Prevalence and Characterization of Multi Drug Resistant Gram Negative Bacilli Isolates from a Tertiary Care Centre of Western U.P., India

Infections with MDR GNB are associated with mortality rates 21% higher than those of non resistant GNB and results in longer in patient stays and higher treatment costs. Several Indian studies have reported prevalence of carbapenemase producing Enterobacteriaceae, Pseudomonas and Acinetobacter species in a range of 11% to 81%, because of ample variation reported in prevalence and incidence of carbapenemases reported from different geographical region from time to time, we aimed to determine prevalence of carbapenemase producing organism and carbapenemase encoding genes among clinical MDR-GNB isolates from our area and also to assess the performance of the phenotypic tests. This was a cross sectional study. A total of 510 multi drug resistant isolates included were subjected to MHT and MBL E strip Test to detect carbapenamase production. In addition these isolates were subjected to PCR assay to confirm presence of carbapenamase genes encoding for these enzymes. The study found carbapenemase prevalence of 58.6% by phenotypic tests. blaNDM was the most common gene (24.7%) found by PCR assay followed by blaKPC (14.9%), blaVIM (9.6%) and blaOXA-48 (8.6%). Awareness of the prevalence and incidence of the carbapenem resistance and carbapenemase enzymes is crucial in the prevention of their spread and selection of appropriate treatment options. Study shows high prevalence rate of carbapenam resistant gram negative bacilli in this area, which indicates danger of limited treatment options and requirement of continuous detection of these cases to limit spread of resistant cases.


INTRODUCTION
Bacterial resistance to anti-microbial treatment is emerging as one of the major public health problem. Carbapenamases may be defined as beta lactamases that significantly hydrolyze at least imipenem or meropenem. Resistant to carbapenams is mostly due to the production of carbapenemases, which are βlactamase enzymes with a capacity to hydrolyze not only the carbapenams but also all the other beta lactam agents 1,2 . The most common carbapenemases include verona integron metallo-betalactamases types (VIM), imipenemase (IMP) types, Klebsiella pneumoniea carbapenemase (KPC), oxacillinase-48 (OXA-48), and New Delhi metallo-beta-lactamase-1 (NDM-1), encoded by carbapenem resistance determining gene bla VIM , bla IMP , bla KPC , bla OXA-48 and bla NDM respectively 1 . Phenotypic assay are used to identify carbapenemase activity while molecular assay have been developed to identify carbapenemase encoding genes 2 . Recently, increasing resistance to carbapenams in health care associated infections has been reported worldwide [3][4][5] . Thus, resistance to carbapenams becomes a real threat to the survival of patients with infections caused by MDR-GNB as mortality in such infections has been reported up to 50% who acquire blood stream infections and overall mortality rates are 21% higher than those of non resistant GNB and results in longer in patient stays and higher treatment costs 1,6 . Several Indian studies have reported prevalence of carbapenemase producing Enterobacteriaceae, Pseudomonas and Acinetobacter species in a range of 11% to 81% [7][8][9][10][11] . This study set out to determine the burden of carbapenam resistance, prevalence of carbapenemase producing organism and carbapenemase encoding gene among clinical MDR-GNB isolates obtained from patients. We also aimed to determine performance of Modified Hodge test (MHT) and Metallo β-lactamase (MBL) E Test by comparing them with results of Polymerase chain reaction (PCR) assay.

MATERIALS AND METHODS Study design and setting
This was a cross sectional laboratory based prospective study which was carried out in Microbiology department of Teerthankar  13 . This was in order to find out MDR Strain and also to find relation between resistance to these drugs and carriage of carbapenemase gene. Multi Drug Resistant (MDR) strains were differentiated according to criteria given by Mattner et al. 6 In brief, isolates that were resistant to three different classes of antibacterials but sensitive to carbapenems were included and isolates that were resistant to any one carbapenem but sensitive to other anti-bacterials were also included.

Detection of carbapenemase production
Phenotypic detection of carbapenemase production was done by MHT and MBL E Test. MHT was performed and interpreted according to guideline provided by CDC 14 . MBL E test strip were obtained by HiMedia Pvt. Ltd and test was performed and interpreted according to kit insert provided with kit 15 .

PCR amplification for carbapenemase genes
The entire molecular / PCR test (DNA extraction, amplification and gel electro-phoresis) were done in molecular laboratory of Subharti Medical College, Meerut.
DNA extraction was done using Qiagen DNeasy blood and tissue kit following manufacturer's instructions 16 . for reaction mixture preparation, commercially available Genei® Master Mix kit was used. Manufacturer's instruction manual was followed for using the kit. Primers of PrimeX targeting bla VIM , bla KPC , bla OXA-48 and bla NDM were obtained from Valine Life Sciences, India, as described in study by Asthana S et al. 17 For reaction mix preparations following contents were added Molecular grade water 15µl, Master Mix. in kit 25µl, Primers 0.2µl and Template DNA 10µl. The amplification was done using Applied Biosystem Veriti 96 well thermal cycler. For bla KPC , bla OXA-48 and bla NDM the programme was initial denaturation at 94 o C for 5 minutes followed by 30 cycles of 30 seconds denaturation at 94 o C, annealing at 55 o C for 30 seconds and extension at 72 o C for 1 minute. An additional final extension step was performed at 72 o C for 7 minute. For bla VIM the same programme was used except that annealing temperature was adjusted to 45 o C for 60 seconds and had a final extension step of 72 o C for 10 minutes.
5µl of PCR product were analyzed by electrophoresis in 1.0% agarose stained with ethidium bromide to detect the specific amplified product by comparing with 100 base-pair standard DNA ladder and visualized under gel doc. system. For quality control, of MHT well characterized strains were used. E. coli ATCC 25922 was used as a susceptible strain, K. pneumoniae ATCC BAA-1705 as a positive control while K. pneumoniae ATCC BAA-1706 was used as a negative control and for PCR tests, the following control strains were used; K. pneumoniae ATCC strain BAA-1705 for bla KPC , K. pneumoniae ATCC strain BAA-2146 for bla NDM , and E. coli ATCC BAA-2523 for bla OXA-48.

ethical Approval
The study protocol was carefully reviewed and approved by the Institutional Ethics Committee of the Teerthankar Mahaveer Medical College and Research Centre.

Data Analysis
Data analysis was done using SPSS ver. 16.0. All categorical variables were represented by percentages and Comparison of categorical variables was done by Chi-square test. A p value of <0.05 was considered as evidence of significant statistical difference.

Correlation of phenotypic and genotypic tests
Out of 80 isolates positive by MHT 51 isolates showed presence of one or more gene and in 29 isolates no gene was detected. Out of 120 isolates positive by MBL E test genes were detected in 104 samples and in rest of 16 isolates which were phenotypically positive but no gene

DISCUSSION
Antibiotic resistance to reserve antibiotic class is on a continuous rise among gram negative bacteria especially in the family Enterobacteriaceae and among species of Acinetobacter and Pseudomonas (EPA Species) 1,2 .
Recently, a newspaper article reported 13% of mortality rate in India is due to antibiotic resistant cases, which is more than double when compared to developed nations where mortality rate due to drug resistant cases is 2-7% 18 . Worldwide several studies had reported increased prevalence of carbapenemase producing organisms 5,19,20 . Our findings show out of 510 MDR strains 52.3% were from males and 47.6% were from females. Ratio of male to female patient in this study was 1:1.09 this shows almost equal distribution of Antibiotic resistant strains among both sexes. We found most common MDR organism was E.coli followed by K. pneumoniae and P. aeruginosa and the hotspot zone of these organism were medical and surgical ICU'S.  8,9,21 . These findings may have vital role in making of hospital infection control policy. This study shows a prevalence rate of carbapenemase enzyme of 58.6% by phenotypic tests among EPA species    7,[27][28][29] . Although in western world most common gene encoding carbapenemase found is bla KPC 19,30 . Whereas a study from Africa reported highest prevalent gene were bla IMP types while another one reported bla VIM as the most common gene encoding for carbapenemase enzyme 1,2 . These findings are suggestive of inter-regional spread of the specific mechanism of carbapenem resistance. In our study we found 58(11.3%) samples were phenotypically positive but no gene was detected in them by PCR. This might be due to limited number of genes targeted in our study as well as to other mechanisms of resistance such as porin loss or mutations.

CONCLUSION
Carbapenemases are globally distributed and their prevalence and incidence vary considerably across each continent, nation, region and even centre to centre, so awareness of the prevalence and incidence of the carbapenem resistance and carbapenemase enzymes is crucial in the prevention of their spread and selection of appropriate treatment options. Our study shows high prevalence rate of carbapenamase producing gram negative bacilli in this area, which indicates danger of limited treatment options and requirement of continuous detection of these cases to limit spread of resistant cases. We also found that combination of two phenotypic tests MHT and E strip Test can be done together to rule out false negative results whereas E Test should be done on regular basis to detect MBL as MBL encoding genes were more prevalent in our region as it is not feasible to do PCR on regular basis on every sample.

ACKNOWLEDGMENTS
Authors would like to acknowledge support of Dr. Sanjay Kumar and Dr. Seema Negi of Subharti Medical College Meerut for their guidance and support in processing of samples for gene detection. SM is thankful to Ms. Sana Nudrat for her valuable help in sample processing and data collection. Authors also acknowledges support of Dr. Umme Afifa for help in statistical analysis of collected data.

CONFLICTS OF INTEREST
The authors declare that there is no conflict of interest.

AUTHOR'S CONTRIBUTION
SM performed the tests, collected data, did data analysis and wrote the manuscript. UF guided the study and reviewed the manuscript. SM and UF approved it for publication.

FUNDING
None.

DATA AVAILABILITy
All datasets generated or analysed during this study are included in the manuscript.

ETHICS STATEMENT
The study protocol was carefully reviewed and approved by the Institutional Ethics Committee of the Teerthankar Mahaveer Medical College and Research Centre, Moradabad U.P. India.