Exploring the Interplay of Resistance Nodulation Division Efflux Pumps,Amp C and Opr D in Antimicrobial Resistance of Burkholderia cepacia Complex in Clinical Isolates

Abstract

Aim:

This study aimed at investigating the association of gene expression of multidrug efflux pumps (MexA, MexC, MexE, and MexX), the outer membrane porin OprD, and the β-lactamase AmpC with the antimicrobial susceptibility among 44 clinical isolates of Burkholderia cepacia complex (Bcc).

Results:

Increased expression of ampC gene showed significant association with reduced susceptibility to chloramphenicol. In fact, reduced susceptibility to chloramphenicol was correlated with overexpression of most genes (ampC, mexC, mexE, and mexX) studied here in majority (>95%) of the Bcc isolates. Increased mexA expression showed significant association with reduced susceptibility to β-lactam antimicrobials (ceftazidime, piperacillin-tazobactam, and meropenem) and co-trimoxazole. Reduced susceptibility to meropenem also showed significant correlation with overexpression of mexC and mexX, whereas reduced susceptibility to ceftazidime was also associated with mexE overexpression. Reduced susceptibility to levofloxacin was significantly associated with overexpression of mexX. The involvement of the efflux pumps in levofloxacin and ceftazidime resistance was further inferred from the finding that the efflux pump inhibitor, carbonyl cyanide m-chlorophenylhydrazone reduced minimum inhibitory concentrations for both the antimicrobials.

Conclusions:

To conclude, this study explored the high-level expression of mexC, mexE, and mexX efflux pumps genes and ampC in the clinical isolates of Bcc, which can be targeted at treating infections caused by Bcc.

Introduction

Burkholderia cepacia complex (Bcc) is a group of closely related non-fermenting, Gram-negative bacilli (NFGNBs) that comprises >20 defined species.^1,2 In all Gram-negative species, antimicrobial susceptibility is greatly defined by synergy between permeability and active efflux barriers.³ Among the various mechanisms of resistance in bacteria, influx and efflux of antimicrobials is of particular importance. Porins are located on the outer membrane and act as selective permeability barriers for antimicrobials and toxic compounds, whereas efflux pumps prevent the accumulation and interaction of antimicrobials with bacterial intracellular targets by pumping out the latter. Efflux pumps represent a serious threat in those pathogenic Gram-negative bacteria that couple efflux with a low-permeable cell membrane. Outer membrane protein (oprD) confers resistance to carbapenem in Gram-negative bacteria such as Pseudomonas aeruginosa, but there is no report of the oprD downregulation in Bcc.⁴ Moreover, the role of different efflux pumps on antimicrobial resistance of Bcc isolates has not been investigated in detail. A similar study has been performed on P. aeruginosa.^5–7 Resistance nodulation division (RND) family of efflux pump plays an important role among mediators of multidrug resistance in Gram-negative pathogens. According to Tseng et al.,⁷ efflux pumps in this study matched with the locus tag of the previously studied efflux pumps such as mexC with RND-1, mexX with RND-3, mexA with RND-4, and mexE (ceoAB-opcM) with RND-10.^6,7 RND efflux pumps, including RND-3, RND-4, RND-9, and RND-10, have shown to facilitate multiple antimicrobial resistance in laboratory reference strains of Bcc.^7,8

The treatment of Bcc infections is difficult, because Bcc often has high-level intrinsic resistance to many commonly used antimicrobials, such as penicillins, first- and second-generation cephalosporins, aminoglycosides, fosfomycin, and the antimicrobials of last resort such as polymyxins.^9,10 “AmpC beta-lactamases” (ampC) is another cause of this antimicrobial resistance in Bcc. The gene responsible for production of AmpC enzymes is found on chromosome 3 of Bcc and propagated to other bacteria by plasmids.¹¹ However, it is difficult to conclude as to which mechanism is responsible for a strain to be a resistant one. A few reports have recently shown the prevalence of different carbapenemases such as class-A KPC carbapenemases, class B metallo-β-lactamases, and class D (OXA) carbapenemases in Bcc, which have been well described in P. aeruginosa and Acinetobacter baumannii.^12–14 Despite clinical significance and antimicrobial resistance of Burkholderia spp., characterization of efflux pumps of Bcc lags behind other non-enteric Gram-negative pathogens.^15,16 We studied the role of Bcc efflux pumps in antimicrobial resistance to various antimicrobials. We also observed the change in minimum inhibitory concentrations (MICs) of Bcc by investigating the synergistic effect of the interaction of levofloxacin and ceftazidime with the carbonyl cyanide m-chlorophenylhydrazone (CCCP), a known efflux pump inhibitor.

To further characterize resistance mechanisms in Bcc, we herein investigated the association of expression of RND family efflux systems with the expression of porins and ampC β-lactamases among Bcc clinical isolates.⁵

Materials and Methods

Bacterial isolates

Forty-four Bcc clinical isolates resistant to any or multiple antimicrobials were analyzed. Forty-one isolates were collected from septicemic and cystic fibrosis (CF) patients who visited or were admitted to the Nehru Hospital, Postgraduate Institute of Medical Education and Research, Chandigarh, over the past 7 years (2005–2011). The source of specimens were blood cultures, pus, respiratory specimens, sterile body fluids, intravenous catheters, and cerebrospinal fluid. One isolate (blood culture, 2012) was from Global Hospitals and Health City, Chennai and two isolates (a blood culture isolate and an unopened injection vial isolate, 2013) were from TNMC and BYL Nair Hospital, Mumbai. Ethical clearance obtained from the Institute Ethics Committee, vide file no. 11/6091. Burkholderia cepacia selective agar (BCSA) was used for the recovery of Bcc isolates from the respiratory specimens of CF patients (diagnosed as per the guidelines of CF Foundation) that included sputum, induced sputum, and broncho-alveolar lavage. After routine processing of these specimens, in positive cases, isolates were identified to species level by conventional biochemical identification tests. Gram-negative, oxidase-positive, motile, NFGNB were selected and the identification of isolates as members of the Bcc was carried out by a triphasic analysis¹⁷; growth on BCSA, positive identification using the conventional biochemical testing system, recA-PCR-Restriction Fragment Length Polymorphism as per the previous study protocol.¹⁸ Further, all the Bcc isolates were identified with Matrix-Assisted Laser Desorption Ionization - Time of Flight Mass Spectrometry and confirmed with expanded multilocus sequence typing (E-MLST) as previously described.¹⁹ All Bcc isolates were lyophilized and stored at 4°C for further study.

Disk diffusion and MIC

Antimicrobial susceptibility testing was done on Mueller-Hinton agar (MHA) by Kirby Bauer's disk diffusion method for co-trimoxazole, meropenem, ceftazidime, levofloxacin, chloramphenicol, minocycline, tetracycline, and piperacillin-tazobactam (Oxoid; Thermo Fisher Scientific India Pvt., Ltd., Mumbai, India) as per Clinical and Laboratory Standards Institute (CLSI) guidelines.²⁰ As there are no breakpoints for Bcc against tetracycline and piperacillin-tazobactam, the breakpoints for P. aeruginosa against the respective antimicrobials were used. For chloramphenicol, levofloxacin, and minocycline, MICs were determined by agar dilution method and categorized as susceptible, intermediate susceptible, and resistant by using the breakpoints as per CLSI guidelines.²⁰

Carbapenemases

Thirty-seven of 44 carbapenem-resistant isolates were screened for bla_KPC-2,²¹ bla_OXA-23²², and bla_NDM-1²³ by PCR using the primers shown in Supplementary Table S1.

Real-time reverse transcription-PCR

Late-log-phase cultures were grown on MHA. Total RNA was isolated from late-log-phase cultures with High Pure RNA Isolation Kit (Roche Diagnostics GmbH, Mannheim, Germany) and treated with DNase (10 U/μL). Extracted RNA was subjected to reverse transcription by using random hexamer primers supplied with Transcriptor First-Strand cDNA Synthesis Kit (Roche Diagnostics GmbH). The resultant complementary DNA (cDNA) were subjected to Real-Time PCR by using the LightCycler 480 II Real-Time PCR Machine (Roche Diagnostics GmbH). Primers detailed in Supplementary Table S2 were used to quantify transcription of genes encoding efflux pump proteins MexA, MexC, MexE, and MexX, the outer membrane protein OprD, and AmpC β-lactamase. Each PCR amplification included amplification of cDNA synthesized as a control without reverse transcriptase to ensure that there was no contaminating DNA molecules and all amplification was a result of cDNA.

Reverse-Transcription Polymerase Chain Reaction (RT-PCR) was performed as biological triplicates (2 μg RNA per reaction mixture) by using Light Cycler 480 SYBR Green I Master (Roche Diagnostics GmbH) using primers mentioned in Supplementary Table 2. Gene expression was normalized with respect to rpoD in the same strain and then calibrated relative to a sensitive Bcc strain by the ΔΔC_t method.²⁴ The control strain used in this study was one of the clinical isolates and was tested susceptible to ceftazidime, co-trimoxazole, meropenem, levofloxacin, and tetracycline (Burkholderia cenocepacia 32170). The Whole Genome Shotgun project of Burkholderia cenocepacia 32170 has been deposited at DDBJ/ENA/GenBank under the accession MKPT00000000. The version described in this article is version MKPT01000000 and available at the NCBI website.* Increases or decreases in gene expression of ≥2-fold were taken as significant. For the ΔΔC_t calculation to be valid, the amplification efficiencies of the target and the endogenous reference must be approximately equal. As the housekeeping gene, rpoD amplification efficiency was not similar to the target, the standard curve method was also performed.

Primer design

The protein sequences of four RND efflux pump genes from P. aeruginosa were aligned with that of Bcc by using Basic Local Alignment Search Tool for Protein (BLASTP). Then, the protein sequence of each gene, which showed maximum similarity with that of P. aeruginosa, was considered. Next, the nucleotide sequence was retrieved for each gene target from the National Center for Biotechnology Information (NCBI) (Supplementary Table S3). The methodology just described was utilized for designing primers for four efflux pump genes (mexA, mexC, mexE, and mexX) by using online software Primer3. Primers for rpoD, ampC, and oprD genes were designed by using a nucleotide sequence of Burkholderia cenocepacia J2315 (Supplementary Table S3). All the designed primers were used in Quantitative Reverse Transcription PCR (RT-qPCR) and listed in Supplementary Table 2. These primers generated from Primer3 were analyzed by using Primer BLAST of NCBI with which the primer sequences and product sizes were matched. Primers were synthesized from IDT (Integrative DNA Technologies), and PCRs were performed for each gene by using cDNA of Bcc. PCR gave the exact product sizes for each gene, which were confirmed later on by sequencing (Sanger's Method).

Phylogenetic tree analysis

A phylogenetic tree was constructed by using the Maximum Likelihood (ML) method based on seven housekeeping genes (atpD, gltB, gyrB, recA, lepA, phaC, and trpB). DNA was isolated by using QIAamp DNA Mini Kit (Qiagen, Hilden, Germany), and PCR was performed for the seven genes of all isolates as described earlier. PCR products were extracted and purified by QIAquick Gel Extraction Kit (Qiagen) and the same were processed for DNA sequencing by using BigDye Terminator ready reaction mix, version 3.1 (Applied Biosystems, Thermo Fisher Scientific Company, Waltham, MA). The ML phylogenetic tree was constructed by using a concatenated nucleotide sequence of seven housekeeping genes of 44 isolates of Bcc, keeping 1,000 bootstrap replications. MEGA (Molecular Evolutionary Genetics Analysis, version 6.06) Software was used to determine the diversity among the isolates and to rule out clonality.

Effect of pump inhibitor on antimicrobial resistance

Using a broth micro-dilution checkerboard method, twofold dilutions of levofloxacin (2–256 mg/L) and ceftazidime (2–256 mg/L) were combined with twofold dilutions of CCCP (1–64 mg/L) in 96-well flat-bottom microplates. A defined inoculum of the Bcc strains (5 × 10⁵ CFU/mL) was used, and microbial growth was assessed spectrophotometrically at 620 nm after 24 hours of incubation at 37°C. The MIC of levofloxacin and ceftazidime alone and in combination with CCCP was calculated as the lowest antimicrobial concentration resulting in <10% growth. Five Bcc isolates resistant to levofloxacin and ceftazidime were tested in triplicate. For each checkerboard, the fractional inhibitory concentration index was calculated as previously described.²⁵ An efflux pump activity was defined when a fourfold reduction in the MIC compared with that of the antimicrobials alone was observed.

Statistical analysis

Relative gene expression was associated with in vitro susceptibility by using univariate and multivariate logistic regression analysis (JMP10; SAS Institute). For this analysis, isolates were divided into two groups: susceptible and non-susceptible (based on MIC). Within these groups, isolates were further separated according to relative expression of an analyzed gene for one antimicrobial at a time. The probability of non-susceptibility was plotted against the relative gene expression and fitted with logistic function. The odds ratio and its 95% confidence interval (CI) for non-susceptibility were calculated by using the regression coefficients obtained from the logistic fit.

Results

In vitro susceptibility

Out of 44 Bcc isolates, 5 out of 44 (11%) were resistant to ceftazidime, 20 out of 44 (45%) to meropenem, 11 out of 44 (25%) to co-trimoxazole, 28 out of 44 (86%) to levofloxacin, 2 out of 44 (5%) to piperacillin-tazobactam, and 33 out of 44 (75%) were resistant to tetracycline. Out of 21 carbapenem-resistant isolates, none was found to be positive for bla_KPC-2, bla_OXA-23, and bla_NDM-1.

Phylogenetic tree

The ML phylogenetic tree (Supplementary Figs. S1 and S2) showed variation in gene sequences, as reflected by multiple lineages with high bootstrap values. At least four major lineages with sub-lineages within two lineages were observed, suggesting ongoing variation and diversity among the 44 Bcc isolates (Supplementary Figs. S1 and S2).

Gene expression

In RT-PCR, for the ΔΔC_t calculation to be valid, under the standard curve method, the plot of cDNA dilution versus ΔC_t was obtained close to zero for all the parameters (results not shown). The associations between gene expression and in vitro susceptibility are shown in Figures 1 –3 and Supplementary Table S4. None of the genes among OprD, ampC, mexA, mexC, mexE, and mexX demonstrated a significant association of the relative messenger RNA expression with all the antimicrobials that were tested. However, a statistically significant association between the relative gene expression was observed with some of them (e.g., gene expression of OprD was found to be significantly associated with in vitro susceptibility to levofloxacin and chloramphenicol; Supplementary Table S4). In addition, increased ampC expression was highly variable with one isolate showing expression up to 8,780-fold although it was susceptible to ceftazidime and piperacillin-tazobactam and resistant to meropenem.

FIG. 1.

Frequency plots of relative gene expression with in vitro susceptibility of selected drugs found to be statistically significant on univariate analysis. CAZ and mexA (A); TZP and mexA (B), LEV and mexX (C); MEM and mexA (D); MEM and mexC (E); CHL and mexC (F); CHL and mexX (G). CAZ, ceftazidime; CHL, chloramphenicol; LEV, levofloxacin; MEM, meropenem; TZP, piperacillin-tazobactam.

FIG. 2.

Logistic fits of in vitro susceptibility versus relative gene expression for ceftazidime and mexA [p = 0.0475, OR = 1.35 (95% CI 0.99–1.85)] (A) and mexE [p = 0.0161, OR = 1.30 (95% CI 0.99–1.71)] (B), trimethoprim-sulfamethoxazole and mexE [p = 0.042, OR = 1.14 (95% CI 1.00–1.31)] (C), and meropenem and mexX (p = 0.009, OR = 0.79 (95% CI 0.65–0.97)] (D). Vertical dotted lines represent the cutoffs of 0.5-, 0-, and 2-fold differences in gene expression. CI, confidence interval; DD, disk diffusion; OR, odds ratio; SXT, trimethoprim-sulfamethoxazole.

FIG. 3.

Logistic fits of in vitro susceptibility versus relative gene expression for piperacillin-tazobactam and mexA [p = 0.0028, OR = 2.27 (95% CI 1.02–5.07)] (A) or mexE [p = 0.0023, OR = 4.13 (95% CI 0.84–20.2)] (B), tetracycline and oprD [p = 0.011, OR = 1.25 (95% CI 1.05–1.53)] (C) or mexE [0.036, OR = 0.87 (95% CI 1.00–0.76)] (D), CHL and mexX [p = 0.002, OR (95% CI) unstable] (E). Vertical dotted lines represent the cutoffs of 0.5- and 2-fold differences in gene expression. CHL, chloramphenicol; MIC, minimum inhibitory concentration; TET, tetracycline; TZP, piperacillin-tazobactam.

On univariate analysis, mexA was significantly associated with reduced susceptibility to ceftazidime (p = 0.005) (Fig. 1A; Supplementary Table S4), piperacillin-tazobactam (p = 0.001) (Fig. 1B; Supplementary Table S4), and meropenem (p = 0.018) (Fig. 1D; Supplementary Table S4). On logistic regression analysis, increased mexA expression was significantly associated with reduced susceptibility to ceftazidime and piperacillin-tazobactam with 96% (26/27) and 100% of susceptible isolates showing <0.5-fold expression of mexA, respectively.

On univariate analysis, mexC overexpression was significantly associated with reduced susceptibility to meropenem (p = 0.045) (Fig. 1E) and chloramphenicol (p = 0.009) (Fig. 1F). Logistic regression analysis showed that mexE expression was associated with reduced susceptibility to ceftazidime (Fig. 2B), piperacillin-tazobactam (Fig. 3B), and trimethoprim-sulfamethoxazole (Fig. 2C); whereas a borderline significant inverse relationship was found for tetracycline (Fig. 3D).

On univariate, multivariate, and logistic regression analysis, mexX expression was significantly associated with in vitro susceptibility data of levofloxacin (p = 0.043) (Fig. 1C) and chloramphenicol (p = 0.004) (Figs. 1G and 3E). In particular, for meropenem, logistic regression analysis revealed an inverted relationship between reduced susceptibility and mexX expression (Fig. 2D) with odds ratio of 0.79, (95% CI 0.65–0.97).

Efflux pump activity

The effect of CCCP on the MICs of levofloxacin and ceftazidime is shown in Supplementary Table S5. The MICs of levofloxacin and ceftazidime ranged from 16 to >256 mg/L and 4 to >256 mg/L, respectively. The CCCP showed antimicrobial activity at concentrations from 8 to >64 mg/L. The combination of CCCP with levofloxacin or ceftazidime was synergistic for 4 out of 5 and 3 out of 5 isolates respectively. The MICs of levofloxacin and ceftazidime were reduced by 1- to 32-fold for both antimicrobials, with 4 out of 5 isolates demonstrating efflux pump activity (≥4-fold MIC reduction).

Discussion

In Bcc, the antibiogram broadly did not agree with the known pattern of OprD and AmpC resistance mechanisms. Reduced susceptibility to any of the β-lactam antimicrobials that included ceftazidime, piperacillin-tazobactam, and meropenem was not significantly associated with increased expression of oprD, ampC, and mexE. Isolates producing AmpC β-lactamases (Chromosome 3) have the potential for developing resistance to carbapenems.¹¹ In P. aeruginosa, it has been suggested that AmpC overproduction alone does not significantly alter susceptibility against carbapenems but could certainly contribute to resistance if it is accompanied by additional resistance mechanisms (e.g., decreased OprD, efflux pumps overproduction, and/or production of a class A/class B carbapenemase).²⁶ Of 32 isolates with ampC overexpression, only 2 isolates showed no expression of OprD and only one was resistant to meropenem. Livermore (1992) showed that loss of oprD resulted in resistance to carbapenems only in case of expressed chromosomal ampC in P. aeruginosa.²⁷ However, this close cooperation between either of these mechanisms along with AmpC overproduction has not been significantly observed in Bcc.²⁸ In this study also, ampC overexpression was not significantly associated with any of the antimicrobial agents (including carbapenems). It may be noted that reduced susceptibility to levofloxacin was observed in a large number of isolates overexpressing ampC (73%) but it was not statistically significant.²⁹ None of the isolates tested for bla_KPC-2, bla_OXA-23, and bla_NDM-1 using PCR showed presence of the concerned genes. However, a recent study showed the presence of various β-lactamases in different species of Bcc.¹⁴

Comparing with the extrusion of antimicrobial agents by various efflux pumps in P. aeruginosa, certain similarities were observed.^30,31 Reduced susceptibility to β-lactams (ceftazidime, piperacillin-tazobactam, and meropenem) was significantly associated with increased mexA expression, and resistance to chloramphenicol and levofloxacin was significantly associated with increased mexX expression. For ceftazidime and levofloxacin, this was further substantiated by the increased susceptibility (≥4-fold MIC reduction) on addition of Efflux Pump Inhibitor (EPI), CCCP in 4 out of 5 isolates tested. This is in contrast to the study by Buroni et al.,¹⁶ where RND-4 showed strongest homology with the P. aeruginosa mexX RND pump and its deletion mutant D4 was shown to have no effect on MIC of ceftazidime and meropenem. Similarity was observed with this study in relation to chloramphenicol and levofloxacin where increased susceptibility was observed in the deletion mutant (entire operon encoding different efflux systems).¹⁶ However, an inverse relationship was observed with mexX overexpressed isolates with more than half of these overexpressing isolates being susceptible to meropenem. A similar inverse relationship was observed for tetracycline and mexE expression where almost half of the isolates with reduced susceptibility were under-expressing mexE. It is a well-known fact that efflux pump presence alone may not necessarily lead to clinical resistance unless associated with an additional resistance mechanism, and reduced susceptibility with under-expressing efflux pumps points out to some other resistance mechanism that has not been studied in the present study.^32,33

In 1996, Burns et al.³⁴ sequenced a 3.4-kb subcloned fragment, and identified one complete and one partial open reading frame, which were homologous with two of three components of a potential antimicrobial efflux operon from P. aeruginosa (mexA-mexB-oprM). It showed a 10-fold decrease of chloramphenicol entry into the resistant strain but mexA (the third component of this efflux pump) was not found to be significantly associated with reduced susceptibility.³⁴ In this study also, reduced susceptibility of chloramphenicol was not significantly associated with mexA only among the four efflux pumps studied. However, a good correlation was observed between reduced susceptibility to chloramphenicol and overexpression of other genes (oprD, ampC, mexC, mexE, and mexX) among majority of the isolates (>95%).

MexE was found to have strong homology with the P. aeruginosa RND pump mexEF-oprN, which confers resistance to chloramphenicol, tetracycline, quinolones, and carbapenems but not conventional β-lactams,³⁵ and termed as ceoAB-opcM in Bcc by Nair et al.³⁶ In the latter study, ceoB (mexB) was present in 6 out of 10 of B. cenocepacia isolates. In this study, mexE was not significantly associated with in vitro susceptibility of any of the antimicrobials.

The genome of Burkholderia cenocepacia J2315 contains coding sequences for all five major families of efflux systems,³⁷ with the RND representing the best characterized efflux system³⁸ Nowadays, 16 genes encoding RND efflux pumps have been identified in Bcc.^15,16 Knock-out studies indicated that B. cenocepacia RND pumps such as RND-3 (BCAL1672–BCAL1676), RND-4 (BCAL2820–BCAL2822), RND-9 (BCAM1945–BCAM1947),^7,16,39,40 and RND efflux pump gene cluster B1004–B1006⁴¹ played a wider role than just in antimicrobial resistance, influencing additional phenotypic traits that are important for pathogenesis.^16,39,40

Low levels of resistance can result from mutations or from a decrease in the porin content of the outer membrane, with consequent decreased tetracycline uptake in various bacteria.⁴² In this study, 86% of overexpressing OprD isolates showed reduced susceptibility to tetracycline. However, as we can understand, increased permeability denoted by OprD overexpression does not confer OprD as the mechanism of resistance for tetracycline.⁴³

Another interesting finding observed among 44 Bcc isolates was that both isolates obtained from Mumbai (that included a blood culture isolate and an injection vial isolate used in that patient) showed the same results on RT-PCR with overexpression in all the six genes. On E-MLST,¹⁹ both the isolates belonged to the same Sequence Type (ST)-814 (novel ST with novel allele gyrB 581).

We used RT-PCR to investigate complex combinations of resistance mechanisms in Bcc. This approach had limitations for analyzing reduced susceptibility at the level of individual strains. Aside from distortions arising through multiple co-resident mechanisms in the same strain, RT-PCR provided only a snapshot of gene expression, which may vary through the growth cycle. The function of RND efflux pumps may be modulated not only by expression of the pump protein, as examined, but also by that of other components and by the architecture and energetic membrane within which it functions, which have yet not been detected in Bcc. To conclude, this study explored and described the high-level expression of other resistance mechanisms besides efflux pumps in a large number of clinical isolates of Bcc, which may be targeted to treat the infections.

Footnotes

Acknowledgments

The authors are grateful to Dr. Sujatha Chandrasekaran from Global Hospitals and Health City, Chennai and Dr. Swapana Mali, Dr. Jayanthi Shastri from TNMC and BYL Nair Hospital, Mumbai for providing them with the strains. They are also indebted to Prof. Narottam Sharma, MA (Eng), Med, MCTE, PGDTE (Hyd) Retired from the Regional Institute of English Chandigarh, UT for proofreading this article.

Disclosure Statement

No competing financial interests exist.

Funding Information

The authors hereby acknowledge the Indian Council of Medical Research for providing the funding for this work (File No. 5/3/3/5/2011-ECD-I).

Supplementary Material

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