Molecular characterization of highly prevalent Escherichia coli and Escherichia marmotae resistant to extended-spectrum cephalosporins in European starlings (Sturnus vulgaris) in Tunisia

ABSTRACT European starlings are widespread migratory birds that have already been described as carrying bacteria resistant to extended-spectrum cephalosporins (ESC-R). These birds are well known in Tunisia because they spend the wintertime in this country and are hunted for human consumption. The goal of our study was to estimate the proportion of ESC-R in these birds and to characterize the collected isolates using whole-genome sequencing. Results showed that 21.5% (42/200) of the birds carried either an extended-spectrum beta-lactamase (ESBL) or an acquired AmpC gene. Diverse bla CTX-M genes were responsible for the ESBL phenotype, bla CTX-M-14 being the most prevalent, while only bla CMY-2 and one bla CMY-62 were found in AmpC-positive isolates. Likewise, different genetic determinants carried these resistance genes, including IncHI2, and IncF plasmids for bla CTX-M genes and IncI1 plasmids for bla CMY-2 genes. Three chromosomally encoded bla CTX-M-15 genes were also identified. Surprisingly, species identification revealed a large proportion (32.7%) of Escherichia marmotae isolates. This species is phenotypically indistinguishable from Escherichia coli and has obviously the same capacity to acquire ESC-R genes. Our data also strongly suggest that at least the IncHI2/pST3 plasmid can spread equally between E. coli and E. marmotae. Given the potential transmission routes between humans and animals, either by direct contact with dejections or through meat preparation, it is important to closely monitor antimicrobial resistance in European starlings in Tunisia and to set up further studies to identify the sources of contamination of these birds. IMPORTANCE The One Health concept highlighted knowledge gaps in the understanding of the transmission routes of resistant bacteria. A major interest was shown in wild migratory birds since they might spread resistant bacteria over long distances. Our study brings further evidence that wild birds, even though they are not directly submitted to antibiotic treatments, can be heavily contaminated by resistant bacteria. Our results identified numerous combinations of resistance genes, genetic supports, and bacterial clones that can spread vertically or horizontally and maintain a high level of resistance in the bird population. Some of these determinants are widespread in humans or animals (IncHI2/pST3 plasmids and pandemic clones), while some others are less frequent (atypical IncI1 plasmid and minor clones). Consequently, it is essential to be aware of the risks of transmission and to take all necessary measures to prevent the proportions of resistant isolates from increasing uncontrollably.

resistant to extended-spectrum cephalosporins (ESC-R) and carbapenems (CP) are among the most significant issues.AMR has long been overlooked in wildlife (1) but, for the last decade, clinically relevant AMR bacteria-including ESC-R and CP-R-have been isolated from various wild bird species on all continents, including vultures, gulls, buzzards, or red kites (2)(3)(4).The presence of resistance genes and antibiotic-resistant bacteria in wildlife is most probably an indicator of anthropogenic contamination rather than resistance selection since wild animals are usually not directly exposed to antibiot ics (5).Consequently, wild birds can be both reservoirs and spreaders of resistance genes or bacteria of human origin.Moreover, migratory birds, through their movements over long distances, can be partly responsible for the worldwide transmission of resistance genes (6).
Starlings (Sturnus vulgaris or European starling) are wild birds native to Europe and South-East Asia that have later reached to North America (7), Australia, New Zealand, and South Africa (8).They are great travelers, migrating over long distances (1,000-1,500 km) searching for food in winter (8).Starlings are so widespread and problematic that they were classified among the three worst invasive birds on the World Conservation Union List (9,10).They can be potential reservoirs of bacteria as they have been recurrently described as carrying Campylobacter jejuni isolates (11,12).Cefotaxime-and ciprofloxa cinresistant Escherichia coli were also reported from European starlings in Canada, and one ESC-R SHV-12-producing E. coli was reported from a Spotless starling in Spain (13)(14)(15).
Tunisia is on the migratory route of starlings that are traveling from Europe for the winter season before leaving in spring.During this 6-month period, starlings are traditionally hunted for human consumption and sold alive on local markets.While consumption of meat produced in intensive farms (such as poultry meat) where animals are treated with antibiotics has been widely studied, the risk of consuming wild animal meat has been much less evaluated.Our study thus aimed at isolating and characteriz ing ESC-R and CP-R Enterobacterales, in order to determine whether these birds carry resistant bacteria, which could be further transmitted through the food chain or by fecal contamination of domestic animals and the environment.

Prevalence and genetic diversity of ESBL/AmpC-producing isolates
Over the 200 samples tested, corresponding to 200 birds, 42 (42/200, 21.5%) presented growth on plates containing cefotaxime, while none of them grew on imipenem-contain ing plates.One isolate was picked per plate/bird, except for one plate that presented two colony morphologies, leading to a final collection of 43 Enterobacterales.
Isolates were identified as E. coli (n = 27, 62.8%) and Escherichia marmotae (n = 16, 37.2%).All E. marmotae isolates belonged to ST133, except for two presenting a common unknown sequence type (ST) (Table S1; Fig. 1).ST133 isolates were genetically linked and clustered according to their geographical origin; isolates from Gabès were nearly identical with 0-4 single-nucleotide polymorphism (SNP) differences, but differed by 42 SNPs from the Bizerte isolate and by 192-198 SNPs from the Zaghouan isolates (Table S2; Fig. S1).Only one isolate from Gabès (#60301) was genetically closer to the Bizerte isolate (#60314) than from the other isolates from Gabès.On the contrary, E. coli isolates belonged to 20 different sequence types, including 1 unknown and 19 already described STs: ST5451 was found in three different samples, while ST126, ST162, ST973, and ST1177 were each found in two samples.Three STs belonged to pandemic clones, namely ST38, ST155, and ST162.

Characterization of the genetic determinants carrying ESBL/AmpC genes
Combined analysis of Southern blots, short-read, and long-read sequences proved that the plasmid-borne bla CTX-M genes were carried by large IncHI2 or IncF plasmids.The bla CTX-M-15 gene was carried on the chromosome in three isolates, and on an undeter mined genetic determinant in the fourth one.All four bla CTX-M-15 genes were preceded by the ISEcp1 element and the two chromosomally encoded genes were followed by a tryptophan synthase beta chain gene in a genetic environment similar to the one described by Guenther et al. in the ST38 IMT37356 isolate (16).Alternatively, bla CMY-2 genes were carried either on IncI1 plasmids or on the chromosome.

bla CTX-M-27 -carrying plasmids
The bla CTX-M-27 gene was found in four E. coli isolates, including the two clonal ST5451 ones (0 SNP difference).It was carried on IncF/F2:A-:B10 plasmids in three isolates and on the chromosome in the fourth one.In this latter isolate, the bla CTX-M-27 gene was preceded by an IS908B element and the isolate additionally presented the aadA1, aph(6)-Id, aph(3″)-Ib, tet(A), sul3, floR, and dfrA1 resistance genes.The two fully sequenced p60294-CTX-M-27 (113,851 bp) and p60304-CTX-M-27 (109,448 bp) were highly similar (>99% identity, GC content around 51.9%) and co-harbored the aph( 6)-Id, aph(3″)-Ib, tet(A), and sul2 resistance genes (Fig. 2B).In both plasmids, the bla CTX-M-27 gene was flanked by two copies of the IS26, while the downstream IS26 and a third IS26 surroun ded the sul2-aph(3″)-Ib-aph( 6)-Id-tet(A) genes.Comparisons with the NCBI database showed that both plasmids shared >95% identity and 99.9% coverage with two plasmids (OX359170.1 and OX359164.1)found in E. coli strains isolated from human urine samples in a hospital in Norway (Fig. 3B).These two plasmids contained the same resistance genes found in the plasmids of the present study as well as an additional sul2 gene and shared the same genetic organization with flanking IS26 elements.

bla CTX-M-1 -carrying plasmid
The bla CTX-M-1 gene was carried by a large (251,934 bp) IncHI2/ST4 plasmid named p60280-CTX-M-1 which co-harbored the bla TEM-1B , mph(B), two copies of aadA1, dfrA1, catA1, tet(A), and a truncated mcr-1 gene (Fig. 2C).The bla CTX-M-1 gene was preceded by an ISEcp1 insertion sequence, but not located close to other resistance genes.p60280-CTX-M-1 shared 99.9% sequence identity and 87% coverage with the 240,662 bp 180-PT54 plasmid (CP015833.1)which was found in an E. coli O157 strain isolated from a human fecal sample with diarrhea in 2012 in the United Kingdom (20).The genetic backbones of these two plasmids were similar but they differed in the resistance region, since only the dfrA1 and tet(A) genes were shared (Fig. 3C).

bla CMY-2 -carrying plasmids
The 21 bla CMY-2 genes and the unique bla CMY-62 gene were identified in six E. marmotae ST133 and 16 E. coli belonging to 15 different STs (two isolates belonged to ST162).

DISCUSSION
ESC-R E. coli have spread in all One Health sectors including wildlife (23), so that they have become a key indicator in the global monitoring of AMR and a marker of environ mental contamination related to human activity (24)(25)(26).ESBL-producing E. coli isolates were first described from wild birds in 2006 in Portugal (27) and numerous cases from a wide variety of bird species have been reported since then throughout the world (28).In this study, we observed a high prevalence of ESC-R Escherichia spp.isolates (21.5%) in European starlings, due to the presence of both ESBL-conferring (11.5%) and AmpCconferring (11.0%) resistance genes.However, the origin of these resistance genes and resistant bacteria remains unknown; indeed, they might have been acquired in Tunisia as well as on the birds' migratory routes.This is the first description of ESC-R-positive European starlings in North Africa, after their report in North America where 4% of the birds tested were ESC-resistant (13), and the detection of one SHV-12-positive E. coli in a Spotless starling in Spain (15).The carriage of ESC-R determinants in these widespread migratory birds is of concern and further studies are needed to decipher the routes of contamination, including through meat consumption, and the risk of contamination for humans and animals in contact.Also, other factors that play an important role in the evolution of the bird microbiota, such as diet, sex, age, feeding habits, and geographical location-which were not recorded here and are often overlooked-should be taken into account in future studies.
Here, 62.8% of the isolates were identified as E. coli (n = 27).These isolates were genetically highly diverse since they belonged to 20 different STs, indicating the absence of the large-scale spread of one or two successful clones among these birds that live in large groups.Only ST38, ST155, and ST162 belonged to pandemic clones.The remain ing 37.2% (n=16) were identified as E. marmotae, among which 14/16 belonged to ST133.These isolates mainly clustered according to their city of origin, differing by 0-5 SNPs when originating from the same city and by 45 or 198 SNPs when originating from different cities (Table S2).This strongly suggests inter-individual transmissions and micro-evolutions at the city level.Only one E. marmotae from Gabès (#60313) clustered with the unique isolate collected from Bizerte (#60314); this genetic proximity cannot be explained since the two cities are 475 km apart.E. marmotae, renamed in 2015 after its host Marmota himalayana (29), was formerly known as E.coli cryptic clade V, a clade especially associated with birds (30).This new species, which is phenotypically identical to E. coli, has since then been reported as a bla CTX-M-1 carrier from an Alpine marmot in a Belgian zoo (31), and as a bla CTX-M-32 carrier from a red deer in Poland (32); these reports, in addition to the starlings' samples here, suggest that wild animals are preferential hosts of E. marmotae.A recent publication analyzing the genomes of 41 E. marmotae isolates suggested that this species can be pathogenic for humans but is still carrying few resistance determinants, even if sporadic cases of ESBL-and carbapenemase-producing isolates were reported (33).Our study revealed that E. marmotae can be a frequent carrier of AmpC-and ESBL-conferring genes that can be shared with E. coli isolates present in the same niche.
The ESBL phenotype was due to the presence of four different genes (bla CTX-M-1 , bla CTX-M-15 , bla CTX-M-14 , and bla CTX-M-27 ) but only bla CTX-M-14 was identified in E. marmotae.The bla CTX-M-14 gene was carried by an IncHI2/ST3 plasmid in all E. coli and E. marmotae isolates, suggesting that this plasmid can spread easily and equally in both Escherichia species.This plasmid was also geographically widespread since it has been isolated in all three cities where birds were captured, even though it was much less present in Zaghouan.The backbone of this IncHI2 plasmid has already been described in E. coli and Salmonella Schwarzengrund isolates displaying different resistance genes.The resistance gene modules aph( 6)-Id-aph(3″)-Ib and bla CTX-M-14 -fos3 described here were each flanked by insertion sequences and displayed genetic organizations already identified in other non-identical IncHI2/pST3 plasmids, indicating the plasticity of the resistance region.The presence of the fosA3 gene is of concern since fosfomycin is one of the last resort antibiotics to treat carbapenemase-producing Gram-negative bacteria.The fosA3 gene has often been associated with ESBL-conferring genes (34), including on IncHI2 plasmids in the same genetic context as described here in both E. coli and E. marmotae (17,35), but it has only been reported twice in wild birds: in a German black kite and a frigate bird in a pristine Brazilian atoll (36,37).Its presence in migratory birds living in large colonies favoring close contacts and genetic transfers should thus be monitored.
The second genetic determinants shared between E. coli and E. marmotae were the IncI1/bla CMY-2 plasmids, which have only been identified in Zaghouan.These plasmids only carried the bla CMY-2 gene with no additional gene conferring resistance to nonβ-lactam antibiotics, as often seen in IncI1/pST2 and pST12 subtypes (38).The ISEcp1bla CMY-2-blc-sugE fragment, which is a common genetic environment for bla CMY-2 , was always found in the finQ gene (38,39).All plasmids were genetically highly similar, except that those identified in E. coli lacked the repI1 allele.Given the rare allelic profile identified in both E. coli and E. marmotae for the four other genes of the pMLST scheme, we can hypothesize that the IncI1 plasmid with the complete pMLST profile is the common ancestor, which then underwent recombination and excision before spreading in E. coli.Interestingly, plasmids showing the closest pMLST types all originated from humans or pigs in Taiwan, except two originating from a dog in the UK and from an unknown origin in Sweden.
In conclusion, our study revealed a surprisingly high prevalence of ESC-R isolates in European starlings in Tunisia.This was mostly due to the epidemic success of the bla CTX-M-14 /IncHI2/pST3 mostly in Bizerte and Gabès, and bla CMY-2 /IncI1 plasmids exclusively in Zaghouan.In addition, a few more sporadic resistance determinants were found, such as bla CTX-M-27 /IncF/F2:A-:B10, bla CTX-M-1 /IncHI2/ST4, and the chromoso mally encoded bla CTX-M-15 genes.Our results also highlighted the importance of E. marmotae-and notably the ST133 lineage-as an ESC-R commensal bacterial species in wild birds, in coherence with other reports of E. marmotae in wildlife.The absence of data on the resistance genes/plasmids found in humans, animals, and the environment of these three cities, and especially in the olive farms that are seasonally invaded by starlings, is a limitation of this study-as well as a potential perspective for further studies, since the real risk of AMR transmission cannot be assessed.

Wild bird sampling and bacterial isolation
Wild birds (n = 200) were purchased alive on the Sousse market, Tunisia, between January and February 2022 during the legal hunting period.Birds were caught in Bizerte, Zaghouan, or Gabès (Fig. S1), and kept in aviaries for as short a time as possible, without being fed.The intestine of each bird was collected immediately after the death of the bird using a sterile scalpel and stored at −20°C.Intestinal content was emptied in 10 mL of Trypto-casein soy broth (Biokar), homogenized and incubated for 18-24 h at 37°C.Overnight cultures were inoculated on selective MacConkey agar plates supplemented with cefotaxime or imipenem (2 mg/L), for the detection of ESC-or CP-resistant Enterobacterales.One colony per morphology and per plate was picked up.Identification was performed using API20E galleries (bioMérieux).

MinION long-read sequencing
MinION long-read sequencing libraries were prepared according to the Oxford Nanopore Technologies using the native barcoding expansion kit (EXP-NBD104) and the ligation sequencing kit (SQK-LSK109).Sequencing was performed on a MinION sequencer using a SpotON Mk 1 R9 version flow cell (FLO-MIN106D).Assembly of both Illumina and Nanopore reads was performed using Unicycler.The assembled contigs were annotated using Bakta (Web version 1.7.0/DB:5.0.0)(41).

Molecular characterization
The genetic determinants carrying the ESBL/AmpC genes were detected by Southern blot on I-Ceu1or S1-digested DNA Pulsed-Field Gel Electrophoresis (PFGE) as already described (42), using the DIG DNA Labeling and Detection Kit (Roche Diagnostics, Meylan, France) according to the manufacturer's instructions.

In silico characterization
The PROKSEE server (https://proksee.ca/)was used to generate high-quality navigable maps of each circular plasmid described and the visualization of genetic environment of chromosomal genes (43).Circular comparison between the plasmids belonging to the same incompatibility group and the closest plasmids found in the NCBI database was carried out on the PROKSEE server using BLAST analysis (BLAST+ 2.12.0).

a
Tests were performed using disc diffusion, except for colistin, for which the minimum inhibitory concentration was determined using broth microdilution.

FIG 3
FIG 3 Comparison of plasmids carrying the bla CTX-M-14 (A), bla CTX-M-27 (B), bla CTX-M-1 (C), and the bla CMY-2 (D) genes with the closest plasmids found in the publically available databases using the PROKSEE tool.

TABLE 1
Antimicrobial susceptibility phenotypes of all 43 E. coli and E. marmotae isolates characterized in this study a