Plasmid-Mediated Resistance to Extended-Spectrum Cephalosporins and Resistance to Fluoroquinolones in Escherichia Coli Isolates from Black-headed Gulls (Larus

Although resistance to uoroquinolones is common in E. coli isolates from farm and game animals in Serbia, currently no data are accessible on the occurrence of antibacterial resistances in E. coli isolates from gulls. Therefore, 45 cloacal swabs and 50 fecal samples from black-headed gulls were investigated for the presence of Escherichia coli isolates resistant to antibiotics. Multidrug resistance was detected in 22 E. coli isolates. High level resistance to uoroquinolones was found in ten isolates with MIC values of ciprooxacin ranging from 4 to 32 mg/L. Genotyping revealed single or double mutations in the quinolone resistance determining region (QRDR) of the gyrA or gyrA, parC and parE genes, respectively. Ten isolates showed resistance to extended-spectrum cephalosporin antibiotics. These ten isolates belonged to phylogenetic group B2 (ve isolates), group D (four isolates) and group B1 (one isolate). An extended-spectrum β-lactamase resistance phenotype was detected in one isolate which carried the bla CTX-M-1 gene on a plasmid of the I2/FIB replicon type. Nine isolates carried bla CMY-2 genes, which were detected on conjugative plasmids in seven isolates. One transconjugant also carried hly, iroN, iss, ompT and cvaC virulence genes on the plasmid. Five different sequence types (ST38, ST2307, ST224, ST162 and ST34) were detected in E. coli isolates with ESBL or AmpC phenotype and genotype. The resistant to extended-spectrum cephalosporins in mating experiments with the recipient strain E. coli HK225. For these experiments, Luria medium (Becton Dickinson, MD, was supplemented with 2 mg/L cefotaxime and 100 mg/L rifampicin. The isolates were shaken in the liquid medium for 30 minutes and the obtained transconjugants were used for further analysis. DNA preparation and restriction enzyme digestion for Pulsed-eld Gel Electrophoresis (PFGE) of seven E. coli isolates, the recipient strain and the resulting transconjugants were performed as previously described, (Jovcic et al., 2011). The DNA was either digested with the restriction enzyme XbaI or with S1 nuclease or was not digested. PFGE was performed with a 2015 Pulsafor unit (LKB Instruments, Bromma, Sweden) equipped with a hexagonal electrode array for 18 h at 300V at 9ºC. The gels were stained with ethidium bromide and photographed under UV illumination. Transconjugants were conrmed based on the comparisons of XbaI macrorestriction proles and the presence of plasmid bands in the S1 nuclease assay. The eciency of conjugation was estimated according to Phornphisutthimas et al. (2007). qnrA, qnrB, qnrC,qnrD, aac-Ib-cr,qepA, oqxA, oqxB) were not identied in E. coli isolates. Sequencing of the QRDR was not done in isolates 1774/18 and 1776/13 resistant to CIP and in isolates resistant to NAL (1773/59, 1774/1, 1774/14, 1774/18, 1774/25, and 1776/4). out of 96 isolates. However, ve isolates which were resistant only to beta-lactam antibiotics carried the bla CMY−2 gene on transferable plasmids. Virulence genes (hly, iroN, iss, ompT and cvaC) were detected on one conjugative CMY plasmid as well. In isolates with high resistance to uoroquinolones, the resistance was due to multiple mutations in the topoisomerase genes. This result may have been caused by environmental contamination in Serbia.


Resistance and virulence gene screening and sequencing
The primer sequences, annealing temperatures and references used for the resistance and virulence gene screening including primers used for phylogenetic and replicon typing, by PCR are presented in Supplementary Table 1. The master mix for beta-lactam gene detection was prepared by using a commercial kit One Taq Hot Start 2x Master Mix M0484, (New England BioLabs, Ipswich, MA, USA) and amplicons were puri ed using the commercial kit Monarch, PCR and DNA Clean up Kit (New England, BioLabs, Ipswich, MA, USA) and then sent to Macrogen, Amsterdam, Holland for sequencing of both strands. For sequencing the quinolone determining regions (QRDRs) of the topoisomerase genes, the Qiagen Hot Star Taq Master Mix commercial kit (Qiagen, Hilden, Germany) was used for DNA extraction followed by a DNA puri cation step using the QiAquick PCR puri cation kit of the same manufacturer. The sequences of the bla CMY−2 and bla CTX−M−1 genes and of the QRDR genes were analyzed with the Basic Local Alignment Search Tool-nucleotide program-BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn).
Mating experiment and S1 nuclease assay The isolates that were resistant to extended-spectrum cephalosporins were included in mating experiments with the recipient strain E. coli HK225. For these experiments, Luria Bertani medium (Becton Dickinson, Sparks, MD, Le Pont de Claix, France) was supplemented with 2 mg/L cefotaxime and 100 mg/L rifampicin. The isolates were shaken in the liquid medium for 30 minutes and the obtained transconjugants were used for further analysis. DNA preparation and restriction enzyme digestion for Pulsed-eld Gel Electrophoresis (PFGE) of seven E. coli isolates, the recipient strain and the resulting transconjugants were performed as previously described, (Jovcic et al., 2011). The DNA was either digested with the restriction enzyme XbaI or with S1 nuclease or was not digested. PFGE was performed with a 2015 Pulsafor unit (LKB Instruments, Bromma, Sweden) equipped with a hexagonal electrode array for 18 h at 300V at 9ºC. The gels were stained with ethidium bromide and photographed under UV illumination. Transconjugants were con rmed based on the comparisons of XbaI macrorestriction pro les and the presence of plasmid bands in the S1 nuclease assay. The e ciency of conjugation was estimated according to Phornphisutthimas et al. (2007).

Multilocus sequence typing of transconjugants
Multilocus sequence typing (MLST) was performed by PCR ampli cation and sequencing of seven housekeeping genes (adk, fumC, gyrB, icd, mdh, purA and recA) using primers and conditions de ned at the EnteroBase E. coli MLST database (https://enterobase.readthedocs.io/en/latest/mlst/mlst-legacy-infoecoli.html) (Wirth et al., 2006). According to the allele pro le, the isolates were assigned to a speci c sequence type (ST) using the EnteroBase database (Supplementary Table 2). Sequence type designation was not possible for two isolates (1773/47 and 1773/64), which means that these may belong to some new sequence type and thus must be analyzed by the whole genome sequencing approach for the nal MLST con rmation.

Results
Multidrug resistance in E. coli isolates from black-headed gulls Resistance to antibiotics was detected in 44 out of 95 E. coli isolates from black-headed gulls (Table 1). Resistance to three or more antibiotics of different classes was identi ed in 24 isolates. However, resistance to colistin was not found (Table 1). A class 1 integron was detected only in one isolate (1773/30) which was resistant to six different antibiotics including one combination of them: AMP, CHL, STR, SA, TET, TMP/SXT. Resistance to ampicillin was most frequently detected -in 34 isolates in total-and was associated with resistance to streptomycin, sulfonamides and trimethoprim in 15 isolates. The most frequently detected gene that was responsible for resistance to beta-lactams was bla TEM (24 isolates), followed by bla CMY−2 (9 isolates), while bla CTX−M−1 was con rmed in only one isolate. The second most common resistance was found to tetracyclines, which was conferred by tet(A) or tet(B) genes alone (in 20 isolates and 4 isolates, respectively) or by both of them (2 isolates). Some E. coli were found to be resistant to streptomycin (20 isolates), which was mediated by the combination of strA and strB genes, which encode aminoglycoside phosphotransferases APH(3″)-Ib and APH(6)-Id, respectively. Resistance to trimethoprim was detected in 14 isolates among which ve carried dihydrofolate reductase genes of type dfrA7/17 ordfrA5/14, whose products catalyse the reduction of dihydrofolate to tetrahydrofolate. Two other genes, dfrA1 and dfrA12, coding for dihydrofolate reductase enzymes, were detected in three and one isolates, respectively. Resistance to chloramphenicol was mediated by the gene cat1 (four isolates), which encodes a chloramphenicol acetyltransferase protein that inactivates the antibiotic by an acetylation mechanism. Another isolate carried the cmlA gene encoding a speci c transporter protein. The genes involved in resistance to sulphonamides were either sul2 (in 14 isolates) or sul3 (two isolates), while in four isolates both sul1 and sul2 genes were identi ed. In addition, the int1 gene coding for class 1 integrase enzymes was found in six isolates ( Table 1).
Characterization of E. coli isolates resistant to extended-spectrum beta-lactam antibiotics Ten E. coli isolates were resistant to extended-spectrum cephalosporin antibiotics (nine isolates were resistant to cefotaxime, ceftazidime, cefpodoxime and cefoxitin, while one isolate (1773/80) was resistant to cefotaxime and cefpodoxime ( Table 2). Out of these ten isolates, three (1773/50, 1773/52, 1774/1) were co-resistant to quinolones and tetracyclines and were therefore classi ed as multidrug-resistant. Six of the E. coli isolates carried the bla CMY−2 gene and exhibited a resistance phenotype with resistance only to beta-lactam antibiotics. The remaining isolate 1773/80 carried the bla CTX−M−1 gene. However, the plasmid Inc I1/FIB carrying this resistance gene was not transferred to the recipient strain in the conjugation experiments (Table 2). Furthermore, this isolate did not harbor any of the seven APEC virulence genes. In contrast, in the mating experiments, seven of the nine E. coli isolates carrying a bla CMY−2 gene were able to transfer the corresponding bla CMY−2 -bearing plasmid to the recipient strain. It was shown that isolate 1773/50 carried a 95 kb conjugative plasmid, replicon type Inc I1/ FIB, with the bla CMY−2 gene and additional hly, iroN, iss, ompT and cvaC virulence genes ( Table 2). The other conjugative plasmids were either of replicon type I1 or I1/FIB but did not carry any of the virulence genes tested. The conjugation e ciency of these IncI or IncI/FIB type plasmids was moderate to high for isolates 1773/7, 1773/67 and 1775/17 (3.36x10 3 to 2.66x10 1 cfu), while for the rest of the isolates the conjugation transfer was lower (Table 2).  Table 2) Phylogenetic analysis was carried out on all the isolates conferring resistance to extended-spectrum cephalosporins. Five isolates belonged to the phylogenetic-group B2, four isolates belonged to group D and one isolate was assigned to the B1 group. Five different sequence types (ST38, ST2307, ST224, ST162 and ST34) were detected in these E. coli isolates.

Resistance to uoroquinolones (FQ)
In this study, high-level resistance to uoroquinolones was detected in ten isolates. The MIC values in FQ resistant strains ranged from 4 to 32 mg/L (Table 3). Mutations in the quinolone resistance determining region (QRDR) of the topoisomerase genes were investigated in a few selected isolates (Table 3). For these isolates it was shown that high MIC values of CIP were achieved due to multiple mutations in the gyrA, parC and/or parE genes (Table 3), while single point mutations in the gyrA gene were detected in isolates resistant to NAL, resulting in amino acid transitions Ser83→Leu or Asp87→Asn. Plasmid-mediated resistance (PMQR) determinants were not found.

Discussion
In the course of the present study, it was shown that almost half of the black-headed gulls living in the wild carried resistant or multi-resistant E. coli. The resistance patterns, but also the resistance genes detected in the E. coli isolates were similar to those of other research studies, in which fecal samples from waterfowl were taken (Dolejska et al., 2007;Poeta et al., 2008;Dolejska et al., 2009;Literak et al., 2010;Tausova et al., 2012). However, the signi cant resistance to uoroquinolone in E. coli isolates from gulls in Serbia implies the overuse of these antibiotics in human and veterinary medicine and the unsafe disposal of communal and medical waste in Serbia.
In the present collection of isolates, resistance to extended-spectrum cephalosporins was mediated by the plasmid-borne resistance gene bla CMY−2 except in one case where the bla CTX−M−1 gene was identi ed. Often the CMY-2 plasmid carriers were resistant only to beta-lactam antibiotics. A similar result was obtained in another study, in which only three out of eight cephalosporin-resistant E. coli isolates with a bla CMY−2 gene from gulls and bald eagles from Alaska were multidrug-resistant (Ahlstrom et al., 2018). However, in E. coli isolates from food-producing animals, the bla CTX−M−1 gene is widely distributed in the Mediterranean area (Dandachi et al., 2018). The CTX-M family is also prevalent in Enterobacteriaceae in many European countries, mainly due to the epidemic spread of resistance plasmids. It is important that CTX-M carriers were found not only in E. coli and Klebsiella pneumoniae isolates from hospital patients but also in patients with community-acquired infections (D' Andrea et al., 2013;Canton et al., 2014). The bla CTX−M−1 gene has also been detected in E. coli isolates from Serbia from cases of clinical bovine mastitis, from diseased pigs and wildlife Velhner et al., 2018), and in this work also in an isolate from a gull.
We detected several important virulence genes in the E. coli isolates conferring resistance to beta-lactam antibiotics. These virulence genes comprised genes encoding a siderophore receptor for the iron acquisition mechanism (iroN), an episomal outer membrane protease (ompT), a putative avian hemolysin (hly), a serum survival protein (iss) and the colicin V structural gene (cvaC), which is important for increasing the adhesion and invasiveness of E. coli and other species of the Enterobacteriaceae family and which are frequently identi ed in avian pathogenic E. coli-APEC isolates (Gilson et al., 1987;Johnson et al., 2008;Johnson et al., 2010).
In the analyzed collection of isolates, only those with the CMY plasmid carried APEC virulence genes. Our results are therefore similar to those presented in the research by Touzain et al. (2018). In their work none of the E. coli isolates from diseased broilers, which contained the bla CTX−M−1 gene on IncI1/ST3 conjugative plasmids, carried APEC virulence genes. However, virulence genes were found on bla CMY−2 plasmid of the IncF replicon type (Touzian et al., 2018).
The genotypes of the ESBL-producing E. coli from gulls living in the vicinity to a dense human population were similar to the genotypes found in human isolates in southern France (Bonnedahl et al., 2009) and Sweden (Bonnedahl et al., 2010). Therefore, E. coli from gulls can serve as a biological indicator of environmental contamination due to their habits of living near humans and feeding on land lls along or off the coast (Bonnedahl et al., 2009;Bonnedahl et al., 2014).
Twelve E. coli isolates from gulls in Barrow, Alaska were of sequence type ST38 and carried the bla CTX−M−14 gene (Bonnedahl et al., 2014). In this work only isolate 1773/7 was identi ed as sequence type 38 and this carried the IncI1/CMY plasmid. In the European Union, the ST38 lineage is considered typical for poultry but has also been found in human E. coli isolates. It was evident that in E. coli isolates from Germany IncK, IncI and IncA/C plasmids most frequently carry the bla CMY−2 gene, while IncA/C plasmids were the most common CMY carriers in North America (Pitech et al., 2018). Horizontal transfer via plasmids is perhaps the most likely mechanism of dissemination of the bla CMY−2 gene although the epidemiological spread of speci c lineages such as CMY-2 producing ST131 is involved in the transmission of CMY plasmid as well. Nevertheless, a common ancestor of E. coli isolates carrying the bla CMY−2 gene has been identi ed in genetically distant strains suggesting that bacteria change over time due to their genome plasticity and diversity (Pitech et al., 2018). It was found that the successes in the proliferation of CMY-2 plasmids depend on the selective pressure posed by the use of antibiotics or on the size of the plasmid since larger plasmids, such as CMY carriers, are associated with a signi cant tness cost (Subbiah et al., 2011). Therefore, the long-term stability of the bla CMY−2 plasmids in E. coli isolates from gulls, which are discussed in this study, should be determined in the future using in vitro experimental approaches.
In this work, we detected high-level resistance to FQ in several isolates. However, resistance to FQ was also observed in E. coli isolates from mallards and hearing gulls residing at the Polish coast of the Baltic sea (Literak et al., 2010) and from feces of gulls, pigeons and birds of prey in Portugal, Sweden and Spain (Vredenburg et al., 2013). Resistance to FQ antibiotics and extended-spectrum cephalosporins was also recently found in extra-intestinal pathogenic E. coli (ExPEC) isolates from silver gulls residing at the coastline in Australia which included pandemic ExPECST131 strains belonging to clade C (C1-H30-R and C2-H30-Rx), (Mukerji et al., 2019).
Resistance to beta-lactam antibiotics and uoroquinolones in bacteria isolated from wild animals, including wild birds, is of concern because the environment appears to be contaminated by anthropogenic activities, affecting the bacterial ora of wildlife (Mukerji et al., 2019). While ESBL-producing E. coli isolates from food-producing animals are often multi-resistant due to co-selection mechanisms (Michael et al., 2017), E. coli isolates from black-headed gulls carrying the plasmid-borne AmpC gene (bla CMY−2 ) appear to be resistant only to beta-lactam antibiotics (Atterby et al., 2016;Touzian et al., 2018). It must also be pointed out that the global spread of epidemic E. coli clonal lineages is of great importance in medicine and the spread of such isolates needs to be closely monitored (Pitout and DeVinney, 2017).

Conclusion
In conclusion, multidrug resistance in E. coli isolates from black-headed gulls residing in the city of Novi Sad was detected in 22 out of 96 isolates. However, ve isolates which were resistant only to beta-lactam antibiotics carried the bla CMY−2 gene on transferable plasmids. Virulence genes (hly, iroN, iss, ompT and cvaC) were detected on one conjugative CMY plasmid as well. In isolates with high resistance to uoroquinolones, the resistance was due to multiple mutations in the topoisomerase genes. This result may have been caused by environmental contamination in Serbia.

Declarations
Author contribution MV, BJ and MK designed the study and analyzed the results, DT, KN and GL performed the sampling and the experiments, MV and BJ wrote the manuscript, CK reviewed the manuscript.

Disclosure statement
No competing nancial interest exists.
Funding Information