Occurrence and Characteristics of ESBL- and Carbapenemase- Producing Escherichia coli from Wild and Feral Birds in Greece

Wild and feral birds are known to be involved in the maintenance and dissemination of clinically-important antimicrobial-resistant pathogens, such as extended-spectrum β-lactamase (ESBL) and carbapenemase-producing Enterobacteriaceae. The aim of our study was to evaluate the presence of ESBL- and carbapenemase-producing Escherichia coli among wild and feral birds from Greece and to describe their antimicrobial resistance characteristics. In this context, fecal samples of 362 birds were collected and cultured. Subsequently, the antimicrobial resistance pheno- and geno-type of all the obtained E. coli isolates were determined. A total of 12 multidrug-resistant (MDR), ESBL-producing E. coli were recovered from eight different wild bird species. Eleven of these isolates carried a blaCTX-M-1 group gene alone or in combination with blaTEM and one carried only blaTEM. AmpC, fluoroquinolone, trimethoprim/sulfamethoxazole, aminoglycoside and macrolide resistance genes were also detected. Additionally, one carbapenemase-producing E. coli was identified, harboring blaNDM along with a combination of additional resistance genes. This report describes the occurrence of ESBL- and carbapenemase-producing E. coli among wild avian species in Greece, emphasizing the importance of incorporating wild birds in the assessment of AMR circulation in non-clinical settings.


Introduction
Antimicrobial resistance (AMR) has recently been declared by the World Health Organization as one of the ten most important public health threats faced by humanity [1]. Among Gram-negative bacteria, the major driving force of resistance is the occurrence units of Greece. Samples were obtained by inserting a sterile cotton swab into recently deposited feces or directly into the cloaca of live, captured wild and feral birds. Birds were captured using Larsen-type traps, Australian-type traps or modified bird-catching nets, and immediately released after sampling, according to Greek legislation. Samples were only collected following bird species identification. The swabs were transported in Amies medium (Transwab ® Amies, Corsham, UK) under refrigeration and arrived in the laboratory within 48 h of their collection.

Isolation, Identification and Antimicrobial-Resistance Phenotype of ESBL-and Carbapenemase-Producing Enterobacteriaceae
Swabs were directly streaked onto both ESBL selective media (CHROMID ® ESBL, BioMérieux, Marcy l'Etoile, France) and CPE selective media (CHROMID ® CARBA SMART, BioMérieux), and the plates were incubated aerobically at 37 • C for 24-48 h. Putative E. coli colonies of pink to burgundy coloration were sub-cultured on MacConkey agar. Identification and antimicrobial susceptibility testing of the isolates were performed using the Vitek-2 system (BioMérieux), according to the manufacturer's instructions. The AST-GN96 card was used in order to determine the minimum inhibitory concentration (MIC) of the following antimicrobials: ampicillin, amoxicillin/clavulanic acid, ticarcillin/clavulanic acid, cefalexin, cefalotin, cefoperazone, ceftiofur, cefquinome, imipenem, gentamicin, neomycin, flumequine, enrofloxacin, marbofloxacin, tetracycline, florfenicol, polymyxin B and trimethoprim/sulfamethoxazole. Results were automatically interpreted using the Vitek-2 software (BioMérieux, system version 8.02). Isolates were considered MDR when they exhibited diminished susceptibility to at least one agent of more than three antimicrobial classes.

Phenotypic Confirmation of ESBL or Carbapenemase Production
E. coli isolates that presented resistance to third generation cephalosporins (cefoperazone, ceftiofur) were subjected to the double disk synergy test (DDST) for the phenotypic confirmation of ESBL production, according to EUCAST guidelines [26]. A positive result was indicated when the inhibition zones around any of the cephalosporin disks were augmented or when a "keyhole" was formed in the direction of the disk containing clavulanic acid.
Microorganisms that were resistant to imipenem were assessed for phenotypic carbapenemase production using MIC test strips containing meropenem plus phenylboronic acid and meropenem plus ethylenediaminetetraacetic acid (EDTA) (Liofilchem). Isolates that had a ratio meropenem/meropenem plus phenylboronic acid ≥8 or meropenem/meropenem plus EDTA ≥8 were considered positive for class A or class B carbapenemases, respectively.

Antimicrobial Resistance Genotyping of the ESBL-and Carbapenemase-Producing E. coli
Isolates that were found positive in the DDST or the phenotypic carbapenemase tests were characterized using the DNA microarray-based assay CarbaResist from InterArray (FZMB GmbH, Bad Langensalza, Germany). Primer and probe sequences have previously been described in detail [27]. In addition, probes for the detection of the colistin resistance gene family mcr were included on the present microarray (see Supplementary File S1). The microarray layout is presented in Supplementary File S2. Protocols and procedures were conducted in accordance with the manufacturer's instructions (https://www.interarray.com/Further-Genotyping-Kits, accessed on 10 May 2022). In brief, bacteria were grown overnight on Columbia blood agar, harvested and enzymatically lysed prior to DNA preparation. Genomic DNA from the bacteria was extracted using the Qiagen blood and tissue kit (Qiagen, Hilden, Germany), following the manufacturer's instructions. The DNA was used in a multiplexed primer elongation, incorporating biotin-16-dUTP. Amplicons were stringently hybridized to the microarray, washed and incubated with a horseradishperoxidase-streptavidin conjugate. Hybridizations were detected by adding a precipitating dye. An image of the microarray was taken for further analysis.

Occurrence and Characteristics of ESBL-Producing E. coli
A total of 12 E. coli was found to be resistant to third-generation cephalosporins and had a positive ESBL-confirmation test (DDST). The isolates were retrieved from 12 of the 362 sampled wild and feral birds (3.3%) and, in particular, from four Eurasian magpies (Pica pica), two Common buzzards (Buteo buteo), one Short-toed snake eagle (Circaetus gallicus), one Eurasian sparrowhawk (Accipiter nisus), one Steppe eagle (Aquila nipalensis), one Grey heron (Ardea cinerea), one Eurasian Scops owl (Otus scops) and one Common swift (Apus apus). These birds originated from three regional units of Greece; Korinthia and Messinia that are located in the Peloponnese region of southern Greece, and Magnesia that is located in central Greece.
The 12 ESBL isolates presented resistance to ampicillin and first-to fourth-generation cephalosporins. One exhibited an intermediate phenotype to ticarcillin/clavulanic acid, while none were resistant to carbapenems. Molecular detection of ESBL genes showed that 11 isolates carried the bla CTX-M-1 group gene alone (n = 4) or in combination with bla TEM (n = 7) and one isolate carried the bla TEM alone. The AmpC gene bla ACT was detected in four of the isolates and the broad spectrum β-lactamase bla OXA-1 in only one.
All the isolates were further categorized as MDR. Reduced susceptibility to aminoglycosides was detected in one of the ESBL E. coli (1/12, 8.3%) which presented resistance to gentamicin, was intermediate to neomycin and harbored aphA. However, the remaining 11 strains were also found to possess aminoglycoside resistance genes and, in particular, aadA2 (n = 7), aadA4 (n = 3) and rmtA (n = 5).
Eleven of the isolates were additionally non-susceptible to fluoroquinolones (11/12, 91.7%), being either resistant or intermediate to both flumequine and enrofloxacin, while four of them were further resistant to marbofloxacin. The plasmid-mediated quinolone resistance gene qnrS was detected in all the fluoroquinolone-resistant E. coli.
Finally, the intI1 gene was detected in two strains. The antimicrobial resistance phenotypes and genotypes of the ESBL-producing E. coli isolates are summarized in Tables 2 and 3. An example of the microarray results is depicted in Figure 1.

Occurrence and Characteristics of Carbapenemase-Producing E. coli
Among the 362 samples tested, only one, originating from a Caspian gull (Larus cachinnans), yielded a positive culture on the CPE selective media (1/362, 0.3%). The isolated E. coli strain was resistant to imipenem and was phenotypically detected to produce MBL in the meropenem-EDTA test.
This isolate was MDR, also being resistant to the other tested β-lactams, as well as to gentamicin, flumequine, enrofloxacin, marbofloxacin and trimethoprim/sulfamethoxazole, while it was susceptible to neomycin and tetracycline.
The antimicrobial resistance phenotype and genotype of the carbapenemase-producing E. coli is presented in Tables 2 and 3.

Occurrence and Characteristics of Carbapenemase-Producing E. coli
Among the 362 samples tested, only one, originating from a Caspian gull (Larus cachinnans), yielded a positive culture on the CPE selective media (1/362, 0.3%). The isolated E. coli strain was resistant to imipenem and was phenotypically detected to produce MBL in the meropenem-EDTA test.
This isolate was MDR, also being resistant to the other tested β-lactams, as well as to gentamicin, flumequine, enrofloxacin, marbofloxacin and trimethoprim/sulfamethoxazole, while it was susceptible to neomycin and tetracycline.
The antimicrobial resistance phenotype and genotype of the carbapenemase-producing E. coli is presented in Tables 2 and 3.
ESBL-producing E. coli was retrieved from eight different wild bird species. Four species; Common buzzard, Short-toed snake eagle, Steppe eagle and Eurasian sparrow hawk, are raptors and two; Steppe eagle and Eurasian magpie, can be considered omnivores and scavengers. Their diet, which is commonly in direct contact with anthropogenic environments and agricultural animals and waste, constitutes a major potential route for ESBL acquisition [41]. Another alternative pathway is the environment inhabited by the aforementioned species, as also applies to the Grey heron, a waterbird. Aquatic associated species have been documented to be important ESBL hosts as a result of human activities in their habitat [36,42]. Of note, ESBL strains were also detected in samples from a Eurasian Scops owl and a Common swift, two insectivorous birds that live in urbanized areas in vicinity to humans, possibly underlining the importance of human-derived environmental contamination in the dissemination of AMR. Nevertheless, a potential transmission via insects, such as houseflies, cannot be ruled out either [43,44]. Four of the birds that were found to be colonized by ESBL E. coli (Short-toed snake eagle, Steppe eagle, Grey heron, Common swift) are also migratory and could thus act as dispersion vectors across long distances [45].
Molecular characterization of the ESBL isolates revealed a dominance of bla CTX-M-1 group, which is consistent with the majority of published studies on wild avian species to date [12,32,[46][47][48][49][50][51]. E. coli producing CTX-M group 1 β-lactamases, especially CTX-M-1 and CTX-M-15, has been described as an alarming cause of human infections globally, while variant CTX-M-1 further prevails among food-producing animals [52][53][54]. Notably, we also detected the acquired AmpC gene bla ACT in four of the isolates, but not bla CMY-2 , even though prior studies worldwide as well as in Greece have described the latter as the most dominant plasmid encoded AmpC beta-lactamase gene in Enterobacteriaceae of wild bird origin [55][56][57]. Co-occurrence of bla ACT and bla CTX-M-1 group has previously been reported in an Escherichia fergusonii isolated from a Cattle egret in Tunisia [12].
Of special interest is the fact that a carbapenemase-producing, bla NDM positive E. coli was identified in the fecal sample of a Caspian gull. The species is one of the most numerous in Greece with a wide range of different feeding habitats. This particular bird was sampled in Porto Lagos, a coastal area in eastern Macedonia, within a wide wetland complex that includes a lagoon, lakes and numerous islands. This site is part of the Ramsar Convention and is regarded as a location of considerable ecological value for breeding and wintering waterbirds and raptors [58]. In Porto Lagos, Caspian gull individuals are either resident or may originate from several other regions, such as the Black Sea, having undertaken a lengthy migration, especially in their first calendar year [59]. Noteworthily, another NDM producing strain, namely a NDM-1 Klebsiella pneumoniae recovered from the feces of a Caspian gull, has recently been reported from the Azov-Black Sea in Ukraine [60]. Therefore, we could hypothesize that this resistance gene has been imported to Greece via migratory birds or alternatively that it circulates in the region due to human contamination. In most parts of the world, NDM-type MBLs are sporadically described among humans [3], however, studies in Greece during the past three years have outlined a wide dissemination and establishment of NDM-producing Enterobacteriaceae [61,62]. Given the fact that Caspian gulls frequently feed on anthropogenic food sources such as trawler discards and refuse dumps or nearby livestock-associated facilities such as slaughterhouses, uptake of the resistant strain or the resistance determinants during feeding could also be possible [63]. NDM-producing E. coli has also been recovered from other gull species and, in particular, from Yellow-legged gulls (Larus michahellis) in Spain, Lesser black-backed gulls (Larus fuscus) in Spain and Franklin's gulls (Larus pipixcan) in Chile [60]. Additionally, Black kites (Milvus migrans) have been found to harbor bla NDM-5 E. coli in Pakistan and bla NDM-1 Salmonella enterica subsp. enterica serovar Corvallis in Germany, the latter being the first ever description of a carbapenemase producer isolated from wild animals [60,64]. Here we report, for the first time in Greece, the detection of a carbapenemase-producing organism from a wild bird, which was identified as NDM positive E. coli.
Although our study focused on β-lactamase mediated resistance (ESBLs and carbapenemases), we additionally identified high rates of non-wild-type susceptibility to almost all the antibiotic classes tested, including tetracyclines, sulfonamides, trimethoprim and quinolones. Microarray analysis confirmed the presence of resistance genes against several antibiotics, with variants sul2, dfrA5, qnrS, mrx and aadA2 being the most frequently detected resistance determinants for sulfonamides, trimethoprim, quinolones, macrolides and aminoglycosides, respectively. The detection of multiple AMR genes in all the isolates probably indicates the wide dissemination of AMR determinants in the environment and could be attributed to the presence and cohabitation of transferable plasmids [17,65]. Moreover, two ESBL-and the carbapenemase-producing isolates carried intI1, a gene encoding an element known to play a crucial role in the recruitment, spread and expression of resistance genes [66]. Numerous earlier studies have shown an association of both bla CTX-M and bla NDM with the variable region of class 1 integrons in E. coli isolates of human and animal origin [67][68][69][70]. Among wild animals, intl1 presence is related to their close contact with humans, farm animals and pets [71]. However, previous research has presented a higher incidence of intl1 among ESBL producers isolated from wildlife varying between 72.4% and 100% [46,50,72], a discrepancy that requires further investigation.

Conclusions
This study provides evidence of wild birds' colonization with ESBL-and carbapenemaseproducing E. coli in Greece. The circulation of bla CTX-M-1 group, bla TEM and bla NDM, along with genes conferring resistance to five classes of non β-lactam antimicrobials (fluoroquinolones, trimethoprim, sulfonamides, aminoglycosides, macrolides), poses a serious threat for the spread of MDR bacteria. Thus, wild birds should be regarded as a reservoir, vehicle and indicator of AMR in the environment. Considering that ESBL-PE and CPE detection rates among humans in Greece remain high, regular surveillance studies are required to fully unveil the extent of wild birds' role in the circulation of such pathogens.  Official permission for capturing and sampling migratory and resident wild birds was provided by the Hellenic Ministry of Environment and Energy (181997/1000/10-5-2019). Capturing, handling and sampling wild birds complied with European and national legislation and the study protocol was approved by the academic board of the Veterinary Faculty of the University of Thessaly, Meeting 51/18.11.19. Data Availability Statement: All data generated for this study are presented within the manuscript and the supplementary files.

Conflicts of Interest:
The authors declare no conflict of interest.