Occurrence of High-Risk Clonal Lineages ST58, ST69, ST224, and ST410 among Extended-Spectrum β-Lactamase-Producing Escherichia coli Isolated from Healthy Free-Range Chickens (Gallus gallus domesticus) in a Rural Region in Tunisia

Antimicrobial-resistant Escherichia coli isolates have emerged in various ecologic compartments and evolved to spread globally. We sought to (1.) investigate the occurrence of ESBL-producing E. coli (ESBL-Ec) in feces from free-range chickens in a rural region and (2.) characterize the genetic background of antimicrobial resistance and the genetic relatedness of collected isolates. Ninety-five feces swabs from free-range chickens associated with two households (House 1/House 2) in a rural region in northern Tunisia were collected. Samples were screened to recover ESBL-Ec, and collected isolates were characterized for phenotype/genotype of antimicrobial resistance, integrons, and molecular typing (pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST)). Overall, 47 ESBL-Ec were identified, with the following genes detected: 35 blaCTX-M-1, 5 blaCTX-M-55, 5 blaCTX-M-15, 1 blaSHV-2, and 1 blaSHV-12. Resistance to fluoroquinolones, tetracycline, sulfonamides, and colistin was encoded by aac(6′)-Ib-cr (n = 21), qnrB (n = 1), and qnrS (n = 2); tetA (n = 17)/tetB (n = 26); sul1 (n = 29)/sul2 (n = 18); and mcr-2 (n = 2) genes, respectively. PFGE and MLST identified genetic homogeneity of isolates in House 1; however, isolates from House 2 were heterogeneous. Notably, among nine identified sequence types, ST58, ST69, ST224, and ST410 belong to pandemic high-risk clonal lineages associated with extrapathogenic E. coli. Minor clones belonging to ST410 and ST471 were shared by chickens from both households. The virulence genes fyuA, fimH, papGIII, and iutA were detected in 35, 47, 17, and 23 isolates, respectively. Findings indicate a high occurrence of ESBL-Ec in free-range chickens and highlight the occurrence of pandemic zoonotic clones.


Introduction
Antimicrobial resistance has emerged as a critical public health challenge globally. It has been associated with hospital (nosocomial) environments; however, antimicrobialresistant bacteria (ARB) are increasingly reported from livestock, animal-derived food products, companion animals, wild animals, aquatic environments, and agricultural and tance, recommending continuous and widespread monitoring of antimicrobial resistance across a diverse range of ecosystems. Accordingly, the current study aimed to investigate the occurrence of ESBL-producing E. coli in feces from free-range chickens (poultry that are raised by free-range farming) in a rural region in northern Tunisia. Collected isolates were characterized via identification of genes encoding antibiotic resistance and virulence factors, integrons, and their phylogenetic grouping, in addition to genetic relatedness via pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST).

Sampling Sites
The presence of ESBL-producing Enterobacteriaceae was investigated using a sample of 95 feces swabs from free-range chickens (Gallus gallus domesticus) owned by two families (House 1: n = 40; House 2: n = 55) in a rural region of northern Tunisia, collected during the period of February-March 2019. The two houses were randomly selected in one of the principal Tunisian regions of wheat crops. The region is under limited anthropogenic impacts or inputs and there were no rivers or riverines (which might contain and bring resistant bacteria or genes encoding antimicrobial resistance). Chickens were raised for supplementary income and/or household consumption, with the households located approximately 300 m apart in an agricultural area. Both subsamples were raised as freeroaming birds and fed locally available grain (wheat and barley) and kitchen scraps. Both families also reared cattle (1-3 per family), ewes (7-15 per family), domestic pets (dogs and cats), and donkeys, with chickens reared in close proximity to these animals. According to both owners, chickens did not previously receive antimicrobial agents for any purpose (therapy or growth promotion).

Feces Swab Samples and Bacterial Identification
The feces swabs were collected from each bird, stored at 4 • C, and transferred to the laboratory within 24 h, with cold chain constantly maintained. Approximately 5 mL of Brain Heart Infusion (BHI) (Oxoid Ltd., Basingstoke, UK) was added to each sample, followed by incubation at 37 • C for 2 h. Subsequently, 1 mL of enriched suspension was streaked onto tryptone bile X-glucuronide agar (TBX agar; Oxoid Ltd., Basingstoke, UK) plates supplemented with 2 mg/L of cefotaxime (Sigma-Aldrich, Munich, Germany) and incubated overnight at 37 • C [26,27] for recovery of cefotaxime-resistant Enterobacteriaceae (potential producers of ESBL and acquired pAmpC). One colony per sample (bird) was randomly selected, identified using API 20E (Bio-Mérieux. La Balme, les Grottes, France), and confirmed as E. coli via species-specific PCR amplification of the uid gene, encoding for β-glucuronidase (primers used: uid-F: 5 ATCACCGTGGTGACGCATGTCGC 3 ; uid-R: 50 CACCACGATGCCATGTTCATCTGC 3 ) [28].

Phylogrouping of E. coli isolates Genetic Relatedness by Pulsed-Field Gel Electrophoresis (PFGE) and Multilocus Sequence Typing (MLST)
Phylogenetic groups (A, B1, B2, C, D, E, F, and Escherichia cryptic clade I) were identified using the phylogroup assignment method of Clermont et al [47]. To assess the clonality of collected isolates, genomic DNA was prepared, digested by XbaI enzyme and analyzed by PFGE, as reported previously [34]. DNA macrorestriction patterns (pulsotypes) were visually analyzed and interpreted according to Tenover et al [48]. Dendrogram for the main phylotypes was performed using GelJ software (version 1.0) [49], based on the Dice similarity coefficient, and clustering by the unweighted pair group method with arithmetic means (UPGMA method) [50]. Bands were selected by the software and then manually annotated. In addition, representative E. coli isolates belonging to relevant PFGE profiles and harboring specific bla and mcr genes or untypeable by PFGE were studied by MLST using PCR amplification and sequencing of seven conserved housekeeping genes (adk, fumC, gyrB, icd, mdh, purA, and recA) [51]. To determine the specific allele combination and sequence type (ST), all amplicons sequences were compared with MLST databases (http://enterobase.warwick.ac.uk/species/ ecoli/allele_st_search, accessed on 25 January 2022).

Occurrence of ESBL-Producing E. coli and Antibiotic Susceptibilities of Isolates
Overall, based on one presumptive E. coli isolate per bird, which was randomly selected and confirmed to the species level by PCR, 47 (49.4%) sampled chickens were colonized by ESBL-producing E. coli isolates. More specifically, 29 (29/40; 72.5%) and 18 (18/55; 32.7%) ESBL-producing isolates were collected from House 1 and House 2, respectively. All isolates were resistant to at least one non-β-lactam antibiotic, with the majority being characterized as MDR, with isolates primarily resistant to nalidixic acid, tetracycline, and trimethoprim-sulfamethoxazole (Table 1). Nalidixic acid resistance was detected in 25 and 13 isolates from House 1 and 2, respectively, with ciprofloxacin resistance concurrently identified in 17 of the 38 nalidixic-acid-resistant isolates. Colistin resistance was observed in three and ten isolates from chickens from House 1 and 2, respectively. Colistin MICs varied from 4 µg/mL to 16 µg/mL. Resistance to carbapenems (imipenem, meropenem, and ertapenem) was not observed.

Genes Encoding ESBL Enzymes and Non-β-Lactam Antibiotics
PCR analyses showed a predominance of bla CTX -type genes; bla CTX-M-1 , bla CTX-M-15 , and bla CTX-M-55 were detected in 35, 5, and 5 isolates, respectively, with bla SHV-2 and bla SHV-12 both detected in one (differing) isolate ( Table 1). The bla OXA-10 gene was concomitantly present with the bla CTX-M-1 gene in three isolates from chickens from House 2 (Table 1), while the bla TEM gene remained undetected.
Among the 38 nalidixic-acid-resistant isolates, 21, 2, and 1 isolates harbored aac(6 )-Ibcr, qnrS, and qnrB genes, respectively. The aac(6 )-Ib-cr gene was found in isolates from both houses (House 1: 18/25; House 2: 3/13); however, the qnrS and qnrB genes were only found in isolates from House 2, and they were associated with the aac(6 )-Ib-cr gene (Table 1). Among the 44 tetracycline-resistant isolates, the tetB gene (n = 26) was more prevalent than tetA (n = 14), with both tet genes found to co-occur in three isolates from House 2. Thirty-two isolates were sulfonamides-resistant and concurred with the presence of sul1 and sul2 genes in 29 and 18 isolates, respectively. Both genes were concomitantly detected in 15 isolates. The mcr-2 gene (the sequence is provided in the Supplementary Materials) was detected in two colistin-resistant E. coli isolates from ten resistant isolates (House 2 only), with mcr-1 not detected. Class 1 integron was detected in 29 isolates, while class 2 integron was detected in just five isolates (Table 1). Both integrons were concurrently present in one isolate (House 2). All int1-positive isolates amplified the conserved 3 region (qac∆E-sul1).

Discussion
The current study collected 95 feces swab samples from two neighboring (≈ 300 M) chicken (G. domesticus)-rearing households in a rural area of northern Tunisia, 47 (49.4%) of which were colonized by ESBL-producing E. coli isolates. Chickens from House 1 exhibited a significantly higher percentage of colonization by ESBL-producing E. coli isolates than House 2 (29 (72.5%) versus 18 (32.7%) isolates). The high occurrence of ESBL-producing E. coli found in the current study mirrors the high rates reported from poultry farms employing intensive production methods, both in Tunisia and further afield [52][53][54]. For example, a recent study by Subramanya et al [55] reported that 19 out of 66 (28.8%) cloacal swab samples from healthy G. domesticus were ESBL positive, of which 16 isolates (16/66; 28.7%) were ESBL-producing E. coli. Conversely, Shoaib et al [56] employed nonselective media for E. coli isolation from 150 sick backyard chickens (free-range), identifying just 8 (5.3%) ESBL-producing E. coli isolates. The majority of isolates from the current study were MDR, a trait commonly reported among ESBL-producing E. coli irrespective of their origin [12,26,27,57]. The high rate of gut

Discussion
The current study collected 95 feces swab samples from two neighboring (≈300 M) chicken (G. domesticus)-rearing households in a rural area of northern Tunisia, 47 (49.4%) of which were colonized by ESBL-producing E. coli isolates. Chickens from House 1 exhibited a significantly higher percentage of colonization by ESBL-producing E. coli isolates than House 2 (29 (72.5%) versus 18 (32.7%) isolates). The high occurrence of ESBL-producing E. coli found in the current study mirrors the high rates reported from poultry farms employing intensive production methods, both in Tunisia and further afield [52][53][54]. For example, a recent study by Subramanya et al [55] reported that 19 out of 66 (28.8%) cloacal swab samples from healthy G. domesticus were ESBL positive, of which 16 isolates (16/66; 28.7%) were ESBL-producing E. coli. Conversely, Shoaib et al [56] employed nonselective media for E. coli isolation from 150 sick backyard chickens (free-range), identifying just 8 (5.3%) ESBL-producing E. coli isolates. The majority of isolates from the current study were MDR, a trait commonly reported among ESBL-producing E. coli irrespective of their origin [12,26,27,57]. The high rate of gut colonization by ESBL-producing E. coli in organically raised chickens was unexpected, considering the (owner-reported) absence of previous antibiotic usage. Additionally, residents from both sampled houses reported that antibiotics had not previously been used for other proximal animals (cows, owe, dogs, pet, and donkeys). Accordingly, the role of antibiotic use among sampled chickens as a driver of multidrug-resistant ESBL-producing E. coli was deemed unlikely, if not impossible; thus, other pathways for resistance must have been present. For example, in both houses, freerange chickens were frequently in contact with household members and (presumably) wild animals, such as birds and rats, which were potentially colonized with ESBL-producing E. coli. Indeed, notwithstanding consumption of kitchen waste and proximity to wastewater, in many rural communities, householders frequently defecate and/or urinate adjacent to areas where chickens move freely. As such, potential transmission of human-or animalderived ESBL-producing E. coli to chickens is plausible. Similarly, free-range chickens are omnivorous and permitted to roam freely and consume wheat grains, a variety of insects, plant debris, and vegetable wastes. Previous studies have reported insects (e.g., flies and cockroaches) as important reservoirs for ESBL-producing E. coli in both urban and rural environments [58][59][60]. Similarly, several studies have reported contamination of vegetables and plant organic matter by ESBL-producing E. coli [61,62]. Given the complexity of antimicrobial resistance and its transmission within and between various ecological niches, it is extremely difficult to confidently trace and identify the origins of antimicrobial resistance acquisition. The limit of this study was the absence of analysis of samples from humans, soil, and animals in both houses; this would have provided important data about the reservoirs/vectors of the collected isolates.
An understanding of the epidemiology of genes encoding ESBL enzymes might be employed to indicate the possible origins of ESBL-producing E. coli isolates. In Tunisia, bla CTX-M-1 has been shown to represent the dominant gene encoding ESBL production in E. coli isolated from poultry samples [13,27,52,53], while bla CTX-M-15 typically predominates among human-derived ESBL-producing E. coli [63,64]. In the current study, bla CTX-M-1 was detected in 35 of 47 isolates, followed by bla CTX-M-15 (n = 5) and bla CTX-M-55 (n = 5). The bla CTX-M-55 gene has been frequently reported among livestock in Asian countries [65]; recent studies from Tunisia have reported increasing emergence among avian ESBLproducing E. coli [27], in addition to recently being found for the first time among E. coli isolates from urban wastewater samples [12,26]. Notably, and to the best of the authors knowledge, this is the first report of the presence of bla OXA-10 , herein found in three isolates in concurrence with bla CTX-M-1 , among ESBL-producing E. coli of animal origin. OXA-10 (PSE-2), belonging to class D β-lactamases, is not an ESBL enzyme but possesses the ability to hydrolyze cephalosporins, hydrolyzing cefotaxime, ceftriaxone, and aztreonam at low levels but sparing ceftazidime, cephamycins, and carbapenems [66]. This enzyme is primarily detected in ESBL-producing Pseudomonas aeruginosa and Acinetobacter baumannii isolates [67,68] but rarely from E. coli [69].
Colistin is one of the "last-resort" antimicrobial agents for treatment of infections caused by ESBL/plasmidic AmpC/Carbpenemase-producing Enterobacteriaceae. Over the past 5-7 years, acquired mobile colistin resistance (mcr) determinants have increasingly been reported worldwide, with at least 10 variants (mcr-1 to mcr-10) having been characterized [70]. In the current study, two of ten colistin-resistant isolates carried the mcr-2 gene, with mcr-1 remaining undetected. To date, while the mcr-1 and mcr-2 genes have never been reported in human-derived enterobacteria in Tunisia, mcr-1 has been increasingly reported among animal-derived ESBL-producing E. coli, including poultry [27,71], dairy cattle [72], camels [53], and wastewater treatment plants [26]. Accordingly, to the best of the authors' knowledge, the current study is the first to report the occurrence of mcr-2 from animal origin in Tunisia.
In the current study, genetic determinants for tetracycline and sulfonamide resistance were predominantly encoded by the globally ubiquitous tetA/tetB and sul1/sul2 genes, respectively [12,26,27,53,72]. With respect to plasmid-mediated quinolone resistance (PMQR) determinants, the aac(6 )-Ib-cr gene dominated, followed by qnrS and qnrB genes, all of which are frequently reported in ESBL-producing E. coli from various origins [5,12,26,27,[72][73][74]. The aforementioned genes were primarily identified as being plasmid borne or a part of integron structures, which enhances their horizontal transfer within strains belonging to the same genera or between different enterobacteria genera. Class 1 integrons were detected in 27 isolates, whereas class 2 integrons were detected in just four isolates. The predominance of class 1 integrons has been well documented [12,[25][26][27]72] and is almost certainly associated with the active integrase gene of class 1 integron, which has the ability to integrate several gene cassettes encoding antibiotic resistance in their variable regions [75].
Genetic relatedness determined by PFGE indicated a low level of isolate heterogeneity within each sampled household, albeit one analogous clone (pulsotype) being identified within each subsample. Within household 1, pulsotype P1 comprised 23 out of 29 isolates, with this clone not detected in household 2. Pulsotype P1 exhibited highly variable resistance profiles, antimicrobial resistance genes, virulence factors, and integron content, thus pointing to a high degree of genetic dynamicity. Three isolates of this pulsotype were types by MLST and revealed two isolates belonging to the ST224 (CTX-M-1/CTX-M-15) and one ST69 (CTX-M-55), which are known as pandemic or international high-risk clonal lineages. ST224 appears to be well adapted to the human-animal interface, being reported globally, including Tunisia (North Africa), mostly in association with plasmid-mediated bla CTX-M -type genes [13,76]. The clonal lineage ST69 (CTX-M-55-positive) has been reported in other studies from clinical, livestock, and environmental samples [12,13,77], and it is one of the most common lineages of extraintestinal pathogenic E coli [78]. In addition, the unique isolate of pulsotype P4 in household 1 was assigned to ST410, which is also known as a high-risk clonal lineage associated with both nosocomial and community-acquired infections and is being increasingly detected from multiple origins worldwide, including Tunisia [13,78]. However, the ST471 (pulosotype 5) and ST617 (untypable by PFGE) lineages have been rarely reported from human and animal origins and are mostly associated with ESBL or carabapenemase production [79,80]. Conversely, despite a lower number of studied isolates, isolates from household 2 were more heterogeneous, with four pulsotypes characterizing more than one isolate, all of them also concurrent with variable phenotypic and genotypic traits. In addition, six different STs were identified (ST46, ST58, ST410, ST471, ST1642, and ST2460). Significantly, ST410 and ST471 were shared by isolates collected from both houses, potentially indicating transmission of ESBL-producing E. coli between chickens reared in the two houses. This may be expected, since in this rural zone, contact between chickens and livestock from differing houses within the same neighborhood is relatively common. It is also plausible that chickens were contaminated by a common vector(s) or source(s) within the general vicinity. A sizeable majority of isolates belonged to phylogroup A (87.2%), followed by phylogroups F (n = 3), C (n = 2), and D (n = 1); however, no isolates from phylogroup B2 were identified. Several studies have shown that avian E. coli strains are primarily assigned to commensal phylogroups A and B1, with a majority of extraintestinal pathogenic E. coli isolates of human origin associated with phylogroup B2, and to a lesser degree, group D [81]. However, the detected virulence genes (fyuA, fimH, papGIII, and iutA) are commonly reported among both avian pathogenic E. coli (APEC) and extrapathogenic E. coli causing human infections [17,82], thus highlighting the zoonotic potential of these isolates to cause human infections, albeit additional virulence genes require future investigation to confirm this hypothesis.

Conclusions
The presented study highlights the significant prevalence of ESBL-E. coli isolates among free-range chickens, the predominance of bla CTX-M-1 gene, and the likely spread of two minor clones, ST410 and ST471, known as international high-risk clonal lineages, between two proximal subsamples within a rural area of northern Tunisia. However, since only limited chicken samples and rural houses were analyzed, it is impossible to deeply understand the real epidemiology of ESBL-producing E. coli isolates from freerange chickens in this area. Many factors can influence the occurrence of such isolates in these chickens, such as colonization of the residents from both sampled houses and animals (domestic and wild) in their vicinity. Therefore, the main limit of this study was the studying of chickens separately from their biotic and abiotic environment in order to accomplish the goal of the "One health" concept. Institutional Review Board Statement: Ethical review and approval were waived for this study since feces samples were collected without handling of chickens.

Informed Consent Statement: Not applicable.
Data Availability Statement: All data generated and analyzed during our study are included in this article.