Antibiotic Resistance and Virulence Profiles of Escherichia coli Strains Isolated from Wild Birds in Poland

Wild animals are increasingly reported as carriers of antibiotic-resistant and pathogenic bacteria including Enterobacteriaceae. However, the role of free-living birds as reservoirs for potentially dangerous microbes is not yet thoroughly understood. In our work, we examined Escherichia coli strains from wild birds in Poland in relation to their antimicrobial agents susceptibility, virulence and phylogenetic affiliation. Identification of E. coli was performed using MALDI-TOF mass spectrometry. The antibiotic susceptibility of the isolates was determined by the broth microdilution method, and resistance and virulence genes were detected by PCR. E. coli bacteria were isolated from 32 of 34 samples. The strains were most often classified into phylogenetic groups B1 (50%) and A (25%). Resistance to tetracycline (50%), ciprofloxacin (46.8%), gentamicin (34.3%) and ampicillin (28.1%) was most frequently reported, and as many as 31.2% of E. coli isolates exhibited a multidrug resistance phenotype. Among resistance genes, sul2 (31.2% of isolates) and blaTEM (28.1%) were identified most frequently, while irp-2 (31.2%) and ompT (28.1%) were the most common virulence-associated genes. Five strains were included in the APEC group. The study indicates that wild birds can be carriers of potentially dangerous E. coli strains and vectors for the spread of resistant bacteria and resistance determinants in the environment.


Introduction
Escherichia coli (E. coli) is a common member of the natural intestinal microflora of humans and animals, including birds. However, in addition to non-pathogenic commensal strains, there are also pathogenic E. coli strains involved in many types of infections.
APEC strains produce a variety of virulence factors facilitating host tissue colonization, including adhesins (encoded by papC and tsh), iron uptake systems (iutA, irp2, sit and iroN), serum resistance (iss, ompT and kpsII), toxins (vat), and others (cvi/cva and etsB) [5]. Many genes coding for these virulence factors are often clustered together on chromosomes and plasmids and can be transmitted by horizontal gene transfer [6]. Moreover, APEC and other ExPEC strains that cause infections in humans, are quite closely phylogenetically related and share some of the same virulence genes. Therefore, APEC strains may hypothetically have zoonotic potential and pose a health risk to humans [7].
In recent years, a significant increase in antibiotic resistance has been noted not only among pathogenic strains, but also among commensal E. coli. Antibiotic-resistant E. coli strains are currently found in a variety of environments, and their spread is promoted by the extreme genomic plasticity of these bacteria [8]. A report from the European Antimicrobial Resistance Surveillance Network (EARS-Net) from 2020 shows that more than half (57.1%) of the E. coli strains from humans reported in 2019 showed resistance to at least one of the groups of antibiotics under surveillance. Resistance to penicillin was most commonly reported (57.1%), followed by fluoroquinolones (23.8%), third-generation cephalosporins (15.1%), and aminoglycosides (10.8%) [9]. A high percentage of antibiotic-resistant E. coli isolates is also noted in farm animals, especially poultry. In Poland, E. coli strains from broiler chickens are most commonly found to be resistant to penicillin, fluor-quinolones and tetracyclines, and antibiotic resistance applies both to isolates from non-clinical birds and to cases of colibacillosis [10].
The occurrence of resistant E. coli bacteria in wild birds, including multidrug resistant strains, has been demonstrated by several authors conducting research in various countries around the world, including Poland [11][12][13][14][15]. They can also be a reservoir of APEC, as well as E. coli strains pathogenic to humans, including E. coli serotype H7:O157 [16,17]. Due to their migratory lifestyle, free-living birds can be contributing vectors for the spread of potentially dangerous E. coli strains between ecosystems.
Information on the characteristics of E. coli strains in free-living birds in Poland is limited. Therefore, we have conducted a study aimed at assessing the occurrence of antibiotic-resistant and virulent E. coli from wild birds inhabiting suburban areas of southeastern Poland. Referring to this fact, the intention of the study was to determine, whether wild birds are carriers and transmitters of dangerous Enterobacteriaceae strains that, when spreading in the environment, may pose a threat to poultry farms.

Antibiotic Resistance of E. coli
Multidrug-resistant (MDR) bacteria show non-susceptibility to at least one agent in three or more antimicrobial categories [18]. Analysis of resistance to antimicrobial agents based on MICs showed that 31.2% (n = 10) of the E. coli strains were resistant to three or more groups of antibiotic and 28.1% (n = 9) were resistant to two groups of antibiotic (Table 1). Only one E. coli isolate (e48) was found to be susceptible to all antimicrobials tested. As many as, 50% of isolates (n = 16) were resistant to tetracycline and 10 strains showed intermediate susceptibility to this antibiotic. Resistance to ciprofloxacin was demonstrated in 46.8% (n = 15) of isolates. Among aminoglycosides, 34.3% (n = 11) of isolates were resistant to gentamicin, and 40.6% (n = 13) showed intermediate susceptibility to this antimicrobial agent, while 18.7% (n = 6) of E. coli strains showed resistance to kanamycin. Lower incidence of resistance was found for trimethoprim/sulfamethoxazole (34.3%, n = 11) and ampicillin (28.1%, n = 9). Resistance to chloramphenicol was noted sporadically, with only two (6.25%) resistant isolates and one with intermediate susceptibility (3.1%) ( Table 2). MDR E. coli isolates came from mallard (Anas platyrhynchos) (n = 6), Eurasian golden oriole (Oriolus oriolus) (n = 1), Eurasian sparrow hawk (Accipiter nisus) (n = 1), common buzzard (Buteo buteo) (n = 1) and little bittern (Ixobrychus minutus) (n = 1). Among the MDR E. coli strains, the most common resistance profile was tetracyclinetrimethoprim/sulfamethoxazole-ampicillin (Table 1).

Detection of Resistance and Virulence Genes
All isolates showing resistance to ampicillin (n = 9) contained the bla TEM gene (encoding β-lactamase TEM capable of inactivating penicillin, including ampicillin), and the aphA1 gene (coding for aminoglycoside 3 -phosphotransferase) was found in all kanamycinresistant E. coli strains (n = 6). The sul2 gene (encoding the mutant dihydropteroate synthase enzyme that does not bind sulfonamides) was detected in 10 of 11 E. coli phenotypically resistant to trimethoprim/sulfamethoxazole, the sul3 gene was detected in three strains, and one strain resistant to these two antibiotics additionally had the dhfrI gene (mediating trimethoprim resistance). The tetA gene was found in four of 16 isolates showing resistance to tetracycline. Regarding streptomycin resistance genes, which belong to aminoglycoside group, aadA was found in two of all examination strains and gene strA/strB was also found in two strains. None of the E. coli isolates contained the tetB, aac(3)-IV, aac(3)-II, aphA2, qnr, catI or sul1 gene ( Table 1).

Determination of E. coli Phylogenetic Groups
Phylogenetic groups of E. coli were determined based on the electrophoretic profiles of multiplex PCR amplicons (yja, TspE4.C2, chuA, svg and uidA). Half (50%) of the isolates (n = 16) belonged to group B1, 25% of isolates (n = 8) were assigned to group A, 12.5% (n = 4) to B2 and 12.5% (n = 4) to group D. None of the isolates belonged to group B2 1 (all isolates were svg-negative) ( Figure 1). Most of the MDR strains belonged to the type A phylogenetic group (seven of 10 MDR strains), two to group B1 and one to group D. Strains containing virulence factors (n = 21.7%) were assigned to various phylogenetic groups, including group A (n = 7), B1 (n = 5), B2 (n = 4) and D (n = 4). As many as 10 of 12 isolates in which no virulence gene was detected represented phylogenetic group B1. In addition, seven isolates in group B1 were negative for both virulence and resistance genes.
The incidence of virulence genes ompT, iutA, and iucD in the phylogenetic group B1 is significantly lower than in A, B2 and D. The observed relationship is at an average level as indicated by the analysis of the contingency coefficient (0.52, 0.47, 0.48, respectively). For these three genes, a significant correlation was also observed in the decrease in virulence with the increase in the number of observations in the B1 = phylogenetic group, which confirms the strength of the relationship observed. Taking into account all the virulence genes tested, the B1 phylogenetic group was significantly less virulent (p = 0.009) than the others (contingency coefficient 0.55).

Discussion
So far, only a few studies have been carried out on antibiotic resistance and the presence of virulence genes in E. coli bacteria from free-living birds in Poland. Antibioticresistant E. coli strains, including MDR, are widely isolated from humans [19], farm animals [20,21] and increasingly from wildlife [22]. Our study has shown that resistant E. coli strains, including MDR, are spread among wild birds. The frequency of resistance is much higher than that observed for E. coli originating in water birds from the Baltic Sea coast in Poland [14] and in birds living in other parts of Europe [11,12,23].
The high frequency of tetracycline resistance (50%) in the strains tested in our study is consistent with the results of Pinto et al. [13] and Radhauani et al. [12], who found that over 70% of E. coli from Portuguese wild birds showed a tetracycline-resistant phenotype. It should be emphasized, however, that several other authors noted less than 22% tetracycline-resistant E. coli from free-living birds [11,[23][24][25][26]. In E. coli isolates from poultry, the frequency of resistance to tetracycline is over 70% [27][28][29]. The tet(A) gene found in several phenotypically resistant strains is often detected in Enterobacteriaceae from poultry [30] and was recorded in E. coli isolates from wild birds of the Azores Archipelago [31].
In the case of ampicillin, the resistance rate in E. coli strains was 28.1%, and the results were similar to those reported by other authors [11,24,26], who identified 18.1-19.5% of isolates as ampicillin-resistant. In poultry, significantly higher resistance to ampicillin (≥63%) is generally found [27,28]. Phenotypic resistance correlated with the presence of the bla TEM gene, that was also previously found in ampicillin-resistant E. coli from buzzards (Buteo buteo) in Portugal and wild birds in Poland [12,14].
The percentage of ciprofloxacin-resistant isolates reported in our study (nearly 50%) is consistent with the results of Radhauani et al. [12], who found a resistance rate of 50% in E. coli isolates from buzzards. Other authors, however, note a much lower frequency of fluoroquinolone resistance in E. coli from wild birds, ranging from 3% to 22% [24,25], and some do not report resistance to this group of antimicrobial agents at all [15,23]. In E. coli from broiler chickens, the percentage of isolates resistant to ciprofloxacin is usually high, above 50% [27,28].
An interesting finding was the high percentage of gentamicin-resistant strains confirmed in our study (75%), which significantly differs from the results of other research projects. Several authors [15,24,26] observed fewer than 1% gentamicin-resistant strains among E. coli isolates from wild birds. To rule out any error in determining the gentamicin MIC value, we additionally used the disc diffusion method (data not shown). Despite the high percentage of gentamicin-resistant strains, the resistance genes that usually determine resistance to gentamicin in Enterobacteriaceae, i.e., aac(3)-II and aac(3)-IV (coding for aminoglycoside acetyltransferase), were not detected. It is therefore possible that resistance to gentamicin in the strains is the result of the production of other aminoglycoside-modifying genes (e.g., ant-2) or other resistance mechanisms [32].
The percentage of trimethoprim/sulfamethoxazole-resistant isolates recorded in our study (over 35%) is similar to the results of Radhouani et al. [12], who classified 22% of E. coli isolates from common buzzards in Portugal as phenotypically resistant. A much lower frequency of trimethoprim/sulfamethoxazole-resistant E. coli strains, of less than 5%, was observed in isolates from European starlings (Sturnus vulgaris) [15]. In line with other studies, we also note the common presence of the sul2 gene in trimethoprim/sulfamethoxazoleresistant E. coli isolates [15].
A high percentage of antibiotic-resistant strains found in wild birds in this study may be the result of acquisition of resistant strains from human sources, farms, or contaminated river water [33]. Wastewater and outflows from fields fertilized with manure make their way into rivers, and fecal bacteria may thus be transported over long distances.
Virulence genes characteristic of APEC, as well as the astA gene, were detected in most of the E. coli isolates. The most frequently identified gene was irp-2, which encodes iron-repressible high-molecular weight protein 2 (IRP-2), involved in iron acquisition. Some authors show a high prevalence of this gene among EAEC strains, although the role of IRP-2 in EAEC pathogenesis in unclear [34]. The irp-2 gene recorded in this study, as well as other genes suggested as predictors of the virulence of APEC strains, i.e., ompT, iutA, iss, pap-C, cva/cvi and tsh, are commonly found in pathogenic E. coli strains from poultry [35], but have also been reported in E. coli from wild birds [36]. Kuczkowski et al. [36] demonstrated the frequent occurrence of the irp-2 and astA genes (in 11.6% of isolates), while the iss, iucD, tsh, pap-C and vat genes were rarely found in E. coli isolates from wild water birds in Poland and the Netherlands. Borges et al. [16] showed that 30.6% of E. coli isolates taken from wild birds in Brazil were positive for at least one virulence gene, the most prevalent being iss, followed by sitA, traT, ompT, fyuA and irp2. Other virulence-associated genes, including iutA, iucC, iucD, tsh and cvaC, were detected sporadically. The astA gene found in several isolates is responsible for the production of heat-stable enterotoxin 1 (EAST1) characteristic of diarrheagenic EAEC strains [37]. The presence of the astA gene has also been confirmed in ExPEC strains derived from both humans (UPEC) and poultry (APEC) [36,38]. However, ExPEC isolates may also contain an astA pseudogene that has not been reported in diarrheagenic E. coli strains [38].
The dominance of E. coli group B1 isolates in this study is consistent with the results of other authors analyzing E. coli from wild birds [36,39]. Commensal E. coli with no pathogenic features which inhabit the gastrointestinal tract most often represent group A or B1, while ExPEC strains (including APEC) are usually assigned to phylogenetic groups B2 and D [2]. The E. coli strains used in these studies were not tested for pathogenicity, but we have noted a significantly lower incidence of virulence genes in isolates of the phylogenetic group B1. Four out of five strains qualified as APEC represented phylogenetic group A, and one strain represented group B1.

Identification of E. coli
The species of the isolates were confirmed by MALDI-TOF mass spectrometry (UltrafleXtreme MALDI-TOF, Bruker Daltonics, Hamburg, Germany) using a standard ethanol/formic acid extraction sample preparation procedure [40]. The mass spectra obtained from each isolate were processed with the MALDI Biotyper 3.0 software package (Bruker Daltonics, Hamburg, Germany), and the results were shown as the top 10 identification matches along with confidence scores ranging from 0.000 to 3.000, according to the manufacturer's criteria (www.bruker.com; accessed on: 19 September 2021).

Antibiotic Susceptibility Testing
Antibiotic profiles of E. coli strains were based on determination of the minimum inhibitory concentration (MIC) of the antibiotic defined by serial microdilution in Mueller-Hinton broth (Oxoid Ltd., Basingstoke, UK) on a 96-well flat-bottomed microplate (Medlab, Raszyn, Poland), according to standards developed by the Clinical and Laboratory Standards Institute [41].
The E. coli colonies grown on Columbia agar with 5% defibrinated horse blood were suspended in 0.85% NaCl solution to obtain a density corresponding to 0.5 on the Mc-Farland scale. Microdilution plates were inoculated with 50 µL of a 1:100-diluted (in Mueller-Hinton broth) inoculum and 50 µL of the appropriate antibiotic concentration (stock solutions were previously dissolved in Mueller-Hinton broth). Plates were incubated at 37 • C for 24 h under aerobic conditions [42]. An Escherichia coli ATCC 25922 reference strain was used as quality control. MIC breakpoint was defined as the lowest concentration of the substance at which no growth of the bacterial strains could be seen. Isolates were classified as susceptible, intermediate and resistant according to the threshold breakpoint proposed by the Clinical and Laboratory Standards Institute [41].

Detection of Resistance Genes
Genomic DNA from E. coli isolates was isolated using a Gene MATRIX Bacterial & Yeast Genomic DNA Purification Kit (Eurx, Gdańsk, Poland) following the manufacturer's instructions. The presence of genes conferring resistance to tetracyclines-tet(A) and tet(B); aminoglycosides-aphA1, aphA2, aac(3)-II and aac(3)-IV; sulfonamides-sul1, sul2 and sul3; β-lactams-bla TEM ; fluoroquinolones-qnr; trimethoprim-dhfrI; and chloramphenicol-catI and cm1A-were determined by PCR using the primers presented in Table A1. The reaction mixture used in the PCR assay contained 2.5 µL of 10× concentrated reaction buffer containing 25 mM MgCl 2 , 1 µL 25mM dNTPs mix, 1U AllegroTaq polymerase (Novazym, Poznań, Poland), 1 µL of each of the primers (10 µM) and 1 µL of the tested E. coli DNA in a total volume of 25 µL for each sample. Amplification reactions were performed in a thermocycler (Eppendorf Mastercycler Gradient, Westbury, NY, USA) using the following program: initial denaturation-1 cycle at 94 • C for 5 min, 30 cycles of 40 s at 94 • C, 40 s at 50-66 • C (according to the annealing temperature for the individual primers; Table A1), and 75 s at 72 • C, followed by 8 min of final extension at 72 • C. The PCR products in a volume of 8 µL were separated by electrophoresis (100 V) on a 1.5% agarose gel in 1 × TBE (Tris-borate-EDTA) buffer and visualized by SimplySafe staining (Eurx, Poland).
A 100-1000 bp molecular standard (Blirt, Gdańsk, Poland) was used to determine the size of the amplification products, using Quantity One software (BioRad, Hercules, CA, USA).

Detection of Virulence Genes
Uni-plex or multiplex PCR, using gene-specific primers (Tables A2 and A3), was used to detect the presence of 24 genes associated with virulence in E. coli strains. Three E. coli strains containing virulence genes were used as positive controls (stx1and stx2-; eaeAand hlyA-; astA-, escVand eaeA-positive strains). Based on criteria described by De Carli et al. [43], isolates containing at least five virulence genes were considered APEC strains and isolates containing fewer than five virulence genes were considered avian non-pathogenic Escherichia coli (non-APEC) strains.

Determination of E. coli Phylogenetic Groups
To determine the phylogenetic groups of the E. coli isolates, five sets of primers for the genes yja, TspE4.C2, chuA, svg and uidA were used in a multiplex PCR, as previously described [44]. PCR products were separated by electrophoresis in 3% (w/v) high resolution agarose (Blirt, Gdańsk, Poland). The phylogenetic groups were determined based on the PCR gel pattern.

Statistical Analysis
In order to identify associations between phylogenetic groups of isolates and the presence of virulence-associated genes, the Chi-square independence test with Yates correction was used. The level of significance was set as p < 0.05. The statistical analysis was performed with the use of Statistica 13 data analysis software system, TIBCO Software Inc. 2017 (Palo Alto, CA, USA).

Conclusions
To sum up, the results of the study indicate that free-living birds can be a reservoir of E. coli strains containing both resistance and virulence genes, and due to their migratory life cycle they can contribute to the spread of resistant microbes between ecosystems. Given the large number of birds migrating in Europe each year, their contribution to the spread of drug-resistant and avian pathogenic strains of E. coli appears to be significant.
Monitoring the presence of antibiotic-resistant and pathogenic microorganisms in wild animals in various geographical areas makes it possible to assess the impact of the spread of resistance genes on humans and animals and enables the implementation of possible measures to control antibiotic resistance.
Further research is needed to clarify the high percentage of gentamicin-resistant E. coli strains not previously found in birds, and the mechanisms of resistance to this antibiotic.  Data Availability Statement: All data generated or analysed during this study are included in this published article and its appendix information files.
Jacek Osek from the National Veterinary Research Institute in Puławy and Doctor Barbara Turchi from the University of Pisa, for positive E. coli strain used in detection of virulence genes.