Manipulating a host-native microbial strain compensates for low microbial diversity by increasing weight gain in a wild bird population.

Empirical studies from laboratory systems and humans show that the gut microbiota is linked to host health. Similar evidence for effects on traits linked to fitness in nature is rare, not least because experimentally manipulating the gut microbiota is challenging. We isolated, characterized, and cultured a bacterial strain, Lactobacillus kimchicus APC4233, directly from a wild bird (the great tit Parus major) and provided it as a self-administered dietary supplement. We assessed the impact of the treatment on the host microbiota community, on weight, and tested whether the treatment affected a previous result linking microbiota alpha diversity to weight in nestlings. The treatment dramatically increased L. kimchicus abundance in the gut microbiota and increased alpha diversity. This effect was strongest in the youngest birds, validating earlier findings pointing to a brief developmental window when the gut microbiota are most sensitive. In time-lagged models, nestling weight was higher in the treatment birds suggesting L. kimchicus may have probiotic potential. There was also a positive time-lagged relationship between diversity and weight in control birds but not in the treatment birds, suggesting L. kimchicus helped birds compensate for low alpha diversity. We discuss why ecological context is likely key when predicting impacts of the microbiome. The manipulation of the gut microbiota with a host native strain in this wild population provides direct evidence for the role of the microbiota in the ecology and evolution of natural populations.

Indirect maternal effects via nest microbiome composition drive gut colonization in altricial chicks.

Gut microbial communities are complex and heterogeneous and play critical roles for animal hosts. Early-life disruptions to microbiome establishment can negatively impact host fitness and development. However, the consequences of such early-life disruptions remain unknown in wild birds. To help fill this gap, we investigated the effect of continuous early-life gut microbiome disruptions on the establishment and development of gut communities in wild Great tit (Parus major) and Blue tit (Cyanistes caeruleus) nestlings by applying antibiotics and probiotics. Treatment neither affected nestling growth nor their gut microbiome composition. Independent of treatment, nestling gut microbiomes of both species grouped by brood, which shared the highest numbers of bacterial taxa with both nest environment and their mother. Although fathers showed different gut communities than their nestlings and nests, they still contributed to structuring chick microbiomes. Lastly, we observed that the distance between nests increased inter-brood microbiome dissimilarity, but only in Great tits, indicating that species-specific foraging behaviour and/or microhabitat influence gut microbiomes. Overall, the strong maternal effect, driven by continuous recolonization from the nest environment and vertical transfer of microbes during feeding, appears to provide resilience towards early-life disruptions in nestling gut microbiomes.

Exploring the faecal microbiome of the Eurasian nuthatch (Sitta europaea).

Gastrointestinal microbiota fulfill pivotal roles in providing a host with nutrition and protection from pathogenic microorganisms. Up to date, most microbiota research has focused on humans and other mammals, whereas birds and especially wild birds lag behind. Within the field of the avian gut microbiome, research is heavily biased towards poultry. In this study, we analyzed the gut microbiome of the Eurasian nuthatch (Sitta europaea), using faecal samples of eight nestlings originating from three nuthatch nests in the south of Ghent (Belgium), using Illumina sequencing of the 16S rRNA gene. Relative frequency analysis showed a dominance of Firmicutes and Actinobacteria and to a lesser extent Proteobacteria. Bacteroidetes and other phyla were relatively rare. At higher taxonomic levels, a high degree of inter-individual variation in terms of overall microbiota community structure as well as dominance of certain bacteria was observed, but with a higher similarity for the nestlings sharing the same nest. When comparing the nuthatch faecal microbiome to that of great tit nestlings that were sampled during the same breeding season and in the same forest fragment, differences in the microbial community structure were observed, revealing distinct dissimilarities in the relative abundancy of taxa between the two bird species. This study is the first report on the nuthatch microbiome and serves as a reference study for nuthatch bacterial diversity and can be used for targeted screening of the composition and general functions of the avian gut microbiome.

Human activity can influence the gut microbiota of Darwin's finches in the Galapagos Islands.

The gut microbiota of animal hosts can be influenced by environmental factors, such as unnatural food items that are introduced by humans. Over the past 30 years, human presence has grown exponentially in the Galapagos Islands, which are home to endemic Darwin's finches. Consequently, humans have changed the environment and diet of Darwin's finches, which in turn, could affect their gut microbiota. In this study, we compared the gut microbiota of two species of Darwin's finches, small ground finches (Geospiza fuliginosa) and medium ground finches (Geospiza fortis), across sites with and without human presence, where finches prefer human-processed and natural food, respectively. We predicted that: (a) finch microbiota would differ between sites with and without humans due to differences in diet, and (b) gut microbiota of each finch species would be most similar where finches have the highest niche overlap (areas with humans) compared to the lowest niche overlap (areas without humans). We found that gut bacterial community structure differed across sites and host species. Gut bacterial diversity was most distinct between the two species at the site with human presence compared to the site without human presence, which contradicted our predictions. Within host species, medium ground finches had lower bacterial diversity at the site with human presence compared to the site without human presence and bacterial diversity of small ground finches did not differ between sites. Our results show that the gut microbiota of Darwin's finches is affected differently across sites with varying human presence.

Methicillin-resistant Staphylococcus aureus and glycopeptide-resistant enterococci in fecal samples of birds from South-Eastern Poland.

BACKGROUND: The incidence of human infection and colonization with methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) has increased in the recent years. Environmental sources, including bird droppings, might play an important role as resistance reservoirs. RESULTS: Fresh fecal samples were collected from rooks and wild-living birds during the autumn-winter period of 2016/2017, and tested for the presence of bacteria associated with human diseases. Besides bacteria representing the genera Enterococcus, Campylobacter, Escherichia, and Staphylococcus, Enterobacter, Citrobacter, Proteus, Hafnia, and Pseudomonas were also identified. The susceptibility of S. aureus and Enterococcus spp. isolates to methicillin, and vancomycin and teicoplanin, respectively, was analyzed to assess the avian wildlife as a reservoir of MRSA and VRE strains. Twenty-two percent of all S. aureus isolates were methicillin-resistant. These strains were screened by polymerase chain reaction (PCR), using the most widely used primer sets specific for the mecA gene. Twenty percent of all Enterococcus strains were phenotypically vancomycin-resistant. The presence of van resistance genes in these strains was investigated by PCR using vanA and vanB gene-specific primers. A good correlation between mecA gene detection and disc diffusion data was observed, while some discrepancy was noted between the PCR data and the vancomycin/teicoplanin phenotypic resistance pattern. The incidence of strains resistant to methicillin and glycopeptide antibiotics in wild-living birds was twice that in rooks. CONCLUSIONS: The study suggests that rooks from urban areas and passerine birds from the natural habitat carry antibiotic-resistant Enterococcus spp. and S. aureus strains, probably reflecting the presence of such isolates in the environmental food sources.

Linked seasonal outbreaks of Salmonella Typhimurium among passerine birds, domestic cats and humans, Sweden, 2009 to 2016.

In 2016, an outbreak of Salmonella Typhimurium (STm) with multilocus variable-number tandem repeat analysis (MLVA) profiles historically associated with passerine birds (2-[11-15]-[3-4]-NA-212) occurred among passerines, cats and humans in Sweden. Our retrospective observational study investigated the outbreak and revisited historical data from 2009-16 to identify seasonality, phylogeography and other characteristics of this STm variant. Outbreak isolates were analysed by whole-genome single nucleotide polymorphism (SNP) typing. The number of notified cases of passerine-associated STm among passerines, cats and humans per month and county, and their MLVA profiles, were compared to birdwatchers' counts of passerines. Seasonal trend decomposition and correlation analysis was performed. Outbreak isolates did not cluster by host on SNP level. Passerine-associated STm was seasonal for birds, cats and humans, with a peak in March. Cases and counts of passerines at bird feeders varied between years. The incidence of passerine-associated STm infections in humans was higher in the boreal north compared with the southern and capital regions, consistent with passerine population densities. Seasonal mass migration of passerines appears to cause STm outbreaks among cats certain years in Sweden, most likely via predation on weakened birds. Outbreaks among humans can follow, presumably caused by contact with cats or environmental contamination.

Chemical regulation of body feather microbiota in a wild bird.

The microbiota has a broad range of impacts on host physiology and behaviour, pointing out the need to improve our comprehension of the drivers of host-microbiota composition. Of particular interest is whether the microbiota is acquired passively, or whether and to what extent hosts themselves shape the acquisition and maintenance of their microbiota. In birds, the uropygial gland produces oily secretions used to coat feathers that have been suggested to act as an antimicrobial defence mechanism regulating body feather microbiota. However, our comprehension of this process is still limited. In this study, we for the first time coupled high-throughput sequencing of the microbiota of both body feathers and the direct environment (i.e., the nest) in great tits with chemical analyses of the composition of uropygial gland secretions to examine whether host chemicals have either specific effects on some bacteria or nonspecific broad-spectrum effects on the body feather microbiota. Using a network approach investigating the patterns of co-occurrence or co-exclusions between chemicals and bacteria within the body feather microbiota, we found no evidence for specific promicrobial or antimicrobial effects of uropygial gland chemicals. However, we found that one group of chemicals was negatively correlated to bacterial richness on body feathers, and a higher production of these chemicals was associated with a poorer body feather bacterial richness compared to the nest microbiota. Our study provides evidence that chemicals produced by the host might function as a nonspecific broad-spectrum antimicrobial defence mechanism limiting colonization and/or maintenance of bacteria on body feathers, providing new insight about the drivers of the host's microbiota composition in wild organisms.

Codiversification of gastrointestinal microbiota and phylogeny in passerines is not explained by ecological divergence.

Vertebrate gut microbiota (GM) is comprised of a taxonomically diverse consortium of symbiotic and commensal microorganisms that have a pronounced effect on host physiology, immune system function and health status. Despite much research on interactions between hosts and their GM, the factors affecting inter- and intraspecific GM variation in wild populations are still poorly known. We analysed data on faecal microbiota composition in 51 passerine species (319 individuals) using Illumina MiSeq sequencing of bacterial 16S rRNA (V3-V4 variable region). Despite pronounced interindividual variation, GM composition exhibited significant differences at the interspecific level, accounting for approximately 20%-30% of total GM variation. We also observed a significant correlation between GM composition divergence and host's phylogenetic divergence, with strength of correlation higher than that of GM vs. ecological or life history traits and geographic variation. The effect of host's phylogeny on GM composition was significant, even after statistical control for these confounding factors. Hence, our data do not support codiversification of GM and passerine phylogeny solely as a by-product of their ecological divergence. Furthermore, our findings do not support that GM vs. host's phylogeny codiversification is driven primarily through trans-generational GM transfer as the GM vs. phylogeny correlation does not increase with higher sequence similarity used when delimiting operational taxonomic units. Instead, we hypothesize that the GM vs. phylogeny correlation may arise as a consequence of interspecific divergence of genes that directly or indirectly modulate composition of GM.

ESTABLISHING NORMAL FECAL FLORA IN WILD AUSTRALIAN PASSERINE BIRDS BY USE OF THE FECAL GRAM STAIN.

The purpose of this study was to determine the normal fecal bacterial and fungal flora and parasite prevalence in wild passerine birds found at the Australian Botanic Garden (Mount Annan, New South Wales). Wild passerine birds (n = 186) from 28 species were captured with mist nets. Fecal Gram stains (n = 155) were made from 26 species and analyzed for bacterial density, Gram stain morphology, and the presence of yeast. Fecal wet preparations (n = 139) were made from 24 passerine species and were analyzed for parasites. Our results showed that 81.9% of passerines sampled had bacteria present in their feces. The bacteria found were entirely Gram positive and consisted predominantly of cocci. Individuals that were caught on multiple occasions were found to have stable bacterial populations, apart from the red-browed finch (Neochmia temporalis). Insectivores had higher bacterial densities and cocci proportions than nectarivores had. Yeasts were rare in most species, with the exception of the bell miner (Manorina melanophrys) and noisy miner (Manorina melanocephala). The yeast, Macrorhabdus ornithogaster, and parasites were not observed in any fecal samples. These results will help practitioners to assess the health of Australian passerine species submitted for care or housed in zoological collections.

Virulence and antimicrobial resistance of Klebsiella pneumoniae isolated from passerine and psittacine birds.

Klebsiella pneumoniae is considered one of the most important Gram-negative opportunistic pathogens. The contact between humans and birds poses health risks to both. The aim of this study was to investigate the resistance and virulence of K. pneumoniae isolates from psittacines and passerines, seized from illegal trade in Brazil. We analysed 32 strains isolated from birds of the orders Psittaciformes and Passeriformes by polymerase chain reaction (PCR) for virulence factor genes. Antibiotic resistance was assessed by disk diffusion assay and PCR. The results indicated that fimH (100%), uge (96.8%), kfu (81.2%) and irp-2 (68.7%) were the most common virulence genes, followed by kpn (46.8%), K2 (43.7%), mrkD (34.3%) and iroN (15.6%). The combination of virulence genes resulted in a great diversity of genotypes and the heterogeneity of the strains is also confirmed in the analysis by amplified fragment length polymorphism. The susceptibility profiles of the K. pneumoniae showed 25% of multiple antibiotic resistance strains. We identified seven strains that presented non-extended spectrum beta lactamase blaSHV variants SHV-1 and SHV-11 and one strain positive to the blaTEM-1 gene. Plasmid-mediated quinolone resistance was present in 10 strains (10/32). The data obtained in this study reveal the pathogenic potential of this pathogen and highlight the need for surveillance and monitoring.

Evolution of pathogen virulence across space during an epidemic.

We explore pathogen virulence evolution during the spatial expansion of an infectious disease epidemic in the presence of a novel host movement trade-off, using a simple, spatially explicit mathematical model. This work is motivated by empirical observations of the Mycoplasma gallisepticum invasion into North American house finch (Haemorhous mexicanus) populations; however, our results likely have important applications to other emerging infectious diseases in mobile hosts. We assume that infection reduces host movement and survival and that across pathogen strains the severity of these reductions increases with pathogen infectiousness. Assuming these trade-offs between pathogen virulence (host mortality), pathogen transmission, and host movement, we find that pathogen virulence levels near the epidemic front (that maximize wave speed) are lower than those that have a short-term growth rate advantage or that ultimately prevail (i.e., are evolutionarily stable) near the epicenter and where infection becomes endemic (i.e., that maximize the pathogen basic reproductive ratio). We predict that, under these trade-offs, less virulent pathogen strains will dominate the periphery of an epidemic and that more virulent strains will increase in frequency after invasion where disease is endemic. These results have important implications for observing and interpreting spatiotemporal epidemic data and may help explain transient virulence dynamics of emerging infectious diseases.

Detection of bacteria and fungi and assessment of the molecular aspects and resistance of Escherichia coli isolated from confiscated passerines intended for reintroduction programs.

Many native bird species are currently considered rare in Brazil because they have been indiscriminately collected by animal traffickers and commercialized, leading to dwindling numbers in their natural habitats. Confiscated animals are at times destined for reintroduction programs that must ensure these animals do not pose a risk to native populations. Healthy or sick wild passerines may carry a great diversity of microorganisms. Therefore, knowledge of the sanitary status of confiscated animals destined for reintroduction is critical to assess whether these animals act as microorganism carriers and to investigate the epidemiology of transmissible diseases, a crucial aspect for animal and human health preservation. This study examined the occurrence of aerobic and facultative anaerobic bacteria and fungi in cloacal swabs collected from wild confiscated passerines intended for reintroduction programs. In vitro susceptibility tests of the most frequent isolates as well as studies of the molecular aspects of Escherichia coli isolates were also performed. There was microorganism growth in 62.5% of 253 samples. The microorganisms that were most frequently isolated were Staphylococcus spp. (15.0%), Micrococcus spp. (11.5%), E. coli (10.7%) and Klebsiella spp. (10.7%). Fifteen bacteria genera and seven fungi genera were isolated. Multidrug-resistance to antimicrobials was observed in Staphylococcus spp., Micrococcus spp., E. coli and Klebsiella spp. isolates. The high occurrence of Enterobacteria observed is possibly related to the sanitary conditions in which confiscated animals are usually kept. One E. coli sample (out of 27 isolates) was positive for the S-fimbrial adhesion encoding gene (sfa). Considering the low occurrence of genes that encode virulence factors, confiscated passerines may represent a low risk for the potential transmission of EPEC, APEC, UPEC and NMEC isolates to other animals or humans. The potential risk of intra- or inter-specific transmission of multidrug-resistant isolates and the introduction of these microorganisms into the environment must be considered, although there are still therapeutic alternatives for treatment of these animals among the antimicrobials which were tested. The stress and poor hygiene conditions imposed on animals during trafficking may have caused their contamination by multidrug-resistant agents transmitted by humans or by the precarious environment to which they were subjected. Risks related to the dissemination of Salmonella spp., Cryptococcus spp. and Candida spp. are low when reintroduction programs are considered.

Manipulation of parental effort affects plumage bacterial load in a wild passerine.

It has been suggested that plumage microorganisms play an important role in shaping the life histories of wild birds. Some bacteria may act as pathogens or cause damage to feathers, and thereby reduce individual fitness. Intense parental care in birds can result in a reduction of self-maintenance and preening behavior in parents and therefore might affect the dynamics of microbiota living on their feathers. However, experimental evidence of this relationship is virtually absent. We manipulated the parental effort of wild breeding pied flycatcher (Ficedula hypoleuca) females by modifying their brood size or temporarily removing male partners. We expected that experimentally decreasing or increasing parental effort would affect feather sanitation in females and therefore also bacterial density on their plumage. In accordance with this hypothesis, manipulation affected the density of free-living bacteria: females with reduced broods had the lowest number of free-living bacteria on their feathers, while females left without male partners had the highest. However, manipulation did not have a significant effect on the densities of attached bacteria. Our results provide experimental evidence that a trade-off between self-maintenance and parental effort affects plumage bacterial densities in birds.

Uropygial gland size and composition varies according to experimentally modified microbiome in Great tits.

BACKGROUND: Parasites exert important selective pressures on host life history traits. In birds, feathers are inhabited by numerous microorganisms, some of them being able to degrade feathers or lead to infections. Preening feathers with secretions of the uropygial gland has been found to act as an antimicrobial defence mechanism, expected to regulate feather microbial communities and thus limit feather abrasion and infections. Here, we used an experimental approach to test whether Great tits (Parus major) modify their investment in the uropygial gland in response to differences in environmental microorganisms. RESULTS: We found that males, but not females, modified the size of their gland when exposed to higher bacterial densities on feathers. We also identified 16 wax esters in the uropygial gland secretions. The relative abundance of some of these esters changed in males and females, while the relative abundance of others changed only in females when exposed to greater bacterial loads on feathers. CONCLUSION: Birds live in a bacterial world composed of commensal and pathogenic microorganisms. This study provides the first experimental evidence for modifications of investment in the defensive trait that is the uropygial gland in response to environmental microorganisms in a wild bird.

Surveillance for microbes and range expansion in house sparrows.

Interactions between hosts and parasites influence the success of host introductions and range expansions post-introduction. However, the physiological mechanisms mediating these outcomes are little known. In some vertebrates, variation in the regulation of inflammation has been implicated, perhaps because inflammation imparts excessive costs, including high resource demands and collateral damage upon encounter with novel parasites. Here, we tested the hypothesis that variation in the regulation of inflammation contributed to the spread of house sparrows (Passer domesticus) across Kenya, one of the world's most recent invasions of this species. Specifically, we asked whether inflammatory gene expression declines with population age (i.e. distance from Mombasa (dfM), the site of introduction around 1950). We compared expression of two microbe surveillance molecules (Toll-like receptors, TLRs-2 and 4) and a proinflammatory cytokine (interleukin-6, IL-6) before and after an injection of an immunogenic component of Gram-negative bacteria (lipopolysaccharide, LPS) among six sparrow populations. We then used a best-subset model selection approach to determine whether population age (dfM) or other factors (e.g. malaria or coccidian infection, sparrow density or genetic group membership) best-explained gene expression. For baseline expression of TLR-2 and TLR-4, population age tended to be the best predictor with expression decreasing with population age, although other factors were also important. Induced expression of TLRs was affected by LPS treatment alone. For induced IL-6, only LPS treatment reliably predicted expression; baseline expression was not explained by any factor. These data suggest that changes in microbe surveillance, more so than downstream control of inflammation via cytokines, might have been important to the house sparrow invasion of Kenya.

Do feather-degrading bacteria actually degrade feather colour? No significant effects of plumage microbiome modifications on feather colouration in wild great tits.

Parasites are known to exert selective pressures on host life history traits since the energy and nutrients needed to mount an immune response are no longer available to invest in other functions. Bird feathers harbour numerous microorganisms, some of which are able to degrade feather keratin (keratinolytic microorganisms) and affect feather integrity and colouration in vitro. Although named "feather-degrading" microorganisms, experimental evidence for their effects on feathers of free-living birds is still lacking. Here, we tested whether (i) keratinolytic microorganisms can degrade feathers in vivo and thus modify the colour of feathers during the nesting period and (ii) whether feather microorganisms have a long-term effect on the investment in colouration of newly moulted feathers. We designed treatments to either favour or inhibit bacterial growth, thus experimentally modifying plumage bacterial communities, in a wild breeding population of great tits (Parus major). Our analyses revealed no significant effects of the treatments on feather colours. Moreover, we found that differences in bacterial exposure during nesting did not significantly affect the colouration of newly moulted feathers. Our results suggest that significant feather degradation obtained during in vitro studies could have led to an overestimation of the potential of keratinolytic microorganisms to shape feather colouration in free-living birds.

Nearly (?) sterile avian egg in a passerine bird.

During early ontogeny, microbiome affects development of the gastrointestinal tract, immunity, and survival in vertebrates. Bird eggs are thought to be (1) initially sterile (sterile egg hypothesis) and (2) colonized after oviposition through horizontal trans-shell migration, or (3) initially seeded with bacteria by vertical transfer from mother oviduct. To date, however, little empirical data illuminate the contribution of these mechanisms to gut microbiota formation in avian embryos. We investigated microbiome of the egg content (day 0; E0-egg), embryonic gut at day 13 (E13) and female faeces in a free-living passerine, the great tit (Parus major), using a methodologically advanced procedure combining 16S rRNA gene sequencing and microbe-specific qPCR assays. Our metabarcoding revealed that the avian egg is (nearly) sterile, but acquires a slightly richer microbiome during the embryonic development. Of the three potentially pathogenic bacteria targeted by qPCR, only Dietzia was found in E0-egg (yet also in negative controls), E13 gut and female samples, which might indicate possible vertical transfer. Unlike in poultry, we have shown that major bacterial colonization of the gut in passerines does not occur before hatching. We emphasize that protocols that carefully check for environmental contamination are critical in studies with low-bacterial biomass samples.

Pollution-related changes in nest microbiota: Implications for growth and fledging in three passerine birds.

Non-ferrous smelters emit toxic metals into the environment, posing a threat to wildlife health. Despite the acknowledged role of microbes in host health, the impact of such emissions on host-associated microbiota, especially in wild birds, remains largely unexplored. This study investigates the associations of metal pollution, fitness, and nest microbiota (serving as a proxy for early-life microbial environment) which may influence the nestling health and development. Our study focuses on three passerine birds, the great tit (Parus major), blue tit (Cyanistes caeruleus), and pied flycatcher (Ficedula hypoleuca), within control and metal-polluted sites around a Finnish copper-nickel smelter. The polluted sites had been contaminated with arsenic (As), cadmium (Cd), copper (Cu), nickel (Ni), and zinc (Zn). We performed bacterial 16S rRNA sequencing and metal analyses on 90 nests and monitored nestling body mass, fledging success, and various biotic and abiotic factors. Our findings revealed species-specific responses to metal exposure in terms of both fitness and nest microbiota. P. major and C. caeruleus showed sensitivity to pollution, with decreased nestling growth and fledging in the polluted zone. This was accompanied by a shift in the bacterial community composition, which was characterized by an increase in some pathogenic bacteria (in P. major and C. caeruleus nests) and by a decrease in plant-associated bacteria (within C. caeruleus nests). Conversely, F. hypoleuca and their nest microbiota showed limited responses to pollution, indicating greater tolerance to pollution-induced environmental changes. Although pollution did not correlate with nest alpha diversity or the most abundant bacterial taxa across all species, certain potential pathogens within the nests were enriched in polluted environments and negatively correlated with nestling fitness parameters. Our results suggest that metal pollution may alter the nest bacterial composition in some bird species, either directly or indirectly through environmental changes, promoting pathogenic bacteria and potentially impacting bird survival.

Antibiotic resistance, virulence genes, and phylogenetic groups of bacteria isolated from wild passerine birds in Iran.

Wild passerine birds may serve as environmental reservoirs and as vectors for the long-distance dispersal of microorganisms and resistance determinants. However, there is no much knowledge on pathogenic bacteria in wild birds in Iran. The present study aimed to analyze antibiotic resistance in wild passerine birds collected from the northeast region of Iran as the rich breeding bird fauna with a special focus on Escherichia coli virulence, integron, and phylogenetic groups. A total of 326 isolates were collected and identified from the cloaca of wild birds using a swab. The results showed a high percentage of resistance to tetracycline (45.8%) and ampicillin (26.7%). The resistance genes, tet(A), tet(B), tet(M), and tet(L) were detected in tetracycline-resistant isolates, while the blaTEM gene was the most prevalent in ampicillin-resistant isolates (38.6%). Out of the 129 E. coli isolates examined, 99 isolates were found to have virulence gene, with the highest prevalence of the fimbriae (fimH) gene (22.4%). Additionally, the E. coli strains were most often classified into phylogenetic groups B1 (48.8%) followed by B2 (19.3%). Also, the highest average frequency of class 1 integron was detected among our isolates. Results indicated that wild birds are reservoirs of multidrug resistance and virulence genes that may have the potential to be transferred to other organisms, including humans.