Occurrence and molecular characterization of Escherichia coli strains isolated from black grouse (Lyrurus tetrix) from the Karkonosze National Park in Poland

The purpose of this study was to characterize Escherichia coli (E. coli) strains isolated from wild black grouse (Lyrurus tetrix), carried out due to the crossing of hiking trails with wild bird habitats from the Karkonosze National Park. Twenty-seven E. coli isolates were obtained from fecal samples collected during the winter months of 2017 and 2018. The strains were assigned to their relevant phylo-groups and the prevalence of virulence genes characteristic of APEC strains (irp2, astA, iss, iucD, papC, tsh, vat, cva/cvi, stx2f) was checked using PCR analysis. In addition, the phenotypic and genotypic resistance to antibiotics was determined. The entire study provided a better understanding of the potential bacteriological threat to wild birds of the Karkonosze National Park. The results showed that 55.6% of the strains belonged to phylo-group B1 (15/27), 33.3% to group B2 (9/27) and 11.1% to group D (3/27). Among the virulence genes tested, irp2 was detected in 25.9% of isolates (7/27), vat in 22.2% (6/27) and iucD in 3.7% (1/27). The tested E. coli strains showed susceptibility to most antimicrobials, only 14 (51.9%) of them were intermediate resistant or resistant to sulfamethoxazole. The presence of none of the tested genes responsible for resistance to selected antibiotics was identified. Our research indicates a low level of transfer of antimicrobial substances to the natural environment and confirms the effectiveness of the Karkonosze National Park’s activities to protect and restore black grouse habitats. Supplementary Information The online version contains supplementary material available at 10.1186/s12917-024-03886-3.


Background
Escherichia coli (E.coli) is a Gram-negative, rod-shaped bacteria belonging to the Enterobacteriaceae family, that is commonly found in both humans and animals (mammals and birds) as either an intestinal commensal or a pathogen [1].Bacteria may transmit among different host species and exchange genes codifying antibiotic-resistance mechanisms are generally thought.The variety of virulence factors produced, as well as their acting mechanisms, makes it possible to distinguish each serotype and allows intraspecies classification of strains.There is a division to intestinal pathogenic strains and those that cause diseases outside the gastrointestinal tract, depending on the location of the infection occurrence [2].
A special group of extraintestinal E. coli strains are APEC (Avian Pathogenic Escherichia coli), which are a component of the natural microflora of the gastrointestinal tract of birds, but they can cause opportunistic infections in the case of the host immunosuppression [3].APEC strains are involved in zoonotic transmission, which may be an important etiological factor and a reservoir of zoonoses, due to their genetic similarity to human strains causing urinary tract infections and meningitis such as UPEC (Uropathogenic E. coli) and NMEC (Neonatal Meningitis E. coli) [4].
Escherichia coli is responsible for both posing a public health risk [5] and for many economic losses in the industry of various livestock species [6,7].The declining efficiency of antimicrobials, correlated with the increasing antibiotic resistance of microorganisms, concerns that the available treatment options for infections caused by bacteria, including E. coli, will be significantly limited.
Wild birds can be a reservoir of antibiotic-resistant bacteria due to the fact that the habitats of these animals are intertwined with human settlements and tourist routes [8,9].Moreover, birds may play an important role in the dissemination of microorganisms during migration [10].The fact that antibiotics enter the environment, e.g.into terrestrial waters, also poses a threat of acquiring drug resistance by bacteria [11].Despite growing interest in studying the occurrence and pathogenicity of bacterial strains isolated from wild birds, there is still little data on their prevalence, transmission routes, and drug resistance.
The black grouse (Lyrurus tetrix) is a widespread species in many countries in Europe and Asia [12].In Poland, it is a protected bird, found in the south-eastern parts of the country, mainly in wetlands.The population is declining as a result of adverse human activities, climate change, and predation by wild mammals [13].In 2017, the State Forests launched a programme to restore the wild population of black grouse, which includes the construction of centres through which young reared birds are released into the wild [14].Ongoing reintroduction programmes may pose the risk of introducing birds that have already come into contact with human-applied therapeutics [15].This wild bird species, despite its limited population, may be a potential reservoir of antimicrobial-resistant microorganisms [16].
The aim of the study was the molecular characterization of Escherichia coli strains isolated from the feces of black grouse (Lyrurus tetrix), birds belonging to the population that inhabits the Karkonosze National Park in Poland.The phylogenetic relationship and origin of the strains -anthropogenic or zoogenic -were determined, and the virulence genes, phenotypic and genotypic sensitivity to selected antimicrobial agents were specified.In addition, this study allows to establish the risk of potential infection, caused by virulent strains of E. coli, for the declining black grouse population.

Discussion
It is well known that increasing human interference in the environment leads to exchange of pathogens between humans and wildlife.There are still some unknowns regarding the transmission of E. coli itself, its genetic diversity, and the routes of infection between humans and wildlife [17].Antimicrobial resistance among the microflora of free-living birds is a growing problem.This is also confirmed by studies conducted in Poland on E. coli strains present in wild birds: aquatic [18,19], as well as terrestrial species [20].Monitoring the increasing resistance of bacteria, which are intestinal commensals, is of particular importance because it can serve as an indicator of the level of environmental pollution and the accumulation of antimicrobial agents in ecosystems.
It also provides a good reference when comparing antibiotic resistance in different animal groups and animal production sectors [21,22].It should be taken into account that bacteria that carry resistance genes can be isolated from slaughter animals and transmitted to humans, among other veterinary staff.In addition, microorganisms are under very strong pressure from the excessive use of bactericidal and bacteriostatic substances [21].
Nearly half (51.9%) of the strains were found to be resistant or intermediate resistant to sulfamethoxazole, but none of the three sulfonamide resistance genes tested -sul1, sul2 or sul3 -were present.On the other hand, Nowaczek et al. [20] reported a lower percentage of resistance for trimethoprim/sulfamethoxazole (34.3%) and confirmed the presence of the sul2 and sul3 gene among strains resistant to this antimicrobial.In our results, the lack of sensitivity to this antibiotic may be due to the presence of another gene variant (sul4), or chromosomal resistance determined by a mutation in the dihydropteroate synthase (folP) enzyme gene [23][24][25].Interestingly, in studies on E. coli in wild birds, sulfonamide resistance has been repeatedly demonstrated, but it was most often correlated with the presence of sulfonamide resistance genes [20,22], which were not detected in the conducted study.
More than half (n = 15) of the E. coli isolates obtained from black grouse belong to phylo-group B1, indicating the dominance of non-pathogenic strains in the study population, while the rest were classified into groups B2 (n = 9) and D (n = 3), comprising potentially virulent strains [26].The results obtained for the phylo-groups correlate with the presence of the virulence genes shown in the study − 6 strains belonging to group B2 had the virulence genes irp2 and vat, and 1 strain from group D had the iucD gene.Despite the assumption that phylo-group B1 contains non-pathogenic strains, in this study 1 of the strains possessed the virulence gene irp2.Differences in pathogenicity within a single phylo-group have also been observed in previous studies on APEC [27].In addition, a similar result was obtained in a study by Nowaczek et al. [20] on wild birds in Poland, where virulence genes were detected in strains belonging to phylo-group B1, however, their frequency was significantly lower than in groups B2 and D. The virulence genes selected for analysis are responsible for the pathogenicity of APEC isolates that cause colibacteriosis in poultry flocks [28].There are also hypotheses, supported by molecular studies, comparing APEC strains with ExPEC (Extraintestinal Pathogenic Escherichia coli).These experiments indicate that a group of extraintestinal E. coli may have zoonotic potential and cause infections in humans [4].On the other hand, there is also the possibility of reverse transmission from humans to animals [29].
The most frequently isolated virulence gene was the irp2 gene, responsible for the production of an iron repressible-protein, and the vat gene, encoding a vacuolating autotransporter toxin.In studies on wild birds in Poland and the Netherlands, the irp2 gene was also shown to be one of the most common virulence genes in E. coli [19,20].Similar studies were performed in a group of wild birds, where the frequent occurrence of virulence genes iss and, less frequently, tsh (also genes characteristic of APEC strains), were demonstrated [30].In the aforementioned work, an examination of eagle and hawk feces also showed the presence of E. coli strains with significant antibiotic resistance.Whereas, in the feces of migratory birds in Bangladesh, E. coli strains possessing iucD and papC genes have been identified, which are also associated with APEC [31].In the same study, these E. coli strains were found to be significantly resistant to ciprofloxacin, tetracycline, or ampicillin.The reports of researchers who obtain these results indicate that there is a ground for further research on other groups of wild birds in different regions of the world.
This study also showed that feces samples collected from black grouses can be fibrous (hard and cylindrical) or soft (without form and semi-liquid).In the winter months, feces with a hard and fibrous structure predominate.Furthermore, it should be noted that significantly fewer strains of E. coli and other bacterial species have been cultured from samples with this structure under laboratory conditions.This decrease in the amount and diversity of the intestinal microbiome may be related to the black grouse diet, which in winter is based on the needles and buds of coniferous trees [32,33].Although the winter months facilitate quick and aseptic sampling for microbiological tests, it is much more difficult to isolate bacteria from the fecal samples of these birds.
The insufficient scientific data in the literature on the gastrointestinal microbiome of black grouse makes it impossible to conduct a comparative analysis of their different populations.Further research is required to determine the composition and interactions between the bacterial species that form the physiological flora of these birds.Additionally, the assessment seasonal changes in intestinal microbes composition depending on changes in wild birds diet should be also performed [32].This knowledge will contribute to protecting the populations of bird species and improving their welfare.

Conclusions
Although there is a possibility that wild birds carry resistant bacteria that are passed on to them by humans or other animals, our study demonstrated the sensitivity of E. coli strains to most selected antibiotics, including the most commonly used in human medicine.First, our results present the low degree of antibacterial substances transmission into the natural habitat of black grouse.Second, the strains isolated from black grouse are unlikely to be of anthropogenic origin.It is possibly due to the Karkonosze National Park's activities about the preservation and restoration of black grouse's habitat, among other things, through the control of forestry works and tourist traffic.

Isolation of E. coli strains
The study included a total of 197 black grouse fecal samples, collected in the Karkonosze National Park of approximately 25 km 2 area and 1000-1250 m above sea level, from places where the birds spent the night, during the winter months of 2017 and 2018.The collected feces samples were placed in sterile containers and immediately transported in an isothermal box with freezing inserts to the "Epi-Vet" Veterinary Diagnostic Laboratory.For microbiological examination, 1 g of fresh feces was incubated for 16-20 h at 37 o C in BHI (Brain Heart Infusion broth) (Oxoid, Hampshire, United Kingdom).The suspension was then inoculated onto McConkey's agar (Oxoid, Hampshire, United Kingdom) and incubated for 18 h at 37 o C. The single, lactose-positive colonies characteristic of E. coli were transferred to nutrient agar medium (Merck, Darmstadt, Germany).The isolates were preserved for further testing at -70 o C, using the Microbank system (Pro-lab Diagnostics, Richmond Hill, Canada).Furthermore, isolated bacteria were identified as E. coli by observing their cultural characteristics, morphology by Gram's stain, oxidase test, EMB (eosin methylene blue) agar and motility test.Presumptive E. coli strains were selected for further PCR analysis.

Antimicrobial-susceptibility testing
All E. coli isolates were subjected to drug resistance testing using the disk diffusion method and the broth microdilution technique for colistin.Escherichia coli ATCC 25,922 was used as an internal control for both the disk diffusion method and the establishment of the minimum inhibitory concentration (MIC) of colistin.The bacteria were inoculated on MacConkey agar (Oxoid, Hampshire, United Kingdom) and incubated for 18 h at 37 °C.Subsequently, bacteria suspensions were prepared in 0.9% NaCl with an optical density of 0.5 McFarland.For the disc diffusion method, the bacteria suspensions were spread onto Mueller-Hinton agar (Oxoid, Hampshire, United Kingdom) using a sterile swab.Susceptibility to the following antimicrobial agents was determined: amoxicillin with clavulanic acid (20/10 µg) (Bio-Rad, California, USA), ciprofloxacin (5 µg), tetracycline (30 µg), sulfamethoxazole (25 µg), gentamicin (10 µg), ampicillin (10 µg) and nalidixic acid (30 µg) (Oxoid, Hampshire, United Kingdom).The inhibition zones were identified and visually interpreted.The minimum inhibitory concentration sensitivities for colistin were determined using a broth microdilution technique with a ComASP Colistin panel (Liofilchem, Via Scozia, Italy) following the suggested manufacturer protocol using Mueller-Hinton broth.The antimicrobial resistance phenotyping of the isolates was performed and interpreted according to the Clinical and Laboratory Standards Institute (CLSI) M100 ED23; 2023 [34].

Molecular analysis
The isolation of genomic DNA was performed using a Genomic Mini kit (A&A Biotechnology, Gdańsk, Poland) according to the instructions provided by the producer.The isolated genetic material was stored at -70 o C for further studies.

Table 1
Affiliation of the studied E. coli strains (n = 27) to phylogroups

Table 2
Occurrence of virulence genes and affiliation of studied E. coli strains (n = 27) to phylo-groups Fig. 1 Sensitivity of E. coli strains (n = 27) to selected antimicrobials