Molecular Characterization of Extended Spectrum β-lactamase and Carbapenemase Producing Klebsiella pneumoniae from a Tertiary Care Hospital

ABSTRACT Objective The extended-spectrum beta-lactamase (ESBL) and carbapenemase producing gram-negative bacteria among the members of Enterobacteriaceae are of major health concern globally. The present study was carried out to determine proportion and genetic characterization of ESBL and carbapenemase producing Klebsiella pneumoniae strains isolated from intensive care units of a tertiary care hospital. Materials and methods A total of 250 non-duplicate K. pneumoniae isolates were recovered from various clinical specimens from our intensive care units from May 2014 to May 2015. Antibiotic susceptibility testing was performed as recommended by Clinical and Laboratory Standard Institute. Phenotypic identification of ESBL and carbapenemase producing isolates were confirmed by the double-disk synergy test, modified Hodge test, imipenem and imipenem-EDTA combined test, respectively. Molecular characterization of β-lactamase genes were performed by polymerase chain reaction. Results Out of 250 Klebsiella pneumonaie, 84% isolates were ESBL producers, 66% were carbapenem resistant based on their reduced susceptibility to imipenem, meropenem and ertapenem. Among these 165 carbapenem resistant isolates, 9.7% were positive for bla NDM-1 and these isolates were also found to be positive for one or more bla genes. Co-carriage of AmpC in ESBL and carbapenem resistant isolates were 7.8% and 3.6%, respectively and were negative for blaKPC genes. Conclusion The study indicated the prevalence of ESBLs and blaNDM-1, with additional bla genes and AmpC among the K. pneumoniae isolates in our intensive care units. NDM-1 producing Enterobacteriaceae is a growing health care problem. Detection of the prevalence of antibacterial resistance pattern helps towards improved antibiotic policy and empirical antibiotic treatment. How to cite this article Beena HB, Shenoy SM, et al. Molecular Characterization of Extended Spectrum β-lactamase and Carbapenemase Producing Klebsiella pneumoniae from a Tertiary Care Hospital. Indian J of Crit Care Med 2019;23(2):61-66.

Carbapenems are most often used in this treatment. The emergence of resistance to these agents has become a serious health concern globally 1 .
Klebsiella pneumoniae is one of the most common Gramnegative bacteria showing resistance to multiple antibiotics. The development of extended-spectrum cephalosporins in the early 1980s was regarded as a major addition to our therapeutic armamentarium in the fight against beta-lactamase mediated bacterial resistance. The emergence of enzymes that have the ability to hydrolyze this cephalosporin's seriously compromised the efficacy of these lifesaving antibiotics. These enzymes were called extended spectrum beta lactamases 2 . Extended spectrum beta-lactamases are plasmid-mediated enzymes that are capable of conferring bacterial resistance to the penicillins, first, second third, fourth generation cephalosporins and aztreonam.
They do this by hydrolysis of these antibiotics but they are inhibited in vitro by beta-lactamase inhibitors 3 .
ESBL is predominantly found in Klebsiella spp. and Escherichia coli, and other members of the Enterobacteriaceae 4 . The most prevalent ESBLs are included in three groups: TEM, SHV and CTX-M 5 . CTX-M type ESBLs show only 40% identity to TEM or SHV ESBLs, but they are closely related to β-lactamases of the Kluyvera spp 6 .
Carbapenems were the drug of choice for the treatment of multidrug resistant gram-negative bacterial infections. Emergence of carbapenem resistant bacteria left limited options in the choice of antibiotics to treat the infections caused by them 7 . These bacteria have the potential to spread rapidly within the hospital environment and also across the continents 8 . Resistance to carbapenem is mostly due to production of enzymes-carbapenemases that hydrolyze carbapenems and other β-lactams. Acquired carbapenemases belong to group A (IMI, NMC, SME GES, and Klebsiella pneumoniae carbapenemase (KPC), group B metallo-β-lactamase (MBLs of VIM, IMP, GIM, NDM, SIM, and DIM series), and group D (carbapenem hydrolyzing oxacillinases e.g. OXA 48 9 . NDM-1 producing bacteria are important because the gene encoding this enzyme is located on a transmissible plasmid (of varying size). It is also associated with other resistant determinants leading to extensive drug resistance which is usually exhibited by a majority of the NDM-1 producing enterobacteriaceae leaving only a few therapeutic options.
Therefore NDM-1 producing organisms are also named as "Super bugs". NDM-1 was first identified and reported in 2009 in Klebsiellae pneumoniae and Escherichia coli. It was isolated from Swedish patient of Indian origin who was previously hospitalized in New Delhi, India 10 . Indian investigators identified E. coli and Klebsiella species containing the gene for NDM-1 in multiple geographic regions in India, Pakistan and Bangladesh 11 .
The objective of this study was molecular characterization of the enzymatic mechanisms of resistance to β-lactam antibiotics in K. pneumoniae. A multiplex polymerase chain reaction (PCR) was setup for the detection of CTX-M, TEM and SHV genes. The reduced susceptibility to carbapenems by disk diffusion test prompted us to determine the molecular assay on these isolates to detect KPC and NDM-1 genes and also to analyze coexistence of AmpC producers among Klebsiella pneumoniae isolates at a tertiary care hospital.

Antimicrobial Susceptibility Testing
The susceptibilities of the different β-lactam and non-β-lactam antibiotics were tested and the results were interpreted as per the Clinical and Laboratory Standards Institute guidelines 12 . Escherichia coli ATCC 25,922 was used as a quality control. The antibiotics were procured from Hi Media, Mumbai, Maharashtra, India.

Minimum Inhibitory Concentration (MIC)
MIC determination was performed for all the isolates by agar dilution method (CLSI) 12 . Among NDM-1 producers, the MIC of meropenem and colistin ranged between 4-32 mg/mL and 0.25-256 mg/mL, respectively.

Detection of Extended Spectrum β-lactamase Producers
Isolates resistant or intermediately resistant to aztreonam, cefotaxime and/or ceftazidime 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 (30/10 µg) 12 . K. pneumoniae ATCC 700603 was used as the ESBL positive control and E. coli ATCC 25,922 was 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 metallocarbapenemase 13 .
MICs of meropenem and colistin (Sigma-Aldrich Corporation, St. Louis, US) were determined by the agar dilution method according to the guidelines from the CLSI 12 . The colistin breakpoint was evaluated using breakpoints for Enterobacteriaceae recommended by the European Committee on Antibiotic Susceptibility Testing. (Resistant: >2 μg/mL; sensitive: ≤2 μg/mL). K. pneumoniae ATCC 700603 was used as a quality control.

Phenotypic Detection of AmpC Production
AmpC phenotype was detected by means of combined disc method using cefoxitin disc (30 μg) (Hi-media Laboratories, Mumbai), alone and in combination with 400 μg of phenylboronic acid (BA) (Sigma-Aldrich, Fluka, China) 14 .

DNA Extraction and Amplification
Total DNA was extracted as described by Lee J H 15 . The extracted DNA was subjected to multiplex PCR for the detection of ESBLs and uniplex PCR for NDM and KPC genes 16,17,18 .
The Universal primers used for PCR amplification were as follows:

results
A total of 250 K. pneumoniae were isolated from different clinical samples. The distribution of these isolates in clinical specimens and in ICUs is shown in Graphs 1 and 2.
Out of 250 K. pneumoniae isolates, a total of 165 (66%) isolates were carbapenem resistant. The distribution of carbapenem resistant K. pneumoniae isolates in clinical samples and in ICUs is given in Graphs 5 and 6.

Prevalence of Carbapenemase Activity Based on Phenotypic tests
The phenotypic detection of carbapenemase production was performed as mentioned in Table 1.

Prevalence and Distribution of Carbapenemase Genes
Plasmid DNA was extracted from all the screened positive isolates 15 . The extracted plasmid DNA of each isolate was subjected to PCR detection of the bla NDM-1 and bla KPC genes by using target specific primers 17,18 , which is given in Table 2 17,18 . Agarose gel showing PCR amplified product of bla NDM-1 genes and susceptibility pattern of bla NDM-1 isolates is shown in Figure 1 and Graph 7.

dIscussIon
Klebsiella has been associated with different types of infections and one of the important aspects of Klebsiella associated infections is the emergence of multidrug resistant strains particularly those involved in nosocomial diseases.
In our study, the prevalence of ESBL production was 84% which was higher when compared to other studies. Sharma 22 . None of the isolates were positive for CTX M gene in our study.
K. pneumoniae have acquired carbapenemases, the enzymes capable of breaking down most β-lactams including carbapenems, and confer resistance to these drugs. Reports indicate that carbapenemase producing enterobacteriaceae isolates seem to be increasing in number in the last few years. In our study, we found 66% of the isolates were carbapenemase producers. Gupta et al., from All India Institute of Medical Sciences, New Delhi in 2006 found that resistance to meropenem was 6.9% 26 . Nagaraj et al observed 75% of the K. pneumoniae isolates were carbapenem resistant in their study 8 . Aseem et al stated that 35.3% of the isolates in their study were resistant to carbapenems 27 .
Genotypic analysis of these carbapenem resistant isolates revealed the prevalence of the bla NDM-1 as 9.7% and none of the isolates were positive for bla KPC in our study. The epidemiology of K. pneumoniae producing KPCs varies geographically. Globally, the highest rate of carbapenem resistance has been reported in Greece with 68% resistance (KPC, OXA-48-like, NDM) followed by India (NDM, OXA-48-like, KPC) and eastern Mediterranean regions (NDM, OXA-48-like) with 54% resistance. USA, China (KPC, NDM, OXA -48-like) and Africa (OXA-48-like, NDM) have low resistance rates with 11, 11 and 4% respectively 28 .
The predominant enzymatic mechanism of resistance in Europe is KPC followed by OXA-48-like and NDM, while in USA; it is KPC followed by NDM and minimal due to OXA-48-like 29 . Several studies in Spain showed that most carbapenemase-producing K. pneumoniae harbored OXA-48-like or class B carbapenemases, and that of KPC-producing K. pneumoniae was very low (2-3%) 30 8,30 .
However, in our study, none of the isolates were positive for bla KPC . This correlates with findings of Nagaraj et al. who has observed 75% of bla NDM but not detected bla KPC from any of the carbapenem resistant isolates 8 . A study from Vellore, by Veeraraghavan et al showed the coexpression of bla NDM and bla OXA48 in 28%, bla NDM 19%, bla OXA48 in 13% and bla KPC was absent among the carbapenem resistant isolates 33 . The endemic spread of NDMproducing K. pneumoniae has also been reported in the UK, which has close relationships with India and Pakistan 32 .
The Balkan states, the Arabian Peninsula, and North African countries have also been recently considered as an additional reservoir of NDM producers [32][33][34][35] . In the Arabian Peninsula, NDM-1 was the most frequently encountered carbapenemase 46.5%, followed by OXA-48-like carbapenemases 32.5% 36 . In India, a study by Deshpande et al, reported NDM-1 was the most common carbapenemase type detected and accounted for 75.2% of the carbapenemase-producing isolates 37 . In another study, an incidence of bla NDM-1 in a single K.pneumoniae isolate was reported from a surgical site infection 38 . In our study we found 9.7% of NDM-1 which is similar to a study from tertiary care hospital of Northeast India where they found 8.67% of NDM pocessing Klebsiella pneumoniae isolates 39 .
It was observed that the coexistence of bla gene along with NDM producers found to be 69% and 50% of SHV and TEM respectively. Previous studies from India have reported the coexistence of bla gene along with NDM producers [39][40][41] . Similar studies from abroad also showed the presence of bla genes along with NDM producers 42,43 .
We found 78.7% of the CRKP were positive for the production of one or more carbapenemase mechanisms phenotypically. It has been observed that among the screened CRKP, 9.7% isolates found to harbor bla NDM-1 gene. Rest of the CRKP isolates did not possess any of the resistant mechanisms tested. The important contributing factors leading to carbapenem resistance in these isolates might be due to the hyper production of ESBL, or other enzymatic mechanisms of carbapenem resistance like, IMP, VIM, and OXA, porin loss or efflux pumps 44,45 . conclusIon In conclusion, carbapenem resistant K. pneumoniae have been considered as one of the greatest threats to the global health care in this century. The prevalence of KPC gene i.e., bla KPC in carbapenem resistant isolates from our geographical area (South Western India) seems to be very less. Pathogens that produce carbapenemases along with an ESBL or AmpC β-lactamases are particularly challenging for clinicians and are a major threat worldwide. Moreover, the widespread dissemination of the new NDM-1 metallo-β-lactamase requires particular attention as the genetic background demonstrates extreme mobility and versatility. The dissemination of such plasmids between different clinically important bacterial species may lead to serious public health issues, as K. pneumoniae accounts for one of the important bacterial species in the dissemination of antibiotic resistance genes; particularly in hospital environments. Therefore, the early detection of the bla NDM-1 possessing K. pneumoniae isolates is necessary with any reduced susceptibility to the carbapenems. Timely intervention in the form of rapid detection, good infection control practices and judicious use of antibiotics will ensure that the spread of drug resistance among bacteria can be kept under control.