susceptible to imipenem and doripenem.
|Table 2: Percentage prevalence of genes detected in the study isolates samples among Klebsiella pneumoniae and Escherichia coli |
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Carbapenemase production confirmatory tests
Phenotypic test for identification of metallo-β-lactamase showed 44/188 (23.4%) of E. coli and 9/53 (16.98%) K. pneumoniae isolates to be modified Hodge test positive while imipenem/imipenem + EDTA disc test showed 41/188 (21.8%) and 13/53 (24.52%) as metallo-β-lactamase producers respectively [Table 2] and [Figure 2].
Multiplex polymerase chain reaction for assay
Lower limit of detection of the multiplex PCR assay in vitro was found to be 10 3 CFU/mL while its sensitivity was 50 ng/mL for CTX-M, TEM and SHV while for NDM the sensitivity was found to be 100 ng/mL.
The percentage of TEM genes detected was significantly higher among K. pneumoniae compared to E. coli (48.5% vs. 30.1%; P = 0.047). Similarly, SHV and NDM genes detected was significantly higher among K. pneumoniae compared to E. coli (33.3% vs. 10.6%; P = 0.001) and (69.2% vs. 18.2%; P = 0.001). In contrast, the percentage of CTX-M gene detected and modified Hodge test were observed to be lower among K. pneumoniae compared to E. coli (42.4% vs. 57.7%; P = 0.117) and (16.9% vs. 23.4%; P = 0.319), however, the differences between the groups did not achieve statistical significance (P > 0.05). The ESBL and metallo-β-lactamase confirmatory tests were found to be similar between K. pneumoniae and E. coli (62.3% vs. 65.4%; P = 0.671) and (24.5% vs. 21.8%; P = 0.675) as shown in [Table 2].
[Table 3] portrays the prevalence of individual ESBL genes and their co-prevalence in only ESBL producing E. coli and K. pneumoniae. The overall prevalence of individual ESBL genes SHV was in 5.03% (95% CI: 2.5, 10.1), TEM 10.79% (95% CI: 6.7, 17.1), and CTX-M 33.09% (95% CI: 25.8, 41.3), respectively. The co-prevalence SHV + TEM + CTX-M (11/139; 7.9%) and TEM + CTX-M (15/139; 10.79%) was observed to be higher compared to co-prevalence SHV + TEM (2/139; 1.43%) and SHV + CTX-M (1/139; 0.72%). The sample size was small and CIs were wide in the K. pneumoniae group, suggesting that the prospective study with larger samples needed for further evaluation.
|Table 3: Prevalence of individual extended spectrum beta - lactamases genes and their co - prevalence in only extended spectrum beta - lactamases producing Escherichia coli and Klebsiella pneumoniae |
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| ~ Discussion|| |
The isolation rate of E. coli as a common causative bacterium in urinary infections is high as compared to K. pneumoniae [Figure 1] which is consistent with the findings of previous studies in which E. coli was the predominant pathogen isolated from patients with urinary tract infections. , Patients whose urine samples yielded K. pneumoniae comprised both hospitalised and out-patients while those that yielded E. coli comprised out-patients or ambulatory patients indicative of community-acquired infection. Of the 17 NDM PCR-positive isolates, nine isolates were collected from out-patients (prevalence, 52.94%), six isolates from in-patients (prevalence, 35.29%) and the remaining two isolates were recovered from patients admitted in the intensive care unit; two of these 17 from paediatric patients. The prevalence of NDM carrying bacterial isolates in the community is indicative of these "superbugs" is common in any natural surrounding; thereby an individual may constrict an infection without being exposed to a hospital environment. This is also observed in the case of the ESBL producers that circulate in the community as well as hospitals [Figure 2]. NDM-producing isolates co-producing CTX-M and TEM β-lactamases was more common as compared to other combinations [Table 3] and [Table 4]; out of the 24 SHV positive isolates 17 showed the co-prevalence of CTX-M and TEM alone and in combination with SHV. The presence of SHV in all K. pneumoniae isolates was not detected despite the primers being designed to target SHV-1 with a prevalence percentage of 33.33% in ESBL producing isolates. This result is comparable to data available from previous publications from Assam, , Kolkata  and Bangladesh  wherein the SHV prevalence is 20-40% in all ESBL producing K. pneumoniae.
|Table 4: Antibiotic resistance profile and the co - prevalence of extended spectrum beta - lactamases genes of all the New Delhi metallo-beta-lactamase positive isolates |
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Each of the NDM-producing isolates have a comparable antibiotic resistance profile showing resistance against third and fourth generation cephalosporins (cefepime, ceftazidime, cefotaxime, cefeperazone and cefexime), β-lactam/β-lactamase inhibitor combinations, aminoglycosides and carbapenems (except imipenem) while trimethoprim-sulfamethoxazole and nitrofurantoin show higher zone of inhibition in these 17 NDM-positive isolates; these NDM-producing isolates are resistant to different number of antibiotics tested whether co-prevalence of ESBL genes are present or not, the resistance profile is described in [Table 4]. Except two isolates of K. pneumoniae, all other isolates have shown resistance against aztreonam. Isolate EC015 which does not carry any other tested ESBL genes but only NDM gene also showed resistance to aztreonam. While isolates KP005, 006 and 008 despite the absence of ESBL genes show similar resistance profile especially between KP006 and 008; these isolates could be inferred to harbour other resistant determinants such as AmpC family or efflux mechanisms. Most of the tested clinical isolates were susceptible to imipenem suggesting possible options, but alternatively are resistant to ertapenem, meropenem and doripenem; ertapenem shows a reduced inhibition against all isolates whether they are carbapenemase-producing or not. High resistance of ertapenem could be a result of combinations of ESBLs in addition to decreased permeability or increased influx. 
Relying on the MIC cut-offs is not always easy taking as the resistance determinants may sometimes confer only a slight increase of MIC values thus the development of a diagnostic method for the detection of ESBL producers is of importance for prevention and drug treatment.  Multiplexing all the four genes enables a quick and easy differentiation of the ESBL and NDM-producing isolates only, among the clinical samples of any geographical area. Further improvisation of this multiplex PCR would involve the addition of other resistant determinant once their prevalence is detected or is important for the given geographical location. The detection of other resistant determinants such as the AmpC family of genes, VIM, IMP, KPC or other region-specific variants is what remains to be carried out as part of the future objective. At present, for the multiplex PCR assay designed positive controls used separately or mixed yielded expected bands and confirmed the specificity of the PCR primers. The primer pairs were tested in uniplex PCR-targeting a single gene, and with a multiplex approach [Figure 3]. The PCR assay resulted in a good specificity in both the approach. The sensitivity or the detection limit enables the assay to be used for a decent cell suspension with anything above or equal to 10 3 CFU/mL.
|Figure 3: Uniplex and multiplex polymerase chain reaction of extended spectrum β-lactamases - CTX-M, TEM, SHV and New Delhi metallo-β-lactamase genes. Lane 1 and 10: DNA ladder of 100 bp. Lanes 2-5: Amplification of CTX-M. Lanes 6-9: Amplification of TEM gene. Lanes 11-14: Amplification of SHV. Lanes 15-16: Multiplex detection of all the four genes. Lanes 17-20: Amplification of New Delhi metallo-β-lactamase gene|
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Having isolated a high percentage of ESBL encoding bacteria, in this study, it may be noted that there is a high possibility of resistance being disseminated among our population. The search for alternative antibiotic agents that could tackle the antibiotic resistant isolates is the need of the hour. Since, more number of genes exhibiting extended spectrum activity against cephalosporins is known; a genotypic method incorporating all the known mechanisms may be helpful, cost effective and will have faster turnaround time (<4 h) for routine diagnostic settings. Some of such kits are available in the Western market, but its use in the developing countries where such bacterial infections are high is restricted by cost factor and/or the need for specialized instruments  such as real-time PCR, microarray analysis or ELISA.
Financial support and sponsorship
Department of Biotechnology, Government of India (Grant no. BT/213/NE/TBP/2011).
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4]