Prevalence and molecular characteristics of multi-resistant Escherichia coli in wild birds

Humans and animals share the same bacterial species including the resistant ones. For that reason, epidemiological studies in domestic and wild animals should be performed on a regular basis. Wild, particularly migratory birds, should be investigated as potential carriers of antimicrobial resistant bacteria that can be spread globally in a short time. The aim of this study was to investigate the prevalence and to characterize multi-resistant Escherichia coli in wild birds. Three hundred and ninety two samples were obtained from different bird species including gulls (Larus spp.), mallards (Anas platyrhynchos), mute swans (Cygnus olor), as well as other species of birds. Phenotypical and genotypical resistance of E. coli was investigated. In total 60 isolates from 179 tested were resistant to three or more antimicrobial classes and treated as multi-resistant (33.5%; 95% CI 21.56–45.44); the isolates were obtained from gulls, mallards, swans, and rooks. All of the strains demonstrated resistance to aztreonam and cefpodoxime. The most frequent resistance prevalence of the above-mentioned isolates in vitro was also demonstrated to ampicillin (82%), ampicillin/sulbactam (68%), cefazolin (66%), ceftriaxone (55%), and ciprofloxacin (47%). All E. coli isolates were susceptible to amikacin. The results of polymerase chain reaction confirmed the presence of the genes encoding resistance to beta-lactams, aminoglycosides, tetracycline, amphenicols, trimethoprim, and sulphonamides. Consequently, wild birds might constitute a potential hazard to human and animal health by transmitting multi-resistant E. coli strains to waterways and other environmental sources via bird faeces. Antimicrobial resistance, genes, migratory birds The resistance of bacteria to antimicrobials becomes a rising health problem in the world. The importance of wild birds as potential vectors of diseases has received renewed empirical interest recently, especially regarding human health. Escherichia coli is found in the environment, food and intestines of people and animals (Benskin et al. 2009; Wasinski et al. 2010). Due to their opportunistic and gregarious nature, gulls may be important reservoirs and vectors for anthropogenically derived faecal pathogens in coastal areas. Moreover, the migratory behaviour of birds has been proposed as a possible dissemination pathway of multi-resistant (MR) bacteria from human influenced habitats to remote places (Nelson et al. 2008; Guenther et al. 2012). Numerous wild bird species are attracted to untreated sewage, garbage dumps and manure, therefore, various pathogens such as Escherichia coli, Salmonella enterica and Campylobacter spp. might be prevalent in those birds (Quessy and Messier 1992; Hatch 1996; Moore et al. 2002; Fogarty et al. 2003; Waldenström et al. 2003). The aim of this study was to investigate the prevalence of and characterize the multi-resistant Escherichia coli in wild birds. ACTA VET. BRNO 2018, 87: 9-17; https://doi.org/10.2754/avb201887010009 Address for correspondence: Lina Merkeviciene Microbiology and Virology Institute Lithuanian University of Health Sciences Tilzes g.18, Kaunas, 47181 Lithuania Phone: +37062475685 E-mail: lina.merkeviciene@lsmuni.lt http://actavet.vfu.cz/ Materials and Methods

The resistance of bacteria to antimicrobials becomes a rising health problem in the world.The importance of wild birds as potential vectors of diseases has received renewed empirical interest recently, especially regarding human health.Escherichia coli is found in the environment, food and intestines of people and animals (Benskin et al. 2009;Wasinski et al. 2010).Due to their opportunistic and gregarious nature, gulls may be important reservoirs and vectors for anthropogenically derived faecal pathogens in coastal areas.Moreover, the migratory behaviour of birds has been proposed as a possible dissemination pathway of multi-resistant (MR) bacteria from human influenced habitats to remote places (Nelson et al. 2008;Guenther et al. 2012).Numerous wild bird species are attracted to untreated sewage, garbage dumps and manure, therefore, various pathogens such as Escherichia coli, Salmonella enterica and Campylobacter spp.might be prevalent in those birds (Quessy and Messier 1992;Hatch 1996;Moore et al. 2002;Fogarty et al. 2003;Waldenström et al. 2003).The aim of this study was to investigate the prevalence of and characterize the multi-resistant Escherichia coli in wild birds.
The ethical approval for this study was granted by the Lithuanian Environmental Protection Agency (permission number A4-8844).

Bacteriological and susceptibility testing
Material was inoculated onto McConkey Agar (Thermo Fisher, UK), for the screening of E. coli.Inoculated plates were incubated at +37 °C for 24 h.After incubation, the plates were screened for presumptive colonies.The randomly selected separate colonies (one colony per sample) were then identified using "Microgen Gram-Negative Plusˮ biochemical identification system (Microgen, UK).
The initial antimicrobial susceptibility testing was performed using the disc diffusion method according to Kirby-Bauer and the results were interpreted according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) clinical breakpoints except for cefazolin as no interpretative criteria were set for this antibiotic.Therefore, the clinical breakpoints for cefazolin were used as per recommendations of the Clinical Laboratory Standards Institute (CLSI 2015).The following discs with antimicrobials (Thermo Fisher, UK) were used: ampicillin (10 μg), gentamicin (10 μg), ciprofloxacin (5 μg), cefoxitin (30 μg), tetracycline (30 μg), imipinem (10 μg) and chloramphenicol (30 μg).Bacterial isolates resistant to three or more antimicrobial classes were then selected for the testing for minimal inhibitory concentrations (MIC's) using Sensititre ® plates and ARIS 2X automated system (Thermo Scientific, UK) for amikacin, ampicillin, ampicillin/sulbactam, aztreonam, cefazolin, cefepime, cefotetan, cefoxitin, cefpodoxime, ceftazidime, ceftriaxone, ciprofloxacin, gatifloxacin, gentamicin, imipenem, nitrofuration, tobramycin and trimethoprim/sulphamethoxazole.Interpretation of the results was carried out using the manufacturer's software (SWIN ® ) adapted to the EUCAST clinical breakpoints.

Molecular testing
DNA (deoxyribonucleic acid) for molecular analysis was obtained after bacterial lysis.The cultures were grown on a Mueller Hinton Agar (Liofilchem, Roseto, Italy) for 24 h and afterwards a loopful of colonies was taken from the surface of the agar and transferred to phosphate buffered saline (pH 7.3).The content was centrifuged for 5 min.The supernatant was discarded and the pellet was re-suspended in TAE (tris-ethylenediaminetetraacetic acid) buffer.The suspension was heated using a thermomixer at 100 °C for 10 min.Boiled suspension was transferred directly on ice and diluted by 1:10 in TAE.
The resistant E. coli isolates were tested by PCR (polymerase chain reaction) for the detection of genes encoding antimicrobial resistance (Table 1).The PCR mix (25 μl) constisted of 12.5 μl of the Dream Taq Green PCR Master Mix (ThermoFisher Scientific, Lithuania), 1 μl of each primer, 2 μl of the DNA template and 8.5 μl of water.The PCR conditions were as follows: initial denaturation for 5 min at 94 °C; 30 cycles of 30 s at 94 °C, 60 s at the temperature indicated for each primer pair in Table 1, 30 s at 72 °C; and a final extension step of 7 min at 72 °C.
The PCR products were determined by electrophoresis of 10 μl of the reaction products on 2% agarose gel with 1X TAE buffer at 105 V for 50 min.

Data analysis
Occurrences of multi-resistant (MR) isolates in faecal specimens were calculated by dividing the number of MR isolates by the total number of investigated specimens.For percentage estimates, Wilson (Score) 95% confidence intervals (CI 95%) and their ranges for true population proportions were calculated.The MR occurrence was determined by bird species as well.Antimicrobial resistance rates for each tested antimicrobial were given as numbers of resistant per total number of MR isolates.

Resistant E. coli prevalence in bird faeces
During the study, E. coli resistant to one or more antimicrobials was recovered from 179 samples out of 392 tested (45.6%; 95% CI 40.67-50.53).Resistant isolates were recovered only exceptionally from gulls, swans, ducks and rooks (Fig. 1).No resistant isolates were detected in the faeces of small passerine birds.

Genes encoding resistance
The genes encoding antimicrobial resistance to antimicrobials are presented in Table 3.

Discussion
The emergence and spread of multi-resistant bacteria in natural environments constitute a serious impact on animal and human health.We have detected 60 multi-resistant E. coli isolates in wild birds out of 176 tested (33.5%).The most prevalent resistance was detected towards aztreonam, cefpodoxime, other beta-lactams and fluoroquinolones.(Middleton et al. 2005;Guenther et al. 2010;Radhouni et al. 2012).The main resistance patterns of the isolates in the above-mentioned studies were resistance combination to ampicillin, streptomycin, and sulphonamides.In Lithuania, the most important species carrying multi-resistant E. coli strains were herring gulls, blackheaded gulls, rooks, hooded crows as well as waterfowl, in particular mallards and mute swans.Such data contradict the results of a previous study performed in Portugal where E. coli isolated from similar species were susceptible to most of the antimicrobials tested (Radhouani et al. 2010).In another study performed by Dolejska et al. (2007) in the Czech Republic, the rate of resistant isolates from black-headed gulls to at least one antimicrobial was 29.2% whereas 6.2% of the isolates were treated as multi-resistant ones.
There is evidence that international human travelling is one of the causes of the spread of multi-resistant E. coli worldwide (Peirano and Pitout 2010).Considering that migratory bird species, particularly gulls, are often carriers of multi-resistant bacteria, it might be assumed that they also pose a risk for the dissemination of multi-resistant isolates throughout different countries or regions.The recent discovery of colistin-resistant E. coli harbouring plasmid-mediated mec-1 gene in herring gulls in Europe (Ruzauskas and Vaskevičiute 2016) and in kelp gulls in South America (Liakopoulos et al. 2016) proves this opinion.The number of migrating birds worldwide has been estimated to be five billion a year (Berhold 2001) so the risk degree for the spread intensity of resistant bacteria is still undervalued.Beta-lactam antibiotics are the most frequently prescribed antibiotics worldwide to treat bacterial infections (Fernández-Aguadob et al. 2014).Therefore, it is not surprising that resistance to this class of antimicrobial agents poses increasingly complex problems for physicians.Extended-spectrum betalactamases (ESBLs) are a rapidly evolving group of beta-lactamases which share the ability to hydrolyze third-generation cephalosporins and aztreonam but are inhibited by clavulanic acid (Philippon et al. 1989).Our results demonstrated high rates of E. coli resistance to the 3 rd generation of cephalosporins and aztreonam.The bla CTX-M gene was detected in 63.2% of the isolates resistant to the 3 rd generation of cephalosporins.
The use of sulphonamides was restricted for food animals in the 1980s after a potential threat to human health from residues in foods of animal origin, and they are currently approved for use in treating calf scours.Resistance to sulphonamides is plasmid mediated but chromosomal mutations for sulphonamide resistance take place very slowly (Prescott et al. 2013).Different genes encoding resistance to this antimicrobial are already described.For instance, sul3 has been detected in E. coli isolates from different animals in Switzerland and Germany (Guerra et al. 2003;Perreten et al. 2003) while sul1 was the most prevalent among human isolates (Hammerum et al. 2006).Our study demonstrated that different sul genes were prevalent in E. coli carried by wild birds including sul1, sul2 and sul3, therefore, it suggests the possibility for birds to acquire antimicrobial resistant bacteria from different sources, including domestic animals and contaminated environment.
Tetracycline-resistance now occurs in anincreasing number of pathogenic, opportunistic, and commensal bacteria.Tetracycline-resistance is often due to the acquisition of new genes, which code for energy-dependent efflux of tetracyclines or for a protein that protects bacterial ribosomes from the action of tetracyclines.A limited number of bacteria acquire resistance by mutations, which alter the permeability of the outer membrane porins and/or lipopolysaccharides in the outer membrane, change the regulation of innate efflux systems, or alter the 16S rRNA (ribosomal ribonucleic acid) (Chopra and Roberts et al. 2001).The detection of tetA gene in almost 85% of tetracycline-resistant isolates in our study shows that the main mechanism of tetracycline resistance in E. coli isolates from wild birds is by active efflux.Similar results were published by Dolejska et al. (2007) where both tetA and tetB genes were detected in E. coli isolates.
This study demonstrated that wild birds, particularly those living in the urban enviroment are carriers of antibiotic resistant bacteria.These bacteria are often resistant to different classes of antimicrobials including those that are critically important for humansaminoglycosides, aminopenicillins and fluoroquinolones.Moreover, microbiota of wild birds share the same antimicrobial resistance genes that are important in aquired resistance of bacteria prevalent in humans and domestic animals.It is still unclear whether E. coli prevalent in birds can colonize humans and domestic animals but there are some studies that show a possible zoonotic transfer of similar E. coli strains among different hosts (Van den Bogaard et al. 2013).
Control measures for disposal of antibiotics should be tightened as residues of antimicrobials and resistant bacteria from the environment can be passed onto wild birds.

Fig. 1 .
Fig. 1.Distribution of 179 E.coli isolates resistant to at least one antimicrobial in different wild bird species (n).

Table 1 .
Antimicrobial resistance genes tested and oligonucleotide primers used in the study.
Other authors have reported data about resistant E. coli isolates obtained from different bird species including Canada geese, common buzzards, black kites, red kites, white-tailed eagles and goshawks

Table 1 (
continued).Antimicrobial resistance genes tested and oligonucleotide primers used in the study.

Table 3 .
Presence of the tested genes encoding antimicrobial resistance in E. coli isolated from wild birds.

Table 3 (
continued).Presence of the tested genes encoding antimicrobial resistance in E. coli isolated from wild birds.