Prevalence and Molecular Epidemiology of Clinical Isolates of Escherichia coli and Klebsiella pneumoniae Harboring Extended-Spectrum Beta-Lactamase and Carbapenemase Genes in Bangladesh

Introduction

Enterobacteriaceae are Gram-negative bacteria that constitute normal intestinal flora and among the most common human pathogens of wide range of both community- and hospital-acquired infections. Escherichia coli and Klebsiella pneumoniae, the representative Enterobacteriaceae causing infectious diseases, have been noted for their acquisition of resistance to beta-lactam antibiotics and spread of the resistant clones. Increased prevalence of the beta-lactam-resistant isolates of these bacterial species raises morbidity and mortality with social costs, posing a global public health concern.^1,2

Beta-lactamases that mediate hydrolysis of beta-lactams are the most common cause of resistance to this class of antimicrobials and have been classified into four major groups: penicillinases, cephalosporinases, extended-spectrum beta-lactamases (ESBLs), and carbapenemases. ³ ESBLs hydrolyze all the beta-lactams except carbapenems and are inactivated by clavulanic acid. ESBL-producing Enterobacteriaceae are globally increasing, through multiple factors, including horizontal gene transfer of plasmids, successful bacterial clones, food animals, and human migration. ⁴ The most common types of ESBL are TEM, SHV, and CTX-M, among which CTX-M is dominant and has become disseminated globally, with CTX-M-14 and CTX-M-15 being the major genotypes. ⁴ Associated with the increased use of carbapenems against ESBL-producing bacteria, emergence and dissemination of carbapenem resistance in Gram-negative rods (GNR) have become remarkable, posing a public health concern. ³ Carbapenem resistance in Enterobacteriaceae is primarily mediated by carbapenemases, together with their synergistic activity with ESBLs or AmpC beta-lactamases and porin loss.^5,6 Carbapenemases identified in GNR have been classified into Ambler class A (KPC, IMI, and GES types), B (NDM, IMP, and VIM types), and D (OXA types) enzymes. ³ Among them, class B carbapenemases (metallo beta-lactamases) exhibit a broad spectrum of activity to all penicillins, cephalosporins, and carbapenems except for aztreonam. NDM has been known as an emerging class B carbapenemase since its first identification in 2008. NDM-producing GNR have been reported almost worldwide, although their prevalence varies in countries and study settings. ⁷

Prevalence of GNR producing ESBL and carbapenemase and their molecular epidemiological features have been studied mostly in developed countries, but limited information is available from developing countries. In Bangladesh, phenotypic and PCR-based detection of ESBLs in clinical isolates of GNR were reported in only some studies, showing variable prevalence.^8–11 Regarding carbapenemase, NDM-1 has been detected in isolates from infected patients^12–15 as well as in environmental specimens,^16,17 while identification of other bla_NDM allele (NDM-7) and bla_OXA-23 was described in our previous study on puerperal infections. ¹⁸ However, genetic types of ESBLs/carbapenemase genes and lineages (i.e., clone) of E. coli and K. pneumoniae clinical isolates with these genes have scarcely been analyzed.

Present study was conducted in Mymensingh, north-central region in Bangladesh, to investigate drug resistance, prevalence and genetic characteristics of recent clinical isolates of E. coli and K. pneumoniae harboring ESBL, and/or carbapenemase genes. We report in this study the first detection of bla_NDM-5 in E. coli and bla_OXA-181 in K. pneumoniae in Bangladesh.

Materials and Methods

Results

Among a total of 233 E. coli isolates, dominant phylogenetic group was B2 (33.5%; 71 and 7 isolates of B2a and B2b, respectively), followed by group A (29.6%) and group D (26.6%). Resistance rates to 18 antimicrobials are summarized in Table 1. E. coli showed significantly higher resistance rates to piperacillin, cephalosporins, and some other antimicrobials than K. pneumoniae (p < 0.01). Resistance rates to cefotiam, cefotaxime, and ceftazidime were 60–76% in E. coli and 35–60% in K. pneumoniae. Groups B2 and D E. coli showed higher resistance rates to cefotaxime (85% and 82%, respectively) than groups A and B1 isolates (65% and 58%, respectively) (p < 0.01), which was similarly found for ceftazidime (p < 0.05). Meropenem-resistance was detected in 8.2% of E. coli (19 isolates) and 3.5% of K. pneumoniae isolates (5 isolates), which were all susceptible to colistin (MIC, 1 μg/mL) and tigecycline (MIC, 0.25–0.5 μg/mL). All the E. coli and K. pneumoniae isolates were susceptible to fosfomycin.

Prevalence of individual beta-lactamase genes are shown in Table 2. While detection rate of bla_TEM was similar between E. coli and K. pneumoniae (41.6% and 49.3%, respectively), bla_SHV and bla_CTX-M were detected at significantly higher rates (p < 0.05) in K. pneumoniae (26.8% and 51.4%, respectively) than in E. coli (1.3% and 34.3%, respectively). Among E. coli, bla_CTX-M was the most prevalent in groups D (41.9%) and B2 (41.0%). Carbapenemase genes were detected in nine and three isolates of E. coli (3.9%) and K. pneumoniae (2.1%), respectively, and identified as genes encoding NDM-1, -5, and 7, and OXA-181. bla_NDM-1 was possessed by groups A and B2b E. coli (one isolate each) and K. pneumoniae (two isolates). bla_NDM-5 was harbored in groups A and B2a E. coli (two isolates each), while bla_NDM-7 in groups B1 and D (two and one isolates, respectively). OXA-181 gene was identified in a K. pneumoniae isolate. Prevalence of AmpC gene (CIT-family) was low in both GNR species (2.9% in total, 11/375). O25b allele was detected in only phylogenetic group B2a isolates (32%; 23/71), all of which showed resistance to levofloxacin (Supplementary Table S4). Among the isolates with O25b allele, fimH30 subtype was the most common (74%, 17/23), while other four subtypes (fimH24, fimH31, fimH54, and fimH191) were identified. PMQR determinant aac-6'-Ib-cr was prevalent in both E. coli (30%) and K. pneumoniae (38.7%), with phylogenetic group B2a of E. coli showing significantly higher positive rate (49.3%) than other groups (p < 0.01).

Representative 26 E. coli isolates from the four phylogenetic groups and 20 K. pneumoniae isolates harboring ESBL/carbapenemase genes were selected for further genetic analysis of beta-lactamase and other traits, including clonal lineage of bacteria (Tables 3 and 4). TEM gene in all the isolates examined was identified as bla_TEM-1. While some isolates of the two GNR species carried bla_SHV-1, bla_SHV-28 was detected in an E. coli isolate and SHV-11, -12, -27, -201, and -202 genes were identified in K. pneumoniae. SHV-201 and -202 were newly identified in the present study and have two and one amino acid substitution compared with SHV-1 sequence, respectively, at positions that are different from those commonly reported for SHV-type ESBLs ⁴⁴ (Supplementary Fig. S1). CTX-M genes were typed as bla_CTX-M-15 in all groups of E. coli and K. pneumoniae, with only exception of bla_CTX-M-27 in a B2a-ST131 isolate. B2a-ST131 isolate EK586 possessed bla_CTX-M-15 and aac6'-Ib-cr, harboring many virulence factor genes. Four D-ST405 isolates possessed bla_CTX-M-15 and eight or more virulence factors, and showed resistance to all the antimicrobials tested except for fosfomycin. In K. pneumoniae, bla_CTX-M-15 was found in 12 STs, including ST11, ST101, and ST395, with ST572 being the most common (four isolates).

Carbapenemase NDM-1 gene was found in A-ST2104, B2b-ST73 E. coli, and ST11 and ST1322 K. pneumoniae isolates. E. coli isolates possessing bla_NDM-5 were classified into A-ST167, B2a-ST2659-SLV (single-locus variant), and B2a-ST38-DVL (double-locus variant), while bla_NDM-7 into B1-ST101, B1-ST224, and D-ST6682. OXA-181 gene was detected in a K. pneumoniae isolate belonging to ST43. All the bla_NDM-positive E. coli isolates carried also bla_CTX-M-15, except for a group B1 isolate. Isolates having eight or more virulence factors were detected mostly in phylogenetic groups B2a and D, irrespective of the presence of beta-lactamase type. AmpC beta-lactamase genes (bla_CMY-2, bla_CMY-27, bla_CMY-42, and bla_CMY-160) were detected in eight E. coli isolates harboring mostly bla_CTX-M-15 and PMQR determinants (aac6'-Ib-cr, qnrB, qnrS, qepA, and oqxAB). CMY-160 was a newly identified CMY allele belonging to CMY-2 group (Supplementary Fig. S2) in a group D-ST466-DLV isolate. While CMY-160 is close to CMY-42 with a single amino acid difference, both CMY enzymes have a common amino acid substitution (V231S) compared with CMY-2 sequence (Supplementary Fig. S3). More than half (11 isolates) of the K. pneumoniae, including four ST572 isolates, possessed oqxAB, although prevalence of virulence factors was generally the same among isolates.

Discussion

The present study first elucidated overall prevalence of ESBL genes represented by bla_CTX-M and carbapenemase genes in recent clinical isolates of E. coli and K. pneumoniae from extraintestinal infections in Bangladesh. TEM type beta-lactamases genetically analyzed were all identified as TEM-1, although nearly half isolates of the two GNR species were bla_TEM-positive. While several different alleles of bla_SHV were detected, ESBLs were only SHV-12 and -27 in two K. pneumoniae isolates among 20 isolates examined. Therefore, major ESBL was considered as CTX-M-1 group, which was present in 34% of E. coli and 51% of K. pneumoniae. These detection rates appear to be comparable to a report of ESBL determined by phenotypic method for hospital isolates in Dhaka (43% and 40% for E. coli and K. pneumoniae, respectively). ⁸ Lower rates (16–19%) ⁹ as well as higher rates with >70% ¹¹ in GNR described in previous studies in Bangladesh may be possibly relevant to specimen selection bias, numbers of isolates, and the use of nongenetical methods. Our study suggested the persistence of ESBL-positive GNR, particularly high prevalence of bla_CTX-M in K. pneumoniae in Bangladesh.

ST131 E. coli, characterized as phylogenetic group B2 with serogroup O25b, has been described as a pandemic clone responsible for high incidence of extraintestinal pathogenic infections and dissemination of multidrug resistance producing ESBL, with other globally distributed lineages as ST38, ST405, and ST648.^31,45 In our present study, B2-O25b allele accounted for about 10% of all E. coli isolates (23/233) and 29% of bla_CTX-M-positive E. coli (23/80), which was similar rate (26%) reported among isolates from puerperal infections in Bangladesh in 2010–2012. ¹⁸ Furthermore, presence of B2-ST131 isolates with bla_CTX-M-15 or bla_CTX-M-27 was confirmed in the present study. These findings suggested that prevalence of B2-ST131 E. coli clone might have been sustained at certain level, while this clone has not yet been predominant among ESBL-producing E. coli. It was of note in our study that all the O25b isolates were resistant to levofloxacin and assigned to the dominant fimH30 subtype and other minor types. ST131 subclone with fimH30, which was revealed to be significantly associated with fluoroquinolone resistance, has spread rapidly in the United States since 2000. ⁴¹ fimH30 subclone is suggested to have been prevalent also in Bangladesh, showing a need to survey fluoroquinolone-resistant fimH30 and other minor fimH subtypes among ST131 E. coli.

Frequent detection of qnrS among isolates with bla_CTX-M-15 suggested dissemination of plasmids containing these genes as reported previously. ⁴⁶ Notably, CTX-M-27 gene was first identified in Bangladesh in this study. Increasing trend of bla_CTX-M-27 has been described in Asia and Europe,^47,48 associated with identification of ST131-O25b E. coli carrying this gene, which poses concern for global spread. ⁴⁹ Therefore, presence of ST131 E. coli with bla_CTX-M-27 as well as bla_CTX-M-15 in Bangladesh warrants more attention.

In the present study, phylogenetic group B2b, which is a variant clone lacking chuA, was revealed to be a minor group (9%) among B2 E. coli. However, most isolates (5/7) harbored bla_CTX-M-15, and two isolates analyzed (ST73 and ST131) showed multidrug resistance having qnrS with either bla_NDM-1 or aac-6'-Ib-cr. Thus, further epidemiological and genetic study may be necessary to understand their significance as drug-resistant subgroup of E. coli. It was remarkable that phylogenetic group D (27% of all E. coli isolates) showed bla_CTX-M-positive rate (42%) as high as that of group B2 (41%), and four group D isolates were classified into ST405 with bla_CTX-M-15 exhibiting multidrug resistance. Because it is suggested that prevalence of the global lineage ST405 appears to be comparable to ST131, further study and attention should be focused on this clone.

Present study revealed prevalence of bla_NDM, including three genotypes in E. coli and K. pneumoniae (3.9% and 1.4%, respectively; overall rate, 2.9%), and showed higher frequencies of NDM-5 and -7 genes than bla_NDM-1 in E. coli. Since the first identification of NDM-1 in 2008, worldwide attention was attracted to this carbapenemase because of its rapid dissemination among Enterobacteriaceae and Acinetobacter spp. clinical isolates, as well as those colonizing humans and contaminating environments.^7,16 Main reservoir of NDM producers is Indian subcontinent, while secondary reservoir is considered the Balkan regions and the Middle East. ⁷ In Bangladesh, NDM-1 was first identified in K. pneumoniae in 2008, ¹² thereafter some reports described detection of bla_NDM-1 and other carbapenemase genes (bla_OXA, bla_KPC, bla_VIM, and bla_IMP) in some GNR species, with bla_NDM-1 being dominant.^14,15 While detection rate of bla_NDM-1 in GNR (3.5%) in a study in 2010¹² seems to be comparable to that in our present study, dominant species was described as K. pneumoniae. Similarly, K. pneumoniae was the most frequently isolated from environmental samples (wastewater and environmental water) among the bla_NDM-1-producing GNR in Dhaka.^16,17 In contrast, our results indicated higher prevalence of NDM genes in E. coli than K. pneumoniae, which may underscore the significance of E. coli as an NDM-producing bacterium in extraintestinal infections in Bangladesh. In addition, NDM genes in nine E. coli isolates were detected in eight different STs, suggesting the spread of bla_NDM to diverse lineages of E. coli.

In the present study, bla_NDM-5 was first identified in Bangladesh, and bla_NDM-7 was described as the second report after our previous study on isolates from puerperal infections. ¹³ Although NDM-1 has been the most dominant in India and other countries among the 17 NDM variants identified to date, NDM-5 has been increasingly detected since 2011 in South Asia,^50,51 East Asia,^52,53 Australia, ⁵⁴ United States, ⁵⁵ Europe,^56,57 and North Africa, ⁵⁸ showing comparable rate to NDM-1 or the second most NDM-type in some reports.^50,59 E. coli harboring bla_NDM-5 has been genotyped as at least 13 STs to date, among which ST167 appears to be the most common. ⁶⁰ In the present study, four bla_NDM-5-carrying E. coli isolates belonged to three STs, including the ST167, suggesting its efficient dissemination. ST167 E. coli is also known as first identified NDM-7-producer that was isolated in a patient having a travel history to Myanmar. ⁶¹ Subsequently, detection of Enterobacteriaceae harboring bla_NDM-7 has been reported in many Asian countries and United States.^51,62,63 While bla_NDM-7 has been detected in E. coli belonging to at least 12 STs, ⁶² three STs (ST101, ST224, and ST6682) identified in the present study have not yet been reported, suggesting spread of this gene to more lineages. Both NDM-5 and NDM-7 have a common amino acid substitution at position 154 (Met→Leu) ⁷ from NDM-1, conferring increased hydrolytic activity toward carbapenems,^60,64 which may be a factor for selective spread of E. coli with the variant enzymes NDM-5 and -7.

Fifteen STs were identified among the 20 isolates of K. pneumoniae in the present study, and included ST11, ST101, and ST395 that are considered as pandemic clones. ⁶⁵ It was remarkable that OXA-181, a variant of class D beta-lactamase OXA-48, was first confirmed in a clinical isolate of K. pneumoniae (ST43) in Bangladesh in the present study. OXA-181 was first identified in India in 2007 and has been found in Enterobacteriaceae almost worldwide and frequently detected in isolates from patients with travel history to Indian subcontinent. ⁶⁶ The OXA-48-like genes have been detected mainly in K pneumoniae and distributed to numerous STs that include dominant types (ST11, ST14, ST101, ST147, and ST231) and also ST43 as one of the minor types. ⁶⁷ In our study, ST11 and ST101 K. pneumoniae were confirmed, thus dissemination of OXA-48-like gene to these clones may be a potential concern.

In summary, we clarified prevalence of ESBL and carbapenemase genes among E. coli and K. pneumoniae, and their molecular epidemiological traits in Bangladesh. The presence of three bla_NDM types and bla_OXA-181 in various clones highlights the need for further surveillance of beta-lactamase genes in GNR to control their dissemination.