The emergence of antimicrobial resistance (AMR) due to several factors has become a crucial multifaced issue, adversely impacting humans, animals, and the environment. Several studies demonstrated that the role of wildlife, which is not directly exposed to antibiotics under normal conditions, should be considered in the emergence of AMR. It was previously reported that migratory birds had the potential to spread resistant bacterial strains, threatening human and animal health, to large geographical areas during their annual migration (Hubálek et al., 2004; Reed et al., 2003, Allen et al. 2010).
The exchange of antibiotic-resistant bacterial strains, with varying prevalences of ESBL, among urban resident aquatic or non-aquatic wild birds feeding on anthropogenic sources such as dump sites and wastewater has been well documented in the literature. The researchers stressed that higher prevalences are more likely detected in big, crowded cities, where residential areas with inadequate infrastructure and health care facilities lacking sufficient sanitation intertwine with industrial zones (Nelson et al. 2008, Poeta et al., 2008, Bonnedahl & Järhult, 2014; Borges et al., 2017, Oteo et al., 2018; Islam et al., 2021,Tapia-Arreola, A. K et al. 2022, Freire, S. et al., 2022). Seagulls were mainly shown to be putatively responsible for the potential spread of multidrug-resistant E. coli strains, particularly in the coastal settlements, due to their feeding habits through animal and human wastes in garbage dumps (Bonnedahl et al., 2009, Simões, 2010, Poirel et al., 2012 Ahlstrom et al. (2019) Ahlstrom et al. (2021) Zeballos-Gross et al. (2021). In another study, AMR-resistant E. coli and AMR-resistant K. pneumoniae were isolated from 30.1% (69/229) and 8.73% (20/229), respectively, from seagull faeces collected in city centres and seasonally-active holiday resorts of ten districts of Western Australia's coastline. All of the AMR isolates were found to have originated from samples that were gathered in urban settings, indicating a clear relationship between the proximity of animals to residential areas inhabited by humans and the transmission of AMR bacterial strains among animals. (Mukerji et al. 2021).
The non-aquatic resident birds inhabiting the cities, such as pigeons and crows, were shown to have higher ESBL prevalences than domestic fowl and other birds, and notably, wild pigeons and crows are recognised presumptive reservoirs of multidrug-resistant and ESBL-producing E. coli, eliciting the spread of the pathogens in urban areas (Parker et al. 2016,Cunha et al., 2019, Ngaiganam vd., 2019).
In the study, 54.4% (37/68) of cefotaxime-resistant E. coli isolated from the waterbirds evaluated in Group 1 revealed phenotypic positivity for ESBL and AmpC, which comprised 51.5% (35/68), represented predominantly by 34 seagulls (29 herring gulls and five little gulls) and a single grey heron, of this group's bird population. That a phenotypic AMR was mainly detected in seagulls in the majority of this species population (34 of 59), which was also compatible with previous studies, is indicative of the possibility that the AMR profile originated in humans or animals, considering the high population of seagulls in the province of Istanbul feeding mostly in garbage dumps, directly consuming human/animal wastes or agricultural waste products.
A prevalence of 20.6% (14/68) for phenotypic ESBL and AmpC positivity in the resident terrestrial birds in Group 2, with a predominance of pigeons (64.3%; 14/68) was suggestive of this species' crucial role in serving as main reservoirs and spreaders of resistant E. coli since urban wild birds that are not exposed to antibiotics under normal conditions, yet have close contact with human and animal wastes by various occasions, such as nesting in resting areas provided with contaminated water sources close to the sewage line and feeding on human wastes through garbage dumps and animal manure as agricultural wastes or grassland invertebrates in contaminated soil in the residential areas with vast human and animal population.
Several studies worldwide have suggested that wild winter or summer migratory birds are the reservoirs of ESBL-producing E. coli. Furthermore, evidence has shown that migratory wild birds can transmit resistant bacterial strains to humans and domestic animals, or vice versa, during their migrations (Fahim, K. M., et.al 2019, Zurfluh, K., ve ark 2019, Athanasakopoulou, Z. et al. 2022, Fuentes-Castillo, D., et.al 2023).
Herein, we detected a prevalence of 10.3% (7/68) for phenotypic ESBL-associated AMR in Group 3's winter migrants and wild predators, with 11.8% (8/68) phenotypically characterized ESBL-positivity in E. coli isolated from this group. On the other hand, the prevalences of phenotypic ESBL associated AMR and ESBL positive E. coli isolates were 4.41% (3/68) in summer migrants and small passerines that comprise Group 4. Even though the bird species evaluated in these two groups are considered the potential carriers of ESBL based AML, the relatively lower prevalences than in the other groups are associated with their less contact with humans and animals, thus less access to waste products such as garbage, sewage, and contaminated water and soil, which is compatible with previous studies.
The most prevalent beta-lactamase gene in ESBL and AmpC positive E. coli isolates obtained from wild birds was introduced as blaCTX−M (Zurfluh, K., et al. 2019, Athanasakopoulou, Z. et al. 2022, Dreyer, S. et al., 2022). Even though the prevalences of gene variants of the CTX-M-type enzymes vastly vary, depending on the geographical sites, the most common ESBL gene variant in humans, domestic animals, and resident and migratory wild birds was discovered to be blaCTX−M−1. (Kahraman B. et al. 2016, Gümüş B. et al. 2017, Sığırcı B et al. 2017, Cormier, A. C., et al. 2022), followed in descending order by blaCTX−M−15 and blaCTX−M−14. As for Beta-lactamase genes, TEM and SHV were less frequently detected (Stedt J et al. 2015, Mohsin M et al. 2017, Athanasakopoulou, Z. et al. 2022, Luo, Y., et al. 2022). The researchers have emphasised that plasmid-mediated AmpC Beta-lactamases, apart from ESBL, are of global concern since they generate resistance to cephalosporin antibiotics and Beta-lactamase inhibitors of substantial clinical significance (Park Y.S. et al. 2009, Haenni M. et al. 2022). The blaCMY−2 variant that ranks among the blaCIT group genes was reported to be the most prevalent AmpC Beta-lactamase gene in the wild birds of Europa, mainly central Europa and North America (Athanasakopoulou Z. et al. 2022, Medvecky M. et al. 2022). Resident and migrant gulls have been indicated to metaphorically serve as "ecological sponges" by carrying bacteria strains with AMR of critical health-threatening significance and potentially transmitting them to different environments, rendering a vicious cycle (Fahim K.M. et al. 2019, Zurfluh K. et al. 2019, Athanasakopoulou Z. et al. 2022, Fuents-Castillo D. et al. 2023). The most prevalent ESBL genes were found to be the blaCTX−M group and its variant blaCTX−M−1 in E. coli isolates with beta-lactamase activity identified from domestic animals and humans in Turkey. Other blaCTX−M gene variants and TEM, SHV, and OXA-10-type variants were also detected (Bonnedahl J. et al. 2014, Day M.J. et al. 2016). The predominance of blaCIT group genes, apart from the presence of blaMOX type genes, was noted in the genotyping studies carried out with AmpC-producing E. coli isolates (Carattoli A. 2008, EFSA 2011, Ewers C. et al. 2012).
The quantity of E. coli isolates in our investigation that carried beta-lactamase genes was 32 in 28 (23 herring gulls, four little gulls, one grey heron) birds of Group 1, with the distribution of genes as blaCTX−M (n = 25), blaSHV (n = 1), blaOXA10 (n = 6), blaMOX (n = 5), and blaCIT (n = 1). Out of the 25 isolates, 15 blaCTX−M genes were found to belong to the blaCTX−M−1 variant. The detected genes occurred as a single group of genes, a simultaneously present set of genes, or coexisting ESBL and AmpC genes. All AMR genes were found in seagull species, with a prevalence of 39.7% (27/68), except for ESBL and/or AmpC and one blaCTX−M group gene. The most prevalent gene group in gulls was blaCTX−M (n = 24), and 14 were in the blaCTX−M−1 group. No blaCTX−M−15 gene was detected in any isolate. The distribution of the primary and subgroups of genes in the genotypically ESBL (n = 15) and AmpC (n = 1) positive isolates of Group 2 was blaCTX−M (n = 15), blaSHV (n = 1), blaOXA10 (n = 1), and blaMOX (n = 1). One E. coli isolate showed the coexistence of the ESBL genes (blaCTX−M, blaSHV, and blaOXA10), and one isolate had a combination of ESBL and AmpC gene groups (blaCTX−M, blaMOX). The blaCTX−M−15 gene, common in Europe and Africa, was undetected in any isolate. To sum up, the genotypic findings were consistent with those of previous studies regarding blaCTX−M and blaCTX−M−1; however, they differed due to the absence blaCTX−M−15.
In a study conducted at Aragorn Wildlife Rehabilitation Center in Spain, with fecal samples of 100 bird species in 15 families, 16 were positive for ESBL. The genotyping revealed the occurrence of 9 blaSHV, three bla CTX−M−1, and five blaTEM positive strains. In another study conducted with predator birds in Rio de Janeiro, all 41 E. coli strains isolated from 14 birds were positive for the blaCTX−M gene. Moreover, the blaCMY−2 gene was found in 2 isolates [51,56]. The phenotypic ESBL resistance was detected in 12 out of 111 resident and migratory birds from Tunisia, and all exhibited the presence of the blaCTX−M−15 gene [57]. In a similar study conducted in Pakistan, blaCTX−M was detected in all 26 ESBL-positive samples isolated from 150 migratory birds, and the presence of the blaTEM gene was noted in 19 isolates [46]. Finally, a joint study in Canada and Chile revealed that 195 ESBL-positive isolates were determined in 400 fecal samples collected from migratory Franklin's gulls. The blaCTX−M gene was detected in 101 isolates in the Chilean part of the study (Bonnedahl J. et al. 2014).
In our study, the distribution of isolates with genotypically detected ESBL (n = 7) genes was blaCTX−M (n = 5) and blaOXA10 (n = 2) in Group 3, which included winter migrants. Neither the coexistence of genes nor an AmpC encoding gene was detected.
As for Group 4, which included summer migrants, the ESBL (n = 5) and AmpC (n = 1) genes were genotypically detected, and the distribution of main and subgroups of genes was blaCTX−M (n = 5), blaOXA10 (n = 1), and blaCIT (n = 1). Furthermore, the coexistence of ESBL and AmpC genes, blaCTX−M, blaOXA10, and blaCIT, was detected in 2 E. coli isolates, and the coexistence of the ESBL genes, blaCTX−M and blaOXA10 in a single one. The blaCTX−M−15 gene, common in Europe and Africa, was found neither in Group 3 nor Group 4. The lower prevalence of E. coli isolates with AMR in wild migratory birds that spend a few months in the migration destination than in the resident wild birds of the region was attributed to having less contact with contaminated birds.
Ultimately, it was shown that wild birds living in the Marmara region or using it as a stopover location during migration harbored ESBL and/or AmpC Beta-lactamase-producing E. coli. The general data suggests that the resident wild bird population has a higher prevalence of ESBL and/or AmpC beta-lactamase-producing E. coli than do migrant birds. This finding can be attributed to the resident birds' increased exposure to humans, animals, and their waste products. Wild urban birds that are thought to be immune-system neutral are frequently in close proximity to people and other animals. Gulls and crows that feed on substantially increasing waste products in the residential districts and pigeons, even though not in direct contact with wastes yet reside in large numbers in contaminated environments, may contribute to spreading infectious agents, becoming a potential source of ESBL genes. Pigeons, mainly long-distance flyers (more than 5 km per day), play an epidemiologically significant role in the widespread spread of antimicrobial-resistant bacteria. Additionally, it was discovered that certain seagull species harbored more antibiotic-resistant E. coli when they nested in recreational areas with high human populations and contaminated water sources, particularly those near sewage sites or garbage dumps, than did birds living near clean water sources. This finding suggests that certain seagull species, acting as reservoirs or carriers of resistant bacteria, are accountable for the inter-ecosystem spread of AMR as a result of migrations from one province to another. On the other hand, given that seagulls primarily eat the wastes of these populations and agriculture, maintaining their crucial role as potential spreaders of antibiotic resistance, the exponential increase in antibiotic use in humans and livestock is another aspect of excessively increased AMR.