Prevalence of Extended-Spectrum Beta-Lactamase/Carbapenemase Genes and Quinolone-Resistance Determinants in Klebsiella pneumoniae Clinical Isolates from Respiratory Infections in Myanmar

Introduction

K lebsiella pneumoniae , a member of Enterobacterales, causes a wide spectrum of infections in humans, including respiratory tract infections, urinary tract infections, and bloodstream infections in hospitalized or immunocompromised patients. In recent years, multidrug-resistant K. pneumoniae is increasing as an important nosocomial pathogen associated with high morbidity and mortality due to limited options of treatment, posing a global public health concern. ¹ Particularly, resistance to beta-lactams and fluoroquinolones (FQs) has been considered noteworthy for this bacterium.^1,2

Beta-lactamases, that is, the most common cause of resistance to beta-lactams, have been classified into four major groups; penicillinases, cephalosporinases, extended-spectrum beta-lactamases (ESBLs), and carbapenemases. ESBLs consist of genetic variants of TEM and SHV and CTX-M, among which CTX-M is dominant globally. ³ In addition to ESBL, plasmid-mediated AmpC (pAmpC) beta-lactamases are also responsible for resistance to broad-spectrum cephalosporins among Enterobacterales. ⁴ Carbapenem resistance in Enterobacterales is primarily mediated by carbapenemases, which have been classified into Ambler class A (KPC, IMI, GES types, etc.), B (NDM, IMP, VIM types, etc.), and D (OXA types) enzymes. ⁵ Among them, class B carbapenemases (metallo beta-lactamases) exhibit a broad spectrum of activity to all penicillins, cephalosporins, and carbapenems except for aztreonam. NDM is recognized as an emerging class B carbapenemase, and NDM-producing Gram-negative bacteria have been reported almost worldwide. ⁶

FQs, that is, new quinolones, are one of the main therapeutic choices for infections caused by bacteria belonging to Enterobacterales. They are the most frequently used antimicrobial agents worldwide due to their broad-spectrum antimicrobial activity. ⁷ However, as their use becomes widespread, FQ resistance has occurred and increased among several clinically important bacteria, including K. pneumoniae.^8,9 FQ resistance is primarily caused by occurrence of mutation(s) in GyrA subunit of DNA gyrase and ParC subunit of topoisomerase IV. ¹⁰ In addition, plasmid-mediated quinolone resistance (PMQR) determinants represented by Qnr, AAC(6′)-Ib-cr, and QepAB/OqxAB confer reduced susceptibility to FQs. ¹¹ It was described in some reports that previous FQ use is an independent risk factor for infections caused by ESBL-producing Escherichia coli, ¹² as well as carbapenem-resistant K. pneumoniae infection. ¹³ This may be partly explained by the fact that multidrug resistance phenotype is possible to be conferred by a single mechanism such as multidrug efflux pumps, which is activated by FQ. ¹⁴ Moreover, when PMQR determinants coexist with ESBLs or carbapenemase genes on the same plasmid that have cross-species/genera transferability, ¹⁵ bacterial strains with resistance to broad spectrum beta-lactams could spread by selective pressure exerted by FQ. Accordingly, it is important to monitor prevalence of FQ resistance along with beta-lactam resistance. However, in Myanmar, such study has scarcely been performed for clinical isolates of K. pneumoniae, while there is only a study on E. coli. ¹⁶ In the present study, the prevalence of beta-lactamases/carbapenemase genes, quinolone-resistance determining region (QRDR) mutations, and PMQR determinants was investigated for clinical K. pneumoniae isolates derived from patients with respiratory tract infection.

Materials and Methods

Results

Among a total of 191 K. pneumoniae isolates, resistance rates to antimicrobials in the decreasing order are as follows: cefuroxime, 75.4%; trimethoprim–sulfamethoxazole, 72.3%; amoxicillin–clavulanic acid, 70.2%; ciprofloxacin, 62.3%; LVX, 62.3%; tetracycline, 61.3%; ceftriaxone, 60.2%; ceftazidime, 60.2%; cefotaxime, 56.5%; ampicillin/sulbactam, 56%; cefepime, 53.9%; piperacillin–tazobactam 52.4%; gentamicin, 45%; aztreonam, 38.2%; cefoperazone/sulbactam, 32.5%; amikacin, 21.5%; meropenem, 14.7%; imipenem, 14.7%; and colistin, 4.7%.

Beta-lactamase genes bla_TEM, bla_SHV, bla_CTX-M, and bla_NDM were identified with detection rates 45.5%, 36.1%, 33.0%, and 7.3%, respectively (Table 1). These beta-lactamase genes were detected mostly within LVX-resistant isolates (n = 119), while LVX-susceptible isolates contained only three isolates with bla_TEM. Among the FQ-resistant isolates, prevalence of bla_CTX-M was 52.9%. According to the genotyping of the beta-lactamase genes, ESBL gene was mostly bla_CTX-M, with CTX-M-15 being dominant. In contrast, except for SHV-27, all the TEM and SHV genes were assigned to non-ESBL types, including dominant types TEM-1 and SHV-28. NDM gene was identified in 14 isolates and assigned into three types, NDM-5 (9 isolates), NDM-1 (4 isolates), and NDM-7 (an isolate), while other carbapenemase genes (bla_VIM, bla_IMP, bla_KPC, and bla_OXA-48) were not detected. No isolates were positive for pAmpC gene. Although nine isolates, including four isolates with NDM-5 and two isolates with NDM-1, showed colistin MIC 4 μg/mL that represent resistance, mcr genes (mcr-1, mcr-2, mcr-3) were not detected in any isolates.

Among the FQ-resistant isolates (n = 119), 106 isolates (89.1%) harbored any of the five PMQR genes (aac-(6)′-Ib-cr, qnrB, qnrS, oqxAB, and qepA). aac-6′-Ib-cr was the most commonly detected (71.4%), followed by qnrB (59.7%), oqxAB (37%), and qnrS (21.8%) (Table 1). In the FQ-susceptible isolates (n = 72), aac-(6)′-Ib-cr, qnrB, and qnrS were found at low rate (4–20%). Among CTX-M-positive isolates, prevalence of aac-(6)′-Ib-cr was 71.4% (45/63), which was higher than those of qnrS, qnrB, and oqxAB (25.4–50.8%) (Supplementary Table S3).

Mutations in QRDR of GyrA were detected in all the 119 isolates showing resistance to LVX (MIC: 8– ≥128 μg/mL) and assigned into any of the 8 patterns at position 83 and/or 87 of GyrA (Table 2), while no mutation was found in ParC. The most common GyrA mutation was a double mutation S83F+D87A (54.6%), followed by S83I (20.2%). All the isolates with any of the three double mutations (S83F+D87A, S83L+D87N, S83Y+D87A) showed ≥64 μg/mL of LVX-MIC, which was higher than most of isolates with single mutation (8–32 μg/mL). Only some isolates with single mutation S83I and S83Y showed high LVX-MIC (≥64 μg/mL).

PMQR genes were detected for 106 FQ-resistant isolates and classified into 15 profiles, among which aac-(6)′-Ib-cr+qnrB (26.9%) and aac-(6)′-Ib-cr+qnrB+oqxAB (19.3%) were the most frequently observed (Table 3). Although relatedness of the presence of individual PMQR genes to LVX-MIC was not generally evident, high MIC to LVX (≥64 μg/mL) was observed for some PMQR gene profiles, that is, aac-(6)′-Ib-cr+qnrB, aac-(6)′-Ib-cr+oqxAB, aac-(6)′-Ib-cr+qnrB+oqxAB, and aac-(6)′-Ib-cr+qnrB+qnrS. Among isolates with all the seven PMQR profiles with aac6′-Ib-cr and any other gene(s), 77.6% (59/76) of isolates were related to higher resistance level to LVX (MIC, ≥64 μg/mL).

STs were determined for the selected 30 FQ-resistant K. pneumoniae isolates with different profiles of beta-lactamase genes and PMQR genes (Table 4). These isolates mostly belonged to ST15 and its single-locus variants (SLVs), that is, ST14, ST975, ST4702, and ST4703, among which ST4702 and ST4703 were novel STs identified in this study. Four isolates with NDM-1 and CTX-M-15 genes were assigned to ST15, while other four isolates were harboring NDM-5, SHV-28, and TEM-1 genes to ST975. One isolate each with bla_NDM-7 and bla_NDM-5 were classified into ST16 and its triple-locus variant (ST4000), respectively. Among the 14 NDM-positive isolates, 9 and 10 isolates possessed bla_CTX-M and aac-(6)′-Ib-cr, respectively, and double mutation in QRDR of GyrA was identified in 9 isolates (Supplementary Table S4). An isolate (ST16) with NDM-7 gene harbored four PMQR genes.

Concatenated sequences of STs identified in the present study were analyzed phylogenetically, together with those of representative drug-resistant clones reported previously.^1,27 As shown in Fig. 1, STs in our study mostly belonged to clonal group (CG) 15 (CG15), CG17, and CG147, among which CG15 and CG17 included STs of carbapenemase-positive isolates.

OPEN IN VIEWER

FIG. 1.

Phylogenetic dendrogram of concatenated sequence of the seven ST loci constructed by the maximum likelihood method using the MEGAX software. Trees was statistically supported by bootstrapping with 1000 replicates, and genetic distances were calculated by Kimura two-parameter model. Variation scale is described at the bottom. Percent bootstrap support is indicated by the values at each node (the values <80 are omitted). Filled circle and open circle indicate STs identified in the present study with and without carbapenemase genes, respectively. Clonal groups are indicated on the right. ST, sequence type.

Discussion

The present study revealed prevalence of ESBL and carbapenemase genes, PMQR genes, and QR-related GyrA mutations for K. pneumoniae first time in Myanmar. The prevalence of ESBL gene (bla_CTX-M) in K. pneumoniae from respiratory infections was 33%, which was higher than overall detection rate of ESBL in K. pneumoniae from intra-abdominal infections (24.3%) in Asia-pacific region (2010–2013). ²⁸ However, the ESBL rate in our study was comparable to those in Thailand (36.5%), Vietnam (30.4%), Malaysia (28.5%), and so forth. ²⁸ Although the TEM and SHV genes showed higher prevalence than CTX-M-type beta-lactamase, they were mostly assigned to non-ESBL type, and bla_CTX-M was exclusive ESBL gene identified, with CTX-M-15 being predominant. This finding may be reflected by the global trend showing the spread of CTX-M-15 among K. pneumoniae through dissemination of plasmids and transposons encoding bla_CTX-M-type ESBLs. ²⁹

Carbapenemases that are produced by K. pneumoniae have been classified into three major groups, that is, KPCs (class A), NDMs (class B), and OXA-48-like (class D), ¹ among which bla_KPC has become the most prevalent globally. While being endemic in the Indian subcontinent, K. pneumoniae harboring bla_NDM has been spreading worldwide. ³⁰ In Myanmar, carbapenemase gene-positive rate in clinical isolates of E. coli was 6.1% (26/426) in our previous study. ¹⁶ Another report in Myanmar described this rate as 3.4% among all the isolates of Enterobacterales. ³¹ In both studies, most of carbapenemases were identified as NDM-type, including NDM-1, NDM-4, NDM-5, and NDM-7, with NDM-5 being dominant.^16,31 Similarly to these findings on E. coli, our study indicated that carbapenemase genes in K. pneumoniae were all identified as bla_NDM with detection rate of 7.3%. Accordingly, it is suggested that NDM-type carbapenemase has been disseminating among Gram-negative pathogenic bacteria in Myanmar. Furthermore, detection of NDM-type beta-lactamase genes was reported in isolates from environment (sewage) and foodstuffs in Yangon, Myanmar.^31,32 These findings suggested the wide distribution of carbapenemase-producing bacteria that may pose an increased risk of refractory infections to local people.

It was of note in our present study that ST15 and its SLVs were identified in most of all the K. pneumoniae isolates harboring ESBL and/or carbapenemase genes, as well as quinolone-resistance determinants. K. pneumoniae strains belonging to CC258 (ST258, ST11, ST340, ST512) have been known as the major global clonal groups with multidrug resistance that carries carbapenemase genes (mainly bla_KPC).^1,27 CC15 (ST14, ST15) is described as the second most prevalent clonal group of multidrug resistant K. pneumoniae that caused outbreaks. ¹ ST15, as well as ST147, K. pneumoniae were shown as those being linked with multidrug resistance worldwide, with geographically diverse distribution, while being most prevalent in Europe. ³³ It was indicated that multidrug-resistant ST15 K. pneumoniae in the UK, Ireland, have genetically divergent nature and are related to global isolates, including those from Asia (Nepal, Singapore, Laos, Indonesia).^33,34 Accordingly, it is suggested that Myanmar might have been also involved in the spread of this clone, due to movement of people and any genetic features of this clone allowing its successful transmission.

NDM-producing K. pneumoniae isolates belong to various STs, including ST14, ST15, ST16, ST17, ST20, ST37, ST101, ST147, ST258, ST340, ST512, and ST972, among which ST14, ST147, and ST340 have been found as those associated with bla_NDM in many countries. ³⁰ Particularly, ST11 (CG258) and ST147 (CG147) were the major clones harboring bla_NDM and found in India. ³⁵ Therefore, ST15 (CG15) and ST16 (CG17), which were identified in our study, are considered minor clones of K. pneumoniae producing NDM-type carbapenemases. However, STs of CG15 were identified most frequently in our present study, irrespective of the presence of carbapenemase genes, suggesting the predominance of CG15 K. pneumoniae in Myanmar. Meanwhile, ST15 K. pneumoniae harboring bla_NDM-1 has been reported in Nepal ³³ and China, ³⁶ thus its potential spread in Asia may be of concern and needs to be monitored.

The high incidence of ST15 and its SLVs among various isolates with different profiles of beta-lactamases and quinolone resistance determinants indicated a clonal dissemination of multiple-drug resistant K. pneumoniae in the hospital of this study. According to the only available information for K. pneumoniae clinical isolates in Myanmar, ST147 and ST101 were major genotypes among bla_NDM-positive isolates, which were mostly derived from blood and urine. ³¹ In contrast, from foodstuffs, ST15, ST16, ST39, ST40, ST395, and ST1537 were reported for bla_NDM-positive isolates. ³² Because only isolates from respiratory infections (sputum) were analyzed in our present study, it is possible that distinct K. pneumoniae clones are prevalent depending on different infection types or specimens. Thus, it remains to be determined whether ST15 K. pneumoniae with bla_NDM is prevalent in diseases other than respiratory infections.

Prevalence of diverse PMQR genes in Gram-negative bacteria has been increasingly associated with rise in quinolone resistance worldwide. ³⁷ PMQR, which causes reduced susceptibility to quinolones, is considered to promote occurrence of mutation in QRDR of GyrA/ParC leading to high level FQ resistance. ³⁸ However, higher prevalence of QNR genes than incidence of the gyrA/parC mutations, presumably due to protection of GyrA/ParC by Qnr, was described in China.^39,40 In contrast, in our present study, mutation(s) in GyrA was detected in all the 119 isolates showing resistance to FQ, among which ∼90% of isolates harbored any of PMQR genes. Similarly high incidence of both GyrA mutations and PMQR genes was reported in a university hospital in Egypt. ⁴¹ These findings indicate that incidence of GyrA mutations is not predictable by prevalence of PMQR genes.

As reported previously, presence of double mutations in GyrA causes higher resistance level to quinolones than single mutation, ⁸ which was also observed in our present study. Furthermore, carriage of qnr alleles or qepA was also implicated in higher FQ resistance level. ⁸ However, PMQR genes were not clearly correlated to higher MIC to LVX in our study, while PMQR profiles with multiple genes with aac(6′)-Ib-cr appeared to be associated. Accordingly, resistance level to FQ may not be defined by PMQR gene alone, but primarily by presence of single or double mutations in GyrA, in combination with PMQR genes. Resistance level defined by PMQR may be variable due to difference in plasmid copy number and gene expression, as suggested previously. ⁸

Among the mutations in QRDR of GyrA, S83L and D87N/A have been the most frequently reported for FQ-resistant K. pneumoniae.^40–42 In the present study, the most common mutations were S83F and D87A, among which S83F had been rarely identified, while there are reports of its detection in China ⁴² and Spain. ⁴³ It is suggested that prevalence of the rare type of GyrA mutation might be caused by clonal spread of a specific ST15 clone. Such rare mutation in this study may be utilized as a marker to distinguish other FQ-resistant strains of different sources, to analyze spread of the ST15 multidrug-resistant K. pneumoniae over other infection types and other areas in Myanmar. Antimicrobial resistance is global concern, and its control needs government sectors and community participation. As described previously, ⁴⁴ an antimicrobial stewardship program (ASP) should include written guidelines for prescription of appropriate antibiotics at correct dosage, shortest duration of treatment, carrier isolation, and rapid diagnosis of viral infections that reduce unnecessary usage of antibiotics. Further promotion of ASP is anticipated in Myanmar for the control of multidrug-resistant bacteria.

This study revealed high prevalence of ESBL and quinolone-resistance determinants, along with emergence of NDM-type carbapenemase genes among K. pneumoniae from respiratory infections. The results underscore the importance of further surveillance of multidrug resistant K. pneumoniae in Myanmar.