High Prevalence of CTX-M Type Extended-Spectrum Beta-Lactamase Genes and Detection of NDM-1 Carbapenemase Gene in Extraintestinal Pathogenic Escherichia coli in Cuba

Increase of extraintestinal pathogenic Escherichia coli (ExPEC) showing resistance to beta-lactams is a major public health concern. This study was conducted as a first molecular epidemiological study on ExPEC in Cuba, regarding prevalence of extended-spectrum beta-lactamases (ESBLs) and carbapenemase genes. A total of 306 ExPEC isolates collected in medical institutions in 16 regions in Cuba (2014–2018) were analyzed for their genotypes and presence of genes encoding ESBL, carbapenemase, plasmid-mediated quinolone resistance (PMQR) determinants by PCR and sequencing. The most common phylogenetic group of ExPEC was B2 (49%), followed by D (23%), A (21%), and B1 (7%). Among ESBL genes detected, blaCTX-M was the most common and detected in 61% of ExPEC, with blaCTX-M-15 being dominant and distributed to all the phylogenetic groups. NDM-1 type carbapenemase gene was identified in two isolates of phylogenetic group B1-ST448. Phylogenetic group B2 ExPEC belonged to mostly ST131 (or its single-locus variant) with O25b allele, harboring blaCTX-M-27, and included an isolate of emerging type ST1193. aac (6’)-Ib-cr was the most prevalent PMQR gene (40.5%), being present in 54.5% of CTX-M-positive isolates. These results indicated high prevalence of CTX-M genes and the emergence of NDM-1 gene among recent ExPEC in Cuba, depicting an alarming situation.


Introduction
Escherichia coli is the most representative Gram-negative bacteria in the intestinal tracts of humans and animals as a commensal organism. However, infectious diseases in humans are caused by a group of E. coli strains, e.g., pathogenic E. coli, which is classified into diarrheagenic E. coli and extraintestinal pathogenic E. coli (ExPEC). While both pathogenic E. coli are distributed globally, ExPEC has been

Beta-Lactamase
Genotypes of E. coli (ST, fimH) and beta-lactamases were determined for a total of 71 ExPEC isolates consisting of 16,11,30, and 14 isolates of phylogenetic groups A, B1, B2, and D, respectively, and summarized in Table 2. These isolates were selected from different provinces and different specimens, in each year, and included ExPEC harboring one of the six CTX-M genes (group 1, bla CTX-M-15 , bla CTX-M-32 , bla CTX-M-55 ; group 2, bla CTX-M-2 ; group 9, bla CTX-M-14, bla CTX-M-27 ), and those with TEM (bla TEM-1 ), CMY (bla CMY-2 ), or NDM (bla NDM-1 ). Common STs of phylogenetic group A isolates were ST10, ST410, and their single-locus variant (SLV) and double-locus variant (DLV), and these isolates harbored mostly bla CTX-M-15 and any of the PMQR genes. ST448 was detected in only phylogenetic group B1 ExPEC, and two ST448 isolates possessed NDM-1 gene. Isolates with ST405 and its variants were commonly identified among phylogenetic group D. Except for only a single isolate of ST1193, all the phylogenetic group B2 isolates belonged to ST131 or its SLVs (five STs), and harbored bla CTX-M-15 or bla CTX-M-27 . Eighteen isolates were positive for O25b allele, and classified into fimH-30 type. Among the five ST131 SLV detected in phylogroup B2, three STs (ST5716, ST5717, ST5718) were newly identified in the present study.     Mutations in QRDR in GyrA and ParC were analyzed for 39 isolates showing resistance to quinolones (Table S2). In most isolates, mutations were detected in both proteins. S83L, D87N mutations in GyrA and S80I, E84V mutations in ParC were the most commonly identified. Among the 22 isolates with double mutations in both GyrA and ParC, 14 isolates harbored aac (6')-Ib-cr. Two isolates belonging to phylogenetic group B2 showed resistance to colistin (MIC, 16 and 8 mg/µL), while mcr-1, mcr-2, mcr-3 genes were not detected by PCR.

Discussion
In the present study, we observed high prevalence of E. coli positive for CTX-M gene (61%) as well as those resistant to ceftazidime (70%) and cefotaxime (76.1%). In a surveillance report from 11 Latin American countries (2011-2014), documented rates of CLSI ESBL screening phenotype in E. coli ranged from 14.7% (Brazil) to 69.9% (Mexico), while overall rate was 37.7% [13]. These rates were higher than previous surveillance (2008)(2009)(2010) in the four Latin American countries representing ESBL rates as 18.1-48.4% [14]. Comparing these CTX-M-positive rates with those in our present study (2014-2018), Cuba is considered one of the countries showing highest prevalence of ESBL in E. coli among Latin America. The early study in Cuba (2002Cuba ( -2004, Havana city) reported that ESBL phenotype rate was 10%, associated with resistance rates to cefotaxime as 14.1% [12]. Accordingly, ESBL-producing E. coli is suggested to have increased drastically during the past decade in Cuba, similarly to the increasing trend in other Latin American countries.
In Cuba, CTX-M type beta-lactamase is considered to be virtually a predominant ESBL, because all the TEM genes analyzed were assigned to non-ESBL genotype (TEM-1). Among the CTX-M type beta-lactamase genes, bla CTX-M-15 was the dominant type, as reported in the United States [15] and Canada [16], and distributed to all the phylogenetic groups. Phylogenetic group B2 included ST131 E. coli with O25b allele harboring bla CTX-M-15 , which is known as the pandemic clonal group of multidrug resistant ExPEC [17], was revealed to be prevalent in Cuba in our present study. Furthermore, it was notable that bla CTX-M-27 was detected in B2-ST131 isolates showing higher incidence than bla CTX-M-15 . ST131 E. coli with bla CTX-M-27 has been rapidly increasing in the past decade in Asia, Europe, and north America [18]. We found unusually high prevalence of ST131 ExPEC with bla CTX-M-27 , suggesting the need for further monitoring of this clone. In addition, rare CTX-M-types, bla CTX-M-32 and bla CTX-M-55 were detected in phylogenetic group A isolates. CTX-M-55 has been reported as an emerging ESBL type among humans, animals, and the environment. [19], while bla CTX-M-32 is often associated with cattle and meat products [20]. Recently in Cuba, bla CTX-M-32 was identified in plasmid of an E. coli strain isolated from a healthy pig [21]. Hence, there may be a possibility that E. coli having CTX-M-32 and -55 genes might be derived from animal or environmental origin.
Since the first identification of NDM-1 in 2008, NDM-type carbapenemase has attracted worldwide attention because of its rapid dissemination among Gram-negative bacteria that caused infections or colonization in human and animals, and also those distributed to environments [22]. Main reservoir of NDM producers is regarded as South Asia, while secondary reservoir is considered the Balkan regions and the Middle East [23]. Among NDM variants that have been discriminated into more than 20 types, NDM-1 is the most common globally, and distributed mainly to specific clones (53 STs) of E. coli with ST101, ST167, ST131, ST405, ST40, and ST648 being more frequently identified. In Latin America, prevalence of carbapenem-resistant E. coli has been extremely low [13,24], and E. coli carrying NDM gene has been rarely reported [25]. Only isolates of ST10 and ST617 E. coli from nosocomial outbreaks were reported to harbor bla NDM-1 in Mexico [25,26]. In our present study, NDM-1 gene was identified in two ExPEC isolates of phylogenetic group B1-ST448 that were isolated from sporadic infections in 2016, representing NDM-detection rate of 0.7% (2/306). This is the first report of NDM-1 gene in E. coli in Cuba, although we identified bla NDM-1 in a rare Acinetobacter species, A. soli in 2011 [27]. It was also remarkable in the present study that bla NDM-1 was detected in a rare E. coli clone, ST448. This clone harboring NDM gene has been identified in only limited reports in Asia (The Middle East and India) and Europe (Spain, Poland), and associated with various NDM-types, with NDM-5 being common [22,28,29]. Furthermore, KPC-3 and VIM-1-producing ST448 E. coli was reported in Spain, as a unique multiresistant clone [30]. Although it is not certain whether the Cuban NDM-producing ST448 E. coli was derived from Europe or autochthonous infection, it is necessary to carefully monitor the trend of this novel clone.
While phylogenetic group B2 isolates were mostly assigned into ST131 or its variant, it was worthy of note that a single isolate (IPK-629) belonged to ST1193, which is described as an emerging clone [31]. ST1193 belongs to the ST14 clonal complex, and shows fluoroquinolone resistance, while resistance profiles were variable depending on isolates [31]. This E. coli clone was documented to show temporal prevalence trend in the US [32], and ST1193 having CTX-M-14 and CTX-M-15 genes were reported in Germany [33], and only the latest report from France described CTX-M-27 gene-positive ST1193 E. coli [34]. IPK-629 in our present study was isolated from lochia in 2018, and had CTX-M-27 gene, and showed resistance to ceftazidime, ciprofloxacin, and trimethoprim-sulfamethoxazole. ST1193 E. coli was suggested to have evolved through frequent gain or loss of resistance gene cassettes [31], and thus, possible to change in its resistance profiles and epidemiological features in the future. Therefore, detection of ST1193 in Cuba may be a concern for the control of ExPEC.
In the present study, high resistance rate was noted also against ciprofloxacin (76.1%), associated with mutations in GyrA and ParC and prevalence of PMQR gene aac (6')-Ib-cr. This finding suggests substantial progress in resistance of E. coli to fluoroquinolone in Cuba. It has been described that PMQR gene aac (6')-Ib-cr is often located on plasmids of the IncF family with bla CTX-M-15 [11]. Considerable rate of aac (6')-Ib-cr among CTX-M-positive E. coli (54.5%) observed in our present study suggest coexistence of these genes on plasmids, which may imply the spread of ESBL associated with quinolone and aminoglycoside resistance, leading to dissemination of multidrug resistant E. coli. While qnrB has been implicated in ISCR-1-linked genes encoding CTX-M and carbapenemases on plasmids [35], prevalence of qnrB was low among CTX-M-positive isolates in our study. Furthermore, among nine qnrS-positive isolates, only two isolates had TEM genes, although association of qnrS1 with bla TEM was documented [35]. These findings suggest the presence of an uncommon or novel type of plasmid containing qnr genes among ExPEC.
Our present study revealed high prevalence of CTX-M type ESBL gene represented by bla CTX-M-15 in ExPEC in Cuba, associated with progress of quinolone resistance, and described also first identification in E. coli of NDM-1 type carbapenemase gene. These findings could be attributable to the presumptive frequent use of third generation cephalosporins and carbapenems in Cuba. Along with continuous surveillance of antimicrobial resistance, a systematic investigation for the usage, i.e., frequency and number of individual antimicrobials used in this country would be necessary for the effective control of antimicrobial resistance in ExPEC.

Bacterial Isolates
A total of 306 non-duplicate clinical isolates of E. coli derived from patients with extraintestinal infections were analyzed. These isolates were collected from hospitals in 16 regions (provinces) in Cuba during a period between 2014 and 2018. The main source of the isolates was urine (49.7%), followed by blood (13.4%), wound (10.8%), tracheal aspirate (9.5%), skin (5.6%), cerebrospinal fluid (2.9%), catheter tip (2.3%), and others (5.8%). Bacterial identification was performed by conventional method. Gram-negative rods grown on MacConkey agar were identified by colonial morphology, motility and a series of biochemical tests (indole test, citrate utilization, urease test, oxidase reaction, lysine and ornithine test and sugar fermentation). Further, E. coli was confirmed by PCR targeting adk using primers employed in multilocus sequence typing (MLST) of this bacterial species [36].

Susceptibility Testing
Minimum inhibitory concentration (MIC) was measured for 18 antimicrobial agents (amikacin, aztreonam, ceftazidime, ciprofloxacin, colistin, cefotaxime, cefuroxime, cefepime, fosfomycin, cefoxitin, gentamicin, imipenem, meropenem, nalidixic acid, norfloxacin, sulfamethoxazole/trimethoprim, tobramycin, piperacillin-tazobactam). E-test was employed for beta-lactams, while disc diffusion test for other antibiotics except for colistin. Broth microdilution test was used for colistin, and also for imipenem and meropenem for confirmation of MIC. Susceptibility to all the antimicrobials except for nalidixic acid was judged according to EUCAST guideline [37]. CLSI guideline [38] was employed for nalidixic acid, of which breakpoint was not assigned in EUCAST guideline.

Molecular Detection of Beta-Lactamase Genes and Plasmid-Mediated Quinolone Resistance (PMQR) Genes
For all the E. coli isolates, presence of beta-lactamase genes bla CTX-M , bla TEM , bla SHV was examined by multiplex PCR as described previously [39], and four bla CTX-M subgroups (group 1, 2, 9 and 8/25/26) were discriminated by multiplex PCR assay [40]. For all the isolates showing resistance to imipenem and/or meropenem, presence of carbapenemase genes (bla NDM , bla VIM , bla IMP , bla KPC , and bla OXA-48 ) were confirmed by multiplex/uniplex PCR using primers and conditions as described previously [41]. Plasmid-mediated AmpC beta-lactamase genes consisting of six families were detected by multiplex PCR according to the scheme described by Perez-Perez and Hanson [42]. Nucleotide sequences of full-length bla TEM , bla CTX-M , carbapenemase genes (bla NDM ), and AmpC genes (bla CMY ) were determined directly from PCR products with primers listed in Table S3, using the BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA, USA) on an automated DNA sequencer (ABI PRISM 3100). Subtypes of beta-lactamase genes were determined by using standard nucleotide BLAST (Basic Local Alignment Search Tool) available at the NCBI website [43]. Identification of plasmid-mediated quinolone resistance (PMQR) genes (aac (6')-Ib-cr, qnrA, qnrB, qnrC, qnrD, qnrS, oqxAB and qepA) was also performed by multiplex PCR using primers and conditions as described previously [44].

Genetic Analysis of E. coli
Four main phylogenetic groups of E. coli (A, B1, B2, and D) were discriminated by triplex PCR method described by Clermont et al. [45]. Sequence type (ST) of E. coli based on Achtman scheme of MLST was assigned by determination of partial sequence of seven housekeeping genes (adk, fumC, gyrB, icd, mdh, purA and recA) [36]. Presence of O25b allele was confirmed by PCR as described previously [46], and isolates with O25b allele were further analyzed for genotype based on fimH (type 1 fimbrial adhesin gene) by PCR and direct sequencing [47], using the FimTyper 1.0 web-based tool. Presence of mutation in quinolone-resistance determining region (QRDR) of DNA gyrase (GyrA) and topoisomerase IV (ParC) was analyzed for selected quinolone-resistant isolates by PCR and direct sequencing. Primer sequences for PCR are as follows: gyrA, forward 5 -ACGTACTAGGCAATGACTGG-3 , reverse 5 -AGTCGCCGTCGATAGAA-3 ; parC, forward 5 -TGTATGCGATGTCTGAACTG-3 , reverse 5 -CTCAATAGCAGCTCGGAATA-3 . For isolates showing resistance to colistin, detection of mcr genes was attempted by PCR, as described previously [24].