Molecular characterization of extended-spectrum beta-lactamases and carbapenemases producing Enterobacteriaceae isolated from North Eastern region of India

INTRODUCTION

The increasing rate of antimicrobial resistance (AMR) has become a severe threat to public health globally. The emergence of extended-spectrum β-lactamases (ESBLs) producing Enterobacteriaceae (ESBL-E) and carbapenemase-producing Enterobacteriaceae (CPE) is responsible for higher morbidity and mortality rates worldwide.^[1] The World Health Organization included ESBL-E and CPE in the prioritized pathogen group.^[2] ESBLs are plasmid mediated and capable of hydrolyzing the amide bond of four membered β-lactamase ring, inactivates the wide variety of beta-lactams including penicillins, cephalosporins, and monobactams except for cephamycins and carbapenems but are inhibited by clavulanic acid.^[3] ESBLs are mostly effective against third-generation cephalosporins,including ceftazidime, ceftriaxone, and cefotaxime. Temoniera (TEM), sulfhydryl reagent variable (SHV) and Cefotaxime-Munich (CFX-M) are the most common families among ESBLs and are associated with the nosocomial infections outbreak.^[4] Several studies have also reported that the genes encoding ESBLs were frequently located on large plasmids, which also carry genes mediating resistance to aminoglycosides and fluoroquinolones.^[5] The increasing rate of ESBL-E strains were treated and controlled using carbapenem antibiotics,including meropenem, imipenem, ertapenem, and doripenem.^[6] However, among Enterobacteriaceae, the emergence of an increasing rate of carbapenem resistance in clinical settings is worrisome, and it is alarming.

Carbapenems are β-lactam antibiotics that have the potential to inhibit transpeptidases enzymes (penicillin-binding proteins), prevent peptidoglycan synthesis, and cause lytic cell death.^[6] Carbapenem resistance is mediated by the production of various carbapenemases enzymes. Carbapenemases are β-lactamases enzymes differentiated into three major classes according to Ambler classification (A, B, and D) based on the hydrolytic mechanisms at their active sites. Carbapenemases hydrolyses β-lactam antibiotics,including penicillins, cephalosporins, monobactams, and carbapenems. Class A carbapenemases include Klebsiella pneumoniae carbapenemase (KPC), which could be inhibited by clavulanic acid. The Metallo-β-lactamases are blaNDM (New Delhi metallo-β-lactamases), blaIMP (imipenemase), and blaVIM(Verona imipenemase) belonging to class B. The class D carbapenemases are referred as Oxacillinase (OXA)- type carbapenemases.^[7] So far, the therapeutic options for Carbapenem-resistant Enterobacteriaceae (CRE) infections remain challenging and are very limited. However, recent studies have reported that novel β-lactamase inhibitor combinations may play an important role in providing new treatment options against CRE infections.^[6] Hence, it is important for clinicians and researchers to screen and highlight the carbapenemase-producing bacteria in clinical settings. Although there are fewer studies on CRE in the North Eastern region of India, Tripura, Nagaland, Meghalaya, and Mizoram have reported <5% of carbapenem resistance prevalence due to poor development in healthcare.^[8] Hence, it is important to clinicians in various regions of North East India for earlier detection of ESBLs and carbapenemases-production in Enterobacteriaceae clinical strains to decide or choose appropriate therapeutic options. Based on this knowledge and background, we aimed to highlight the emergence of ESBLs and carbapenemases production in multidrug-resistant Enterobacteriaceae (MDRE) strains isolated from patients hospitalized in a tertiary care hospital in the North Eastern region of India.

MATERIALS AND METHODS

A total of 210 different MDRE isolates were collected from patients in outpatient departments, different wards, and intensive care units of a tertiary care hospital in Gangtok, Sikkim, for a period from January 2019 to December 2021. This study was reviewed and approved by the Institutional Ethics Committee (IEC), SMIMS (SMIMS/IEC/2018–033). The MDRE strains were found to be resistant to the antibiotics including Ceftriaxone (30 µg), Ciprofloxacin (5 µg), Gentamicin (10 µg), and Amikacin (30 µg). Escherichia coli (E. coli) American type culture collection (ATCC) 25922 and Klebsiella pneumoniae (K. pneumoniae) ATCC 700603 were used as control strains. In addition, out of the total of 210 MDRE, 53 (25.26%), and 55 (26.2%) strains were found to be Imipenem (10 µg) and Meropenem (10 µg) resistant, respectively.^[9] The antimicrobial susceptibility testing assays were carried out by adopting Clinical and Laboratory Standards Institute (CLSI) the described Kirby Bauer disk diffusion method.

RESULTS

A total of 210 MDRE strains were included in this study. All the study isolates were found to be resistant to third-generation cephalosporins, aminoglycosides, and fluoroquinolones. Of the total of 210 MDRE, 39 (18.58%) strains were found to be carbapenemase producer and 153 (72.86%) strains were producing ESBLs phenotypically [Table 3]. Most of the strains were collected from urine (90, 42.86%), followed by sputum (29, 13.81%), pus (13, 6.19%), endotracheal aspirate (12, 5.71%), and blood (9, 4.29%).

ESBLs production was found to be predominant in E. coli (96, 45.71%), followed by K. pneumoniae (45, 21.43%), Morganella morganii (5, 2.38%), Enterobacter cloacae (3, 1.43%), Serratia marcescens (3, 1.43%), and Providencia rettgeri (1, 0.48%). mPCR assays revealed that genes encoding ESBLs were detected in 55.24% (116) of MDRE strains, highlighting that blaTEM (26.67%, 56) was predominant, followed by blaCTX-M (19.52%, 41) and blaSHV (9.05%, 19) [Table 4]. Most of the ESBLs encoding genes were observed in E. coli (blaTEM, 32 (15.24%), blaCTX-M (21, 10%), and blaSHV (12, 5.71%)) and K. pneumoniae (blaTEM, 22 (10.48%), blaCTX-M (20, 9.52%) and blaSHV (7, 3.3%)). Of the total of 55 CRE, 39 (18.58%) strains were found to be producing carbapenemases phenotypically. Among 55 CRE, K. pneumoniae (27, 12.86%) was predominant followed by E. coli (21, 10%), E. cloacae (2, 0.95%), Serratia marcescens (S. marcescens) (2, 0.95%), Providencia rettgeri (P. rettgeri) (1, 0.48%), Morganella morganii (M. morganii) (1, 0.48%), Proteus vulgaris (1, 0.48%), and Shigella sonnei. (1, 0.48%) [Table 5]. Among 39 CRE, blaNDM-1 was observed in 17 (8.10%) strains, blaOXA-48 was observed in 6 (2.86%), and blaVIM was observed in 3 (1.43%). Furthermore, 13 (6.19%) CRE strains were found to be co-occurrence of both blaOXA-48 and blaVIM [Table 4]. We observed that most of the carbapenemases producing strains were found to be K. pneumoniae (22, 10.4%) followed by E. coli (17, 8.0%). Carbapenemases production was not observed in other Enterobacteriaceae species. In addition, efflux pump activity was observed in 5 (2.3%) of CRE isolates K. pneumoniae (3, 1.4%) and E. coli (2, 0.9%).

DISCUSSION

Enterobacteriaceae infections, with the occurrence of multidrug resistance mechanisms, have become an emerging threat in hospital settings. ESBLs and CPE have become a very serious problem, and remain a challenge for diagnostics and therapeutic management. In this investigation, we described the prevalence of ESBLs and carbapenemases production in MDRE strains isolated from North Eastern region of India by phenotypic and genotypic methods. The present study highlighted that 72.86% and 18.58% of MDRE isolates were found to be producing ESBLs and carbapenemases, respectively. This displays an alarming and worrisome scenario. One possible reason could be the upsurge in the pharmaceutical industries in this region, especially in Sikkim, which could have contributed to a higher rate of antibiotic resistance due to the amount of waste reaching the various waterways that may indirectly act as a continuous source of AMR.^[9,13] Other associated reasons could be the increasing rate of diseases, inadequate hospitals or healthcare centers, lack of appropriate diagnostic methods, poor infection control practices, and the affinity of clinicians with the empirical treatment practices may have further supported the global crisis of AMR.^[14] The emergence of ESBLs production in Enterobacteriaceae has become a serious concern worldwide,including India, China, Pakistan, Korea, and Japan.^[15] In India, the prevalence rate of ESBLs ranges from 60 to 80%^[16],and in our study, the magnitude of ESBLs producing MDRE was found to be 72.86%. We also highlighted that the majority of ESBLs producing strains were E. coli (96, 45.71%) followed by K. pneumoniae (45, 21.43%). Similar to our study, various reports from several parts of India highlighted the predominance of ESBLs production in E. coli and K. pneumoniae. In India, a multi-centric study reported that ESBLs production was seen in 33% in E. coli and 42% in K. pneumoniae.^[17] The incidence of ESBLs fluctuates extensively between geographical locations. Low occurrence rates have been reported in USA, Europe, and North America,^[18,19] whereas high rates are generally detected in Asian countries, South America,^[20] and some African countries.^[21] Compared with our investigation, the incidence of ESBLs producing Enterobacteriaceae in Europe is lower; 0.7% in Austria, 23.8% in Turkey,^[22] and 6.3% in Italy.^[23] The difference might be due to infection control strategies in those countries. Other possible reasons which can attribute to the higher incidence of ESBLs and their spread could be non-prescription antimicrobial use, self-medication, poor hygiene, high burden of infectious diseases, consumption of counterfeit drugs, and lack of proper implementation of AMR detection systems.^[24-26] Therefore, it further confirms the spread of such bacteria in this region. In this regard, a conceivable account for such a high magnitude of ESBLs incidence could be due to the selective pressure produced by the significant use of β-lactam antibiotics in this region, where they are commonly anticipated as the first line of therapy for bacterial infections caused by Enterobacteriaceae.^[27] Infectious Diseases Society of America has listed E. coli and K. pneumoniae among six pathogens in urgent need of new drugs to combat the development of drug resistance.^[28] A study from Ethiopia reported the prominence of E. coli 228 (53.5%) and K. pneumoniae 103 (24.1%) among ESBLs producing MDRE.^[29] Mahamat et al. also reported that most E. coli (63.8%) were found to be ESBL-producers followed by K. pneumoniae (21.2%).^[27] In addition, our results highlighted that the ESBLs producing MDRE strains were mainly associated with the occurrence of blaTEM (26.67%, 56), followed by blaCTX-M (19.52%, 41) and blaSHV (9.05%, 19). Similarly, Verma et al. highlighted that blaTEM was most predominant in both E. coli and K. pneumoniae, followed by blaCTX-M and blaSHV.^[30] However, a previous study from North East India reported a higher prevalence of blaSHV (63.4%), followed by blaCTX-M (60.86%) and blaTEM (54.3%).^[31] The spread of mobile genetic elements, mostly conjugative plasmids belonging to classic incompatibility groups, and the diffusion of specific clones have been accountable for the upsurge in ESBL-producing isolates and for the spread of TEM, SHV, and CTX-M, in particular.^[32] CTX-M is being considered endemic in various countries and is swiftly disseminating among different Enterobacteriaceae species.^[27] India, being a densely populated country with meager sanitation and drinking water glitches, denotes the leading reservoir of CTX-M ESBL genes alongside China.^[31] In our study, it was highlighted that most of the ESBLs strains were isolated from urine samples. Similar to this, a study by Shashwati et al. from North India observed that the majority of ESBL-producing strains were obtained from urine samples. In addition, they also revealed that blaTEM gene was predominantly observed in E. coli isolates.^[33] Ravikant et al. observed that blaSHV (63.04%) is predominant in ESBLs producing E. coli followed by blaTEM (60.86%) and blaCTX-M (54.34%).^[31] Urinary tract infection (UTI) is considered as the most recurrent bacterial infection throughout the world in patients with nosocomial and community-acquired infections,and Enterobacteriaceae (primarily E. coli and K. pneumoniae) are commonly the causative agent.^[9,27] In this context, it is pertinent to state that E. coli represents the leading pathogen isolated from urine specimen worldwide that is responsible for UTI infection.^[31] The dissemination of ESBLs compromises the potential of broad-spectrum antibiotics resulting in main therapeutic complications with a substantial impact on the outcomes for patients.^[34] Carbapenem has become the last resort and first-line empirical treatment against ESBLs-producing MDRE. The increasing usage of carbapenem against any known or unknown case of hospital infections leads to the emergence of CRE. The emergence and rapid spread of CRE has become a serious threat in hospital settings. This is worrisome and alarming. In the present study, we highlighted that 18.58% of MDRE was found to be carbapenem-resistance. Among 18.58% of CRE, K. pneumoniae was predominant, followed by E. coli, E. cloacae, S. marcescens, P. rettgeri, M. morganii, P. vulgaris, and S. sonnei. The emergence of novel β-lactamases with direct carbapenem-hydrolyzing activity has contributed to an upsurge in the incidence of CRE. Thus, CRE becomes problematic given the rate with which Enterobacteriaceae cause infections, the high mortality linked with infections caused by CRE, and the potential for extensive spread of carbapenem resistance through mobile genetic elements.^[35] In 2017 and 2018, Modi et al. reported that 34.74% and 29.34% of Enterobacteriaceae strains were found to be carbapenem-resistant, respectively,^[36] which is a much higher percentage compared to our study. Enterobacteriaceae being the popular cause of health care as well as community infections, raises the likelihood of transmission of CRE into the community. These factors, combined with the finite therapeutic options available to treat patients infected with these bacteria, have made CRE troublesome.^[35] A study from North Eastern India by Ralte et al. reported that about 11.3% of Gram-negative strains were found to be carbapenem-resistant in which K. pneumoniae and E. coli were found to be predominant.^[8] Another study from North Eastern India by Chellapandi et al. also revealed the occurrence of 5% of carbapenem resistance, especially in the Enterobacteriaceae.^[37] In our study, it is worth noting that several drug resistance mechanisms have been incorporated in the CRE, including carbapenemases production and efflux pump activity. Our study revealed that blaNDM-1 and blaOXA-48 were mostly detected in CRE strains in our hospital environment and are spreading rapidly, with less occurrence of blaVIM. Several studies from India also reported that blaNDM and blaOXA-48 were the most common carbapenemase genes reported in several states of India. A study by Sharma et al. revealed that about 48% and 19% of E. coli strains were found to be harboring with blaNDM and blaOXA-48, respectively. In addition, 27% and 36% of K. pneumoniae strains were found to be harboring blaNDM and blaOXA-48, respectively.^[38] A study from Mohanty et al. reported that blaNDM (65.6%) and blaOXA-48 (24.7%) were observed among 93 (24.03%) CRE isolates.^[39] In contrast, a study from China by Han et al. observed that blaKPC-2 (51.6%) producing Enterobacteriaceae strains were mostly isolated, followed by blaNDM (35.7%) and blaOXA-48 (7.3%).^[40] Hence, increasing spread of blaNDM and blaOXA-48 like carbapenemases in MDRE is worrisome as they are progressively highlighted with nosocomial infection outbreaks across the world. The epidemiological spreading of carbapenemase producers fluctuates enormously. In Asian subcontinents, blaOXA-48-like and blaNDM-types are more common, while blaKPC and other blaOXA-types are recorded quite often in the European countries.^[8] In India, the incidence of carbapenem-resistant bacterial infections is widespread, especially in the Southern and Northern regions, where the population density is high.^[12] The finding of the present study, wherein the occurrence of 18.58% CRE previously unknown in Sikkim, exemplifies the emergence of CRE in this region that forms an indicator of disseminating resistance to the northeastern parts of India, where the population is not dense. Overuse of antibiotics in healthcare settings leads to selection pressure for resistant strains, whereas lack of early detection of infection with CRE and poor infection control practices promote its spread. Infections with CRE are tough to treat and the limited treatment options as well as the costliness, impose extra financial burden on the patients. Under these circumstances, it becomes important to curb the rise of CRE by formulating and implementing antimicrobial policy that is based on the local antibiogram as well as ensure adherence to infection control practices. In addition to carbapenemases production, we also observed that about 5 (2.3%) of CRE isolates (K. pneumonia (3, 1.4%) and E. coli (2, 0.9%) were found to be exhibiting efflux pump activity. Several studies from India have reported that the efflux pump inhibition has played an important role in decreasing of carbapenems MIC to K. pneumonia and E. coli.^[41]

CONCLUSIONS

Our results highlighted the higher prevalence and rapid emergence of ESBLs and carbapenemases production in clinical MDRE strains isolated from the North Eastern region. The high MDRE rate detected in such a small populated and remote region highlights a peak of danger in bigger populated cities of India. This situation is worrisome. Our study also highlighted the increasing spread of NDM and OXA-48 with co-occurrence of VIM and efflux pump activity among clinical isolates in hospital environment, which remains a challenge for clinicians to choose appropriate treatment against MDRE infections. There are very limited cases of prevalence studies about drug resistance investigated in the North Eastern part of India, which is still underdeveloped in healthcare sectors compared to other parts of India. This finding further reiterates the imperative need for rational use of antibiotics in hospital settings and to develop empirical treatment strategies. In this regard, the present study may contribute to the development of new strategies to control the spread of ESBLs and carbapenemases production in India, including the North Eastern region.