Genetic Diversity of CMY Beta-Lactamase Genes in Clinical Isolates of Escherichia coli in Myanmar: Identification of Three Novel Types and Updated Phylogenetic Classification of bla CMY

Introduction

Occurrence and spread of multidrug resistance among Enterobacteriaceae are a global public health concern. Resistance to broad-spectrum cephalosporins is mediated by extended-spectrum beta-lactamases (ESBLs) and AmpC beta-lactamases. ¹ AmpC enzymes characteristically hydrolyze cephamycins, which are not hydrolyzed by ESBLs and not inhibited by beta-lactamase inhibitors such as clavulanic acid. ² Escherichia coli, a common cause of nosocomial and community-acquired infections, possesses chromosomally encoded AmpC, and is intrinsically susceptible to cephamycins due to low expression level of AmpC. ³ However, since the identification of plasmid-mediated AmpC (pAmpC) enzyme CMY-1 in Klebsiella pneumoniae, ⁴ various types of AmpC beta-lactamases located on plasmid have been increasingly detected worldwide. E. coli and other Enterobacteriaceae species expressing pAmpC beta-lactamases has become a major clinical concern because they are resistant to most beta-lactam antibiotics, except for a few, including carbapenems, and pAmpC is transferable among different species/strains.

CMY-type enzymes are largely classified into two groups, CMY-1-like enzymes and CMY-2-like enzymes, which are related to chromosomally determined AmpC in Aeromonas spp. and Citrobacter freundii, respectively, ² among which CMY-2-like is the most common pAmpC disseminated worldwide. Among several different AmpC beta-lactamases, CMY-type enzymes show the highest genetic diversity, and currently CMY alleles up to number 164 (CMY-164) have been released in the GenBank database. CMY alleles represent cognate genes encoding different CMY amino acid sequences due to substitution, deletion, or insertion. New allele number of CMY is assigned by the U.S. National Center for Biotechnology Information (NCBI) through submission of sequence data according to instructions in the website of Beta-Lactamase Data Resources. A part of CMY enzymes have been revealed to acquire extended-spectrum or enhanced hydrolyzing activity to beta-lactams, through genetic evolution (specific amino acid substitution, insertion, or deletion) from CMY-2.^2,5–11 While prevalence of ESBLs and epidemiological features of ESBL-producing Enterobacteriaceae have been extensively studied in many countries, spread of CMY (CMY alleles) among specific bacterial clones has not yet been well clarified. Moreover, except for the grouping of CMY-1- and CMY-2-like beta-lactamases, genetic relatedness among the considerable number of CMY alleles have not yet been well characterized.

In our previous study in Myanmar, the prevalence of various beta-lactamase genes was investigated for clinical isolates of E. coli, and clonal traits were analyzed for isolates harboring ESBLs and carbapenemase genes. ¹² In the present study, employing the same isolates, AmpC genes were analyzed for their types and E. coli clones with bla_AmpC. To characterize lineages of novel CMY genes identified, all the available bla_CMY sequences were analyzed phylogenetically, presenting an updated, provisional genogrouping scheme.

Materials and Methods

Results

All the 50 AmpC beta-lactamase genes detected among 426 E. coli isolates (11.7%) were assigned to CIT-family, including CMY-, LAT-, BIL-like enzymes by multiplex PCR, and revealed to be bla_CMY by sequencing. These CMY genes were differentiated into 9 types, with CMY-42 being predominant (22 isolates, 44%), followed by CMY-2, CMY-6, CMY-146, and remaining 5 types were identified in a single isolate each (Table 1). Three novel types, CMY-156, CMY-158, and CMY-159, which were assigned by the NCBI, were identified in isolates from 50-year-old female patient (sputum), 77-year-old female patient (sputum), and 25-year-old female patient (wound swab), respectively. E. coli isolates harboring bla_CMY were mostly derived from urine, wound swab, and sputum, which were commonly seen for major CMY types (Supplementary Table S2). While bla_CMY was detected in isolates of all the four phylogenetic groups of E. coli, incidence and rate of bla_CMY was lower in the group B2. CMY-42 gene was mostly detected in E. coli of phylogenetic groups A and B1, belonging to 11 different STs, with A-ST410, A-ST617, B1-ST448, and B1-ST101 being common. A-ST410 E. coli was also common in bla_CMY-2-harboring isolates. Isolates with CMY-6 and CMY-146 genes mostly belonged to D-ST405. Two novel types, CMY-156 and CMY-158, were found in phylogenetic group B2 E. coli with double-locus variants of ST405 and ST69, respectively. CMY-positive ST131 E. coli was only one isolate belonging to phylogenetic group B2, carrying bla_CMY-146 and bla_CTX-M-15.

Prevalence of other beta-lactamase genes and antimicrobial susceptibility of the 50 bla_CMY-positive isolates (39 isolates analyzed in the present study and 11 isolates reported previously ¹² ) are summarized in Table 2. bla_CTX-M-15 was the most common (72%, 36/50), found in most of the isolates with CMY-2 and CMY-146, and just over half of bla_CMY42-positive isolates (52%, 26/50). Carbapenemase genes (NDM-4, NDM-5, and NDM-7) coexisted with mostly bla_CMY-42, whereas some isolates with bla_CMY-2, bla_CMY-6, and bla_CMY-146 harbored NDM-5 or OXA-181 genes. Ten bla_CMY-positive isolates (six bla_CMY-42, two bla_CMY-2, one each of bla_CMY-148 and bla_CMY-158) harbored neither ESBL gene nor carbapenemase gene, having only bla_TEM-1 (data not shown). All the bla_CMY-positive isolates showed resistance to cephalosporins, being mostly resistant to aminoglycosides and levofloxacin. Three isolates harboring novel CMY types were all susceptible to meropenem, and two of them were susceptible to amikacin.

A total of 156 bla_CMY sequences that were available in GenBank database were analyzed phylogenetically. The result revealed the presence of four genetic groups, that is, CMY-1, CMY-2, CMY-70, and CMY-98 groups, because they were supported by high bootstrap values (0.90–0.99) in the dendrogram, with CMY-2 group being separated into two clusters I and II (bootstrap values of 0.90 and 0.99, respectively) (Fig. 1). CMY-70 and CMY-98 groups, and two clusters of CMY-2 were newly described in the present study. CMY-2 group contained 140 types (89.7% of all the CMY types analyzed) (101 and 37 types assigned to cluster I and II, respectively, and 2 unclassified types), whereas CMY-1, CMY-70, and CMY-98 groups had 6, 7, and 2 types, respectively (Supplementary Table S3). CMY-1 group showed 50.1–51.2% nucleotide sequence identity to other groups, in contrast, 87.5–90.1% identity was observed among CMY-2, CMY-70, and CMY-98 groups (Table 3). Between cluster I and II of CMY-2 group, nucleotide identity was 93.9–94.5%. Eight CMY types detected in the present study, including two novel types, CMY-156 and CMY-158, were assigned to CMY-2 group cluster I, whereas a novel type CMY-159 clustered with CMY-98 in the CMY-98 group. According to the annotations of CMY-98 in GenBank database (Accession No. KC603538), this gene was detected in C. freundii isolated from urine in Thailand.

OPEN IN VIEWER

FIG. 1.

Phylogenetic dendrogram of bla_CMY (total 156 sequences available in GenBank database) constructed by maximum likelihood method using MEGA7. Tree was statistically supported by bootstrapping with 1,000 replicates, and genetic distances were calculated by Kimura two-parameter model. Variation scale is provided at the bottom. Percentage bootstrap support is indicated by values at each node (values <80 are omitted). CMY-type detected in the present study are shown with closed squares and circles (novel types). Four groups of CMY genes and clusters I and II of CMY group II are shown on the right.

Alignment of CMY amino acid sequences revealed divergent positions of the newly identified CMY types from the known types (Supplementary Fig. S1). CMY-156 was different from CMY-42 by only one amino acid (phenylalanine) at position 293 located on H-10 helix, having valine at 211 in the omega loop as seen in CMY-42. CMY-158 was identical to CMY-2, except for a single amino acid at position 201. CMY-98 group sequences (CMY-98 and CMY-159) have unique amino acids at nine sites, which are not present in other groups, whereas CMY-159 was different from CMY-98 by five amino acids. Valine at position 298 in the H-10 helix of CMY-159 is found in neither CMY-98 nor other CMY groups (Supplementary Figs. S1 and S2).

Discussion

The present study first elucidated in Myanmar the prevalence of CMY and their individual types among clinical isolates of E. coli in a hospital, revealing the predominance of CMY-42. Overall detection rate of bla_AmpC (bla_CMY) among E. coli clinical isolates was 11.7% (50/426), which was higher than those described in Algeria, ¹⁶ Canada,^17,18 China, ¹⁹ Greece, ²⁰ and Japan ²¹ for isolates before 2010, ranging from 0.09% to 2.6%, with CMY-2 and DHA-1 being commonly identified. In a recent study in Bangladesh, ²² bla_AmpC was detected in 3.8% of E. coli and belonged to CIT family, with bla_CMY-42 being the most common. Similarly, CMY-42 was described as the most common bla_CMY type among Gram-negative bacteria in Egypt, where bla_CMY was detected in 7.1% of isolates. ²³ Accordingly, it is suggested that CMY-42 may be a potentially major type of CMY, surpassing CMY-2 that has predominated originally. ²

CMY-42 is a variant of CMY-2, with a single amino acid substitution at position 211 (V211S) located on omega loop, acquiring increased activity to expanded-spectrum cephalosporins. ⁹ It is suggested that such altered activity of the CMY enzyme may be related to selective spread of CMY-42, rather than CMY-2, through the exposure to cephalosporins. In addition, due to the spread of CMY-42 with expanded activity to cephalosporins, detection probability of CMY may be increased, whereas pAmpC-producing E. coli were not detected by routine diagnosis in earlier times. It was of note that the CMY-156, identified in this study, also has serine at position 211. At the same site (amino acid 211), CMY-30 has glycine, displaying higher hydrolytic efficacy to cephalosporins than CMY-2.^6,24 Moreover, in the present study, alignment of all the CMY types revealed that substitution of valine at position 211 is detected in other CMY types: CMY-139, CMY-141, CMY-145, CMY-146, CMY-147, CMY-148, CMY-154, CMY-160, CMY-163 (serine), CMY-59, and CMY-95 (alanine) (Supplementary Fig. S2 and Supplementary Table S4). In the region of omega loop, which is associated with enzyme/substrate interaction of class C beta-lactamase, ⁶ other mutations implicated in enhanced resistance activity were reported in CMY-32 (G214E substitution) ⁸ and CMY-54 (Glu-Leu^217–218 insertion). ¹¹ In addition, more mutations in the omega loop of CMY-2 group are also identified by sequence alignment (Supplementary Fig. S2): CMY-49, CMY-73, CMY-106, CMY-122, CMY-124 (S212T), CMY-131 (G214R), CMY-32 (G214E), CMY-31 (Q215R), CMY-21 (Q215K), CMY-162 (L216I), CMY-57 (L216F), CMY-38 (D217N), and CMY-111 (A218V).

H-10 helix (R2-loop) is another domain involved in hydrolyzing activity of AmpC, and deletion of amino acids in this domain was implicated in enhanced resistance in CMY-33 and CMY-44.^7,10 Amino acid substitution and insertion in H-10 helix are found in 22 CMY types belonging to CMY-2 group, including the novel CMY-156, and also in CMY-159 (Supplementary Fig. S1, S2 and Supplementary Table S4). Overall, 44 CMY types of CMY-2 group (31%, 44/140) have any mutations in either of the domains. These findings suggested that CMY-2 has been evolving into various bla_CMY types, a portion of which have mutations in the omega loop and/or H-10 helix, acquiring enhanced hydrolyzing activity to broad-spectrum cephalosporins. Such genetic variants appear to occur increasingly, expanding their distribution. Accordingly, prevalence of CMY-2 group pAmpC genes should be carefully surveyed, with occurrence of mutations in the two important regions.

bla_CMY has been phylogenetically analyzed and separated largely into CMY-1 group and CMY-2 group, representing their original species.^2,25–27 Although CMY-1 was first identified in K. pneumoniae ²⁶ and CMY-1 variants (e.g., CMY-9, CMY-19) were reported in K. pneumoniae and E. coli, ⁵ CMY-1 group genes are less prevalent among Gram-negative bacteria than CMY-2 group AmpC.^27,28 In addition to the abovementioned groups, the presence of two minor genogroups, that is, CMY-70 and CMY-98 groups, were identified in the present study. Although both groups comprise mostly Citrobacter spp., suggesting their possible relatedness to Citrobacter, CMY-101 (CMY-70 group) was found in an E. coli isolate from a cat, ²⁹ and CMY-159 (CMY-98 group) was identified in E. coli clinical isolate in our present study. CMY-100, CMY-102, and CMY-103 belonging to CMY-2 group were also reported in E. coli and/or C. freundii from dogs or cats, as new bla_CMY types, ²⁹ posing a concern for transmission of new broad-spectrum beta-lactamases from pet animals to humans.

Previously published studies indicated the presence of bla_CMY genes in E. coli of phylogenetic group B1, ³⁰ and mostly in A and D. ²² In agreement with these findings, in our present study, CMY genes were mostly detected in phylogenetic groups A, B1, and D; particularly more frequently in A-ST410 and D-ST405. In our previous study, some of ST405 and ST410 isolates were revealed to harbor bla_CTX-M-15 and carbapenemase (NDM-5 and OXA-181) genes. These STs have been reported worldwide as one of the major clones producing NDM-type carbapenemases.^31–33 Furthermore, ST405 and ST410 E. coli have many virulence factors, which could play important roles in the dissemination of these clones among population. ³⁴ These characteristics, that is, potentially increased drug resistance and virulence, are suggested to be associated with higher prevalence of CMY genes in these clones.

In contrast, group B2 E. coli harbored bla_CMY at a lower rate, whereas phylogenetic group B2 E. coli in Myanmar had been revealed to comprise ST131 (or STs of ST131 clonal complex) clones possessing bla_CTX-M and many virulence factors. ¹² ST131 E. coli is known as a pandemic clone causing extraintestinal infections. ³⁵ The presence of bla_CMY-harboring ST131 E. coli was found in meat products ³⁶ and evolution of CMY-2 to CMY-33 in ST131 E. coli, associated with acquisition of resistance to cefepime was documented in a patient who was treated with cephalosporins. ¹⁰ Transmission and spread of bla_AmpC to ST131 E. coli is a clinical concern, which may enhance their resistance activity, therefore isolates from various sources should be carefully monitored.

The present study indicated relatively high prevalence of bla_CMY in E. coli clinical isolates belonging to multiple clones (STs), frequently associated with other beta-lactamase genes in a hospital in Myanmar. Although the four genogroups (CMY-1, CMY-2, CMY-70, and CMY-98 groups) and two clusters in the CMY-2 group were identified in the present study, their difference in antimicrobial resistance and distribution in the world have not yet been clarified. For the control of drug-resistant E. coli, further surveillance is needed for bla_CMY worldwide, along with a study on mutations in CMY associated with the alteration of resistance level to beta-lactams.