Genetic Features of Extended-Spectrum β-Lactamase-Producing Escherichia coli from Poultry in Mayabeque Province, Cuba

A total of 434 poultry cloacal samples were collected from seven different farms in different years (2013–2015) in the Cuban province of Mayabeque and analyzed for the presence of third-generation cephalosporin-resistant Escherichia coli (3GC-R-Ec). Sixty-two 3GC-R-Ec isolates were recovered in total from the farms, with detection rates of 2.9% in 2013, 10.3% in 2014, and 28.7% in 2015. Characterization of 32 3GC-R-Ec isolates revealed the presence of the extended-spectrum β-lactamase (ESBL) genes blaCTX-M-1 (n = 27), blaCTX-M-15 (n = 4), and blaCTX-M-1 together with blaLAP-2 (n = 1). The isolates also contained different proportions of genes conferring decreased susceptibility to sulfonamides (sul1, sul2, sul3), trimethoprim (dfrA1, dfrA7, dfrA12, dfrA14, dfrA17), tetracyclines (tet(A), tet(B)), aminoglycosides (aac(6′)-Ib-cr, strA, strB), chloramphenicol (cmlA1, floR), macrolides (mph(A), mph(D)), and quinolones (qnrS, qnrB, aac(6′)-Ib-cr) as well as mutations in the fluoroquinolone-resistance determining regions of GyrA (S83L, D87N, D87Y) and ParC (S80I, E84G). The isolates belonged to 23 different sequence types and to phylogroups A (n = 25), B1 (n = 5), and D (n = 2), and they contained plasmid-associated incompatibility groups FII, X1, HI1, HI2, N, FIA, and FIB. These findings reveal a genetically diverse population of multiresistant ESBL-producing E. coli in poultry farms in Cuba, which suggests multiple sources of contamination and the acquisition of antibiotic resistance genes.


Introduction
The overuse and misuse of antimicrobials in animals and humans have favored the spread of antibacterial resistance worldwide [1,2]. Strains of third-generation cephalosporinresistant E. coli (3GC-R-Ec) in humans and food-producing animals have been widely and increasingly reported over the last decade [3,4]. Poultry plays an important role as a reservoir of 3GC-R-Ec in many countries around the world [5,6]. Third-generation cephalosporin resistance in E. coli is mainly mediated by the acquisition of genes encoding Ambler class A extended-spectrum β-lactamases (ESBLs) and class C plasmid-mediated AmpC (pAmpC) enzymes. The two main families of ESBLs in 3GC-R-Ec are the CTX-M and SHV types, whereas CMY-type enzymes are the most common pAmpC enzymes [7,8]. The CTX-M type appears to be the most widely disseminated group in animals and humans worldwide [9][10][11][12]. The ESBL and pAmpC genes are frequently co-located with other antibiotic resistances genes on conjugative plasmids which may be maintained and transferred within the Enterobacterales population by the use of different classes of antibiotics against infection diseases [5,6]. Such bacteria may reach the human gut via the food chain and food preparation, and their plasmids be transferred to human pathogens [3,5,6].
Strains of 3GC-R-Ec are also frequently resistant to fluoroquinolones, with resistance to the latter resulting from either mutations in the quinolone resistance-determining regions (QRDRs) of the chromosomal topoisomerases GyrA and ParC or the acquisition of plasmidmediated quinolone resistance (PMQR) genes [13,14]. Multiple lines of evidence supported by experimental models and studies of genetic relationships indicate that some avian strains of E. coli may share similar pathogenic features to extraintestinal pathogenic E. coli (ExPEC) strains in humans [15,16]. In addition, associations between E. coli colonization and infection in humans and the exposure of E. coli to retail chicken and other food sources have been reported [17,18]. Animals, particularly poultry, carrying 3GC-R-Ec have raised major public health concerns, since the mobile genetic elements and antibiotic resistance genes associated with 3GC-R-Ec have been linked to human isolates that cause infection [19][20][21][22]. In addition, some studies have revealed the roles of highly epidemic clones of 3GC-R-Ec in the dissemination of antimicrobial-resistant genes among humans, animals and the environment [23,24]. For instance, E. coli belonging to sequence type ST410 and phylogroup A and containing the bla CTX-M-15 gene on plasmids of incompatibility (Inc) groups FIA and FIB has been recognized as a successful and epidemic clone spreading in animals, humans and the environment [23,[25][26][27][28]. Additionally, epidemic-resistance plasmids belonging to group IncF with divergent replicon types (e.g., FIA, FIB and FII) have the ability to acquire resistance genes and rapidly disseminate among members of Enterobacteriaceae, especially among certain clones within species [29].
In Cuba, 3GC-R-Ec isolates have been identified in the environment and in pigs and humans, with the CTX-M genotype being predominant [28,[30][31][32]. However, to date, the presence of 3GC-R-Ec in poultry has not been investigated. In the present study, poultry from poultry farms across the province of Mayabeque in Cuba were screened for the presence of 3GC-R-Ec, and a set of isolates were selected for antimicrobial susceptibility testing and characterization of their genetic background.

Genetic Diversity of the 3GC-R-Ec Isolates
Twenty-five of the E. coli isolates belonged to phylogenetic group A, 5 to B1, and 2 to D ( Figure 1). The isolates were highly diverse, exhibiting 29 different PFGE patterns and belonging to 23 different STs, with ST48 (n = 4), ST410 (n = 3), ST155 (n = 2), ST165 (n = 2), ST656 (n = 2) and ST10 (n = 2) identified more than once; the remaining 17 isolates belonged to unique STs ( Figure 1). Similar PFGE patterns were generated for only two isolates belonging to ST410, obtained from two different farms (A, B), and for isolates of ST90 and ST155, obtained from the same farm (F). The other isolates of the same ST exhibited different PFGE patterns. All the isolates of a given ST were from different farms except for two E. coli isolates of ST48, which were from the same farm ( Figure 1).
The isolates harboring the predominant ESBL, CTX-M-1, were highly diverse, exhibited diverse PFGE profiles, belonged to different STs and harbored plasmids of several different Inc groups. They were distributed across the six farms. In contrast, the isolates containing CTX-M-15 belonged to isolates of only two phylogenetically related STs, namely, ST410 and ST90. However, they were obtained from different farms (Figures 1 and 2).
The multiple correspondence analysis (MCA) revealed patterns of similarity among some of the isolates, which were based mainly on their genotype and phenotype of resistance or susceptibility to antibiotics ( Figure 3). This analysis also revealed an association between antimicrobial resistance pattern and the geographic location of the isolates. For instance, all multiresistant isolates harboring CTX-M-15 and some harboring CTX-M-1 belonged to the STs/phylogroups ST10/A, ST156/B1, ST167/A and ST115/D and were most commonly found at farms A, B, C and E. In contrast, isolates exhibiting less resistance (i.e., those belonging to ST68/D, ST1716/A, ST155/B1 and ST165/A) were predominantly found at farms D, F and G. The MCA also indicated that combined resistance to 3GCs, fluoroquinolones and tetracycline is a common resistance trait for most of the E. coli strains from poultry in the region of Mayabeque.

Discussion
This study provides the first insight into the distribution and genetic features of 3GC-R-Ec in poultry in Cuba. Although the sampling was performed at different farms each year, the percentage of detected ESBL-producing E. coli isolates increased each year, increasing from 2.9% in 2013 to 28.7% in 2015. These results are inconclusive but are consistent with the epidemiological situation of 3GC-R-Ec worldwide, where an increase in the prevalence of ESBL-producing E. coli has been observed in both animals and humans [3,24,33]. The high frequency of 3GC-R-Ec at some of the investigated farms highlights the need for nationwide monitoring of antibiotic resistance in food-producing animals in Cuba, as has been established in several other countries [34,35].
The phylogenetic group A was the most common group, followed by phylogenetic group B1. The phylogenetic groups A and B1 generally do not include pathogenic E. coli and have been reported to be more common in animals than in humans in tropical areas [36,37]. Two isolates belonged to phylogenetic group D; this group, together with group B2, is associated with ExPEC infections in humans [38]. Nevertheless, in recent years, a successful clone of ST410 belonging to phylogenetic group A and disseminated worldwide in animals and environments has been found to be associated with ExPEC infections in humans [23,26,37,39,40]. The detection of such a 3GC-R-Ec clone in different farms and in different years underlines the possible spread and persistence of multiresistant isolates with pathogenic potential in poultry production and emphasizes the importance of implementing a One Health-based surveillance system for antibiotic-resistant bacteria. Indeed, the mechanism of transmission of β-lactamases in poultry farms in Cuba is not well elucidated, and multiple sources of contamination need to be considered, such as various environments, the flies and feces of other animals (i.e., rats, wild birds, dogs, cats, and other food-producing animals), and poultry workers [11,41,42].
In addition to exhibiting resistance to 3rd-generation cephalosporins, the majority of the isolates exhibited resistance to other classes of antibiotics, as has been observed for E. coli from poultry in other regions around the world [6,[43][44][45]. In our study, the most common trait associated with resistance to third-generation cephalosporins was the presence of the CTX-M-1 ESBL. This enzyme is also the most common ESBL type in E. coli from poultry in regions worldwide [6,46] [44,45,47,48]. The isolates of ST410 in this study contained CTX-M-15, which is the predominant ESBL type in human E. coli worldwide [49]. Both CTX-M-1 and CTX-M-15 have been detected in 3GC-R-Ec isolates from patients of Cuban hospitals [28,32]. Nevertheless, the genetic relatedness between isolates and the CTX-M-containing genetic elements has not yet been investigated in Cuba; such investigation would require the whole genome-based characterization of bacterial isolates and large-scale epidemiological information [50,51]. However, our study showed that only the E. coli isolates of ST410 shared the same STs, phylogenetic groups and CTX-M determinant (ST410/A/CTX-M-15) as clinical E. coli isolates obtained from hospitalized patients in Cuba [28]. In addition, E. coli of ST10/A and ST167/A has been found in both poultry and humans in Cuba. However, the poultry isolates contained CTX-M-1, whereas the human isolates contained CTX-M-15 [28].
All of the 3GC-R-Ec isolates except two exhibited resistance to at least three antibiotic classes, with acquired resistances to tetracyclines, quinolones (nalidixic acid and ciprofloxacin), and sulfonamides being the most frequent combination. While the resistances in the majority of the isolates were linked to the acquisition of a specific resistance gene, the high levels of resistance to fluoroquinolones observed in 15 isolates were attributed to known mutations in the quinolone resistance-determining regions (QRDRs) of ParC and GyrA [13]. Of note, other isolates that contained only aac(6)-Ib-cr or qnr without mutations in the QRDRs of ParC and GyrA exhibited low-level resistance to fluoroquinolones, as has been reported previously [13,53,54]. In addition, the presence of aac(6)-Ib-cr and qnr genes was recently reported in ESBL-producing ExPEC strains from clinical settings in Cuba [28]. Although it is difficult to determine the driving force for the selection of these genes in bacteria from poultry, their presence may be related to the use of quinolones in poultry production in Cuba [55]. While antibiotics are not used as growth promoters and as prophylactics in poultry in Cuba, some antimicrobials belonging to the fluoroquinolones, tetracyclines, β-lactams, macrolides, aminoglycosides, sulfonamides as well as colistin are approved for use in poultry for the treatment of respiratory and intestinal tract infections [55]. Their use may have contributed to the selection and maintenance of 3GC-R-Ec and their resistance genes on plasmids in poultry in Cuba, emphasizing the prudent use of these antibiotic classes in poultry. Plasmids encoding ESBL genes may also carry genes for resistance to other antimicrobial agents, such as aminoglycosides, trimethoprim, sulfonamides, tetracyclines and chloramphenicol [8,56]. The coselection phenomenon in ESBL-producing E. coli from aviculture and humans has been suspected as a possible cause for the spread of multiresistant strains [57][58][59]. It is, therefore, strongly recommended to limit the use of antibiotics that are critically important for human health in animals [60,61].

Sampling
During the 2013-2015 period, a total of 434 cloacal swab samples were collected from healthy birds from six farms rearing laying hens in cages and one farm producing broilers via floor rearing for meat production. The farms were located in different regions of Mayabeque Province, Cuba. The cloacal samples from 2013 (n = 139) and 2014 (n = 145) were collected at farms A-B and C-E, respectively, and those from 2015 (n = 150) were from farms F-G (Figure 4). The samples were collected by veterinarians according to an approved procedure [62]. Only one sample per animal was taken. The samples were directly placed into 1 mL Tryptone Soy Broth (Oxoid, Basingstoke, UK) medium and kept cool during transport to the laboratory, where they were processed immediately.

Isolation and Identification of 3GC-R-Ec
The inoculated Tryptone Soy Broth (Oxoid) medium was transferred into 5 mL of MacConkey Broth (Oxoid) containing 8 mg/L of ceftazidime (Sigma-Aldrich, Merck, St. Louis, MO, USA) and incubated at 37 • C for 24 h under agitation. Then, a loopful (10 µL) of the mixture was plated onto MacConkey Agar (BioCen, BioCubaFarma, Bejucal, Cuba) supplemented with 8 mg/L of ceftazidime (Sigma-Aldrich) for the screening of 3GCs-R Enterobacteriaceae and reincubated overnight. Lactose-positive (pink colonies) were selected and streaked three times on selective MacConkey Agar plates to obtain pure culture. Single pink colonies from each selective plate were streaked onto Tryptone Soy Agar plates containing 5% sheep blood (TSA-SB; Becton Dickinson, Franklin Lakes, NJ, USA) and incubated overnight at 37 • C. The colonies were identified using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS, Microflex LT; Bruker Daltonics, Billerica, MA, USA) and frozen at −80 • C in glycerol stocks.

Rep-PCR-Based Genetic Relatedness and Selection of 3GCs-R-Ec Isolates
All 3GCs-R-Ec isolates were submitted to rep-PCR as described previously [63]. A dendrogram was generated using BioNumerics software v.7.6 (Applied Maths, Sint-Martens-Latem, Belgium). The isolates showing more than 90% rep-PCR pattern similarity were considered to belong to the same clonal group generating 39 different lineages ( Figure S1). Out of them, thirty-two isolates, which were either nonclonal or which were clonal but came from a different farm, were selected for further antimicrobial susceptibility testing and molecular characterization. Only one isolate per clonal group was selected from the same farm.

Detection and Identification of Antibiotic-Resistance Genes
DNA was obtained by incubating half a loop full of bacteria in 400 µL of lysis buffer (0.1 M Tris-HCl pH 8.5, 0.05% Tween 20, and 0.24 mg/mL proteinase K) for 45 min at 60 • C followed by 15 min at 95 • C [43]. Antibiotic resistance and virulence factor genes were detected using AMR08 ArrayStrip microarrays and the Hybridization Plus (+) Kit (Alere Technologies GmbH, Jena, Germany); a signal intensity of 0.1 or higher was considered positive. The presence of the β-lactam resistance genes bla CTX-M-1 , bla CTX-M-15 and bla LAP-2 was confirmed by PCR and DNA sequencing [43,66]. The amino acid substitutions in the quinolone resistance-determining regions of GyrA and ParC were detected as described previously [67].

Multilocus Sequence Typing (MLST) and Determination of Phylogenetic Group
MLST was determined using the Achtman scheme and the E. coli database at Enterobase (https://enterobase.warwick.ac.uk/) [68]. The minimum spanning tree of MLST was constructed with Bionumerics 7.6 (Applied Maths). The phylogenetic groups of the isolates were determined using the triplex PCR method as previously described [69].

Pulsed-Field Gel Electrophoresis (PFGE)
The genetic similarity of the 3GCs-R-Ec isolates was determined by PFGE using XbaI digests and conditions as previously described [43]. The definition of a PFGE cluster was based on a similarity cut-off of 90% (Dice coefficient, represented by unweighted pair-group method with arithmetic mean (UPGMA), 1.5% optimization and 1% tolerance). The dendrogram was constructed with Bionumerics 7.6 (Applied Maths).

PCR-Based Replicon Typing (PBRT) of Plasmids
The incompatibility groups of plasmids were investigated and characterized using the PBRT Kit (DIATHEVA, Cartoceto, Italy) as previously described [29].

Statistical Analysis
Multiple correspondence analysis (MCA) was used to analyze the patterns of association between the characteristics of the isolates (ST, phylogenetic group, genotypic and phenotypic resistance or susceptibility to different antibiotics) and their distributions across the farms. The inertia values were calculated by the standard "Burt matrix" method. The analyses were performed using the statistical software package Statgraphics Centurion v. 16.1.03 (StatPoint Technologies Inc, Warrenton, VA, USA).

Conclusions
This study demonstrated, for the first time, the presence of 3GC-R and multiresistant E. coli in poultry in Cuba, and it revealed some of the genetic characteristics of the isolates.
These findings represent a baseline for the establishment of a nation-wide system of surveillance of resistance in food-producing animals and the implementation of strategies for the prudent use of antibiotics in poultry production in Cuba. The results also emphasize the importance of strengthening good hygiene and sanitation practices to prevent the spread of antimicrobial resistance in animals, the environment and humans.
Supplementary Materials: The following are available online at https://www.mdpi.com/2079 -6382/10/2/107/s1, Figure S1: Cluster analysis of rep-PCR fingerprints of the 3rd-generation cephalosporin-resistant Escherichia coli (3GC-R-Ec) isolated from poultry in the province of Mayabeque, Cuba, Table S1  Data Availability Statement: Data is available within the article as well as in Supplementary Table S1 and Figure S1.