Connectivity at the human-wildlife interface: starling movements relate to carriage of E. coli.

Synanthropic bird species in human, poultry or livestock environments can increase the spread of pathogens and antibiotic-resistant bacteria between wild and domestic animals. We present the first telemetry-based spatial networks for a small songbird. We quantified landscape connectivity exerted by spotless starling movements, and aimed to determine if connectivity patterns were related to carriage of potential pathogens. We captured 28 starlings on a partridge farm in 2020 and tested them for Avian influenza virus, West Nile virus WNV, Avian orthoavulavirus 1, Coronavirus, Salmonella spp. and Escherichia coli. We did not detect any viruses or Salmonella, but one individual had antibodies against WNV or cross-reacting Flaviviruses. We found E. coli in 61 % (17 of 28) of starlings, 76 % (13 of 17) of which were resistant to gentamicin, 12 % (2 of 17) to cefotaxime/enrofloxacin and 6 % (1 of 17) were phenotypic extended spectrum beta-lactamase (ESBL) carriers. We GPS-tracked 17 starlings and constructed spatial networks showing how their movements (i.e. links) connect different farms with nearby urban and natural habitats (i.e. nodes with different attributes). Using E. coli carriage as a proxy for acquisition/dispersal of bacteria, we found differences across spatial networks constructed for E. coli positive (n = 7) and E. coli negative (n = 9) starlings. We used Exponential Random Graph Models to reveal significant differences between networks. In particular, an urban roost was more connected to other sites by movements of E. coli positive than by movements of E. coli negative starlings. Furthermore, an open pine forest used mainly for roosting was more connected to other sites by movements of E. coli negative than by movements of E. coli positive starlings. Using E. coli as a proxy for a potential pathogen carried by starlings, we reveal the pathways of spread that starlings could provide between farms, urban and natural habitats.

Seasonal changes in bird communities on poultry farms and house sparrow-wild bird contacts revealed by camera trapping.

Introduction: Wild birds are considered reservoirs of poultry pathogens although transmission routes have not been conclusively established. Here we use camera trapping to study wild bird communities on commercial layer and red-legged partridge farms over a one-year timeframe. We also analyze direct and indirect interactions of other bird species with the house sparrow (Passer domesticus), a potential bridge host. Methods: We conducted camera trapping events between January 2018 and October 2019, in two caged layer farms, one free-range layer farm, and two red-legged partridge farms in South-Central Spain. Results and Discussion: We observed wild bird visits on all types of farms, with the significantly highest occurrence on red-legged partridge farms where food and water are more easily accessible, followed by commercial caged layer farms, and free-range chicken farms. The house sparrow (Passer domesticus) followed by spotless starlings (Sturnus unicolor) was the most encountered species on all farms, with the highest frequency in caged layer farms. On partridge farms, the house sparrow accounted for 58% of the wild bird detections, while on the free-range chicken farm, it made up 11% of the detections. Notably, the breeding season, when food and water are scarce in Mediterranean climates, saw the highest number of wild bird visits to the farms. Our findings confirm that the house sparrow, is in direct and indirect contact with layers and red-legged partridges and other wild birds independent of the type of farm. Contacts between house sparrows and other bird species were most frequent during the breeding season followed by the spring migration period. The species most frequently involved in interactions with the house sparrow belonged to the order Passeriformes. The study provides a comparative description of the composition and seasonal variations of bird communities in different types of layer/ poultry farms in Southern Spain i.e. a Mediterranean climate. It confirms the effectiveness of biosecurity measures that restrict access to feed and water. Additionally, it underscores the importance of synanthropic species, particularly the house sparrow, as potential bridge vector of avian pathogens.

Detection and genetic characterization of blaESBL-carrying plasmids of cloacal Escherichia coli isolates from white stork nestlings (Ciconia ciconia) in Spain.

OBJECTIVES: This study aimed to characterize Escherichia coli isolates from cloacal samples of white stork nestlings, with a special focus on extended-spectrum ß-lactamases (ESBLs)-producing E. coli isolates and their plasmid content. METHODS: Cloacal samples of 88 animals were seeded on MacConkey-agar and chromogenic-ESBL plates to recover E. coli and ESBL-producing E. coli. Antimicrobial susceptibility was screened using the disc diffusion method, and the genotypic characterization was performed by polymerase chain reaction (PCR) and subsequent sequencing. S1 nuclease Pulsed-Field-Gel-Electrophoresis (PFGE), Southern blotting, and conjugation essays were performed on ESBL-producing E. coli, as well as whole-genome sequencing by short- and long-reads. The four blaESBL-carrying plasmids were completely sequenced. RESULTS: A total of 113 non-ESBL-producing E. coli isolates were collected on antibiotic-free MacConkey-agar, of which 27 (23.9%) showed a multidrug-resistance (MDR) phenotype, mainly associated with ß-lactam-phenicol-sulfonamide resistance (blaTEM/cmlA/floR/sul1/sul2/sul3). Moreover, four white stork nestlings carried ESBL-producing E. coli (4.5%) with the following characteristics: blaSHV-12/ST38-D, blaSHV-12/ST58-B1, blaCTX-M-1/ST162-B1, and blaCTX-M-32/ST155-B1. Whole-genome sequencing followed by Southern blot hybridizations on S1-PFGE gels in ESBL-positive isolates proved that the blaCTX-M-1 gene and one of the blaSHV-12 genes were carried by IncI1/pST3 plasmids, while the second blaSHV-12 gene and the blaCTX-M-32 gene were located on IncF plasmids. The two blaSHV-12 genes and the two blaCTX-M genes had similar but non-identical close genetic environments, as all four genes were flanked by a variety of insertion sequences. CONCLUSION: The role played by several genetic platforms in the mobility of ESBL genes allows for interchangeability on a remarkably small scale (gene-plasmid-clones), which may support the spread of ESBL genes.

Antimicrobial resistome of coagulase-negative staphylococci from nasotracheal cavities of nestlings of Ciconia ciconia in Southern Spain: Detection of mecC-SCCmec type-XI-carrying S. lentus.

The antimicrobial resistance (AMR) genes of 268 non-duplicated coagulase-negative staphylococci (CoNS) previously obtained from nasotracheal cavities of nestling storks were characterized. They included S. sciuri isolates (n = 191), and non-sciuri-CoNS isolates (NSc-CoNS, n = 77). All S. sciuri carried the intrinsic salA gene (for clindamycin-resistance) and so, clindamycin was not considered for general analysis in this species. About 71.7%/41.6% of the S. sciuri/NSc-CoNS isolates were susceptible to all antibiotics tested; moreover, 14.1%/16.9% and 3.1%/20.8% of S. sciuri/NSc-CoNS showed single antibiotic resistance and multidrug resistance (MDR) phenotype, respectively. Of the ten mecA-positive CoNS isolates, six were associated with SCCmec types-III, -IV or -V elements. Remarkably was the detection of one MDR-S. lentus isolate carrying both mecA and mecC genes, as well as the SCCmec type-XI element. MDR-CoNS was relatively higher in nestlings of parent storks foraging in landfills (21.3%) than those in natural areas (9.7%) (χ2 = 3.421, df=1, p = 0.064). AMR phenotypes (and genes detected) include penicillin (blaZ, blaARL), erythromycin-clindamycin-constitutive (ermA, ermC, ermT), clindamycin (lnuA, salA, vgaA), erythromycin (msrA, mphC), tetracycline (tetK, tetL, tetM), tobramycin (ant4'), tobramycin-gentamicin (aac6'-aph2″), sulfamethoxazole-trimethoprim (dfrA, dfrG, dfrK), chloramphenicol (fexA, fexB, catPC221), and mupirocin (mupA). Interestingly, one S. epidermidis isolate carried the ermT gene. About 29.9% of nestlings harboured more than one non-duplicated CoNS (with varied 2-5 AMR profiles). This study demonstrated that most of the CoNS isolates were susceptible to all the antibiotics tested (63.1%). However, AMR genes of public health importance were found, including the mecC-mediated methicillin resistance trait.

Nasotracheal Microbiota of Nestlings of Parent White storks with Different Foraging Habits in Spain.

Migratory storks could be vectors of transmission of bacteria of public health concern mediated by the colonization, persistence and excretion of such bacteria. This study aims to determine genera/species diversity, prevalence, and co-colonization indices of bacteria obtained from tracheal (T) and nasal (N) samples from storks in relation to exposure to point sources through foraging. One-hundred and thirty-six samples from 87 nestlings of colonies of parent white storks with different foraging habits (natural habitat and landfills) were obtained (84 T-samples and 52 N-samples) and processed. Morphologically distinct colonies (up to 12/sample) were randomly selected and identified by MALDI-TOF-MS. About 87.2% of the total 806 isolates recovered were identified: 398 from T-samples (56.6%) and 305 from N-samples (43.4%). Among identified isolates, 17 genera and 46 species of Gram-positive and Gram-negative bacteria were detected, Staphylococcus (58.0%) and Enterococcus (20.5%) being the most prevalent genera. S. sciuri was the most prevalent species from T (36.7%) and N (34.4%) cavities of total isolates, followed by E. faecalis (11.1% each from T and N), and S. aureus [T (6.5%), N (13.4%)]. Of N-samples, E. faecium was significantly associated with nestlings of parent storks foraging in landfills (p = 0.018). S. sciuri (p = 0.0034) and M. caseolyticus (p = 0.032) from T-samples were significantly higher among nestlings of parent storks foraging in natural habitats. More than 80% of bacterial species in the T and N cavities showed 1-10% co-colonization indices with one another, but few had ≥ 40% indices. S. sciuri and E. faecalis were the most frequent species identified in the stork nestlings. Moreover, they were highly colonized by other diverse and potentially pathogenic bacteria. Thus, storks could be sentinels of point sources and vehicles of bacterial transmission across the "One Health" ecosystems.

Staphylococcus aureus Carriage in the Nasotracheal Cavities of White Stork Nestlings (Ciconia ciconia) in Spain: Genetic Diversity, Resistomes and Virulence Factors.

The molecular ecology of Staphylococcus aureus in migratory birds (such as white storks) is necessary to understand their relevance in the "One Health" ecosystems. This study determined the nasotracheal carriage rates of S. aureus from white storks in Southern Spain and genetically characterized the within-host diversity. A collection of 67 S. aureus strains, previously obtained from 87 white stork nestlings (52 nasal and 85 tracheal samples) fed by their parents with food foraged in natural and landfill habitats, were tested for their antimicrobial resistance (AMR) phenotypes. Moreover, the AMR genotypes, immune evasion cluster (IEC), virulence genes and the detection of CC398 lineage were studied by PCR. The spa types and multilocus-sequencing-typing (MLST) were also determined by PCR and sequencing. Staphylococcus aureus carriage was found in 31% of storks (36.5%/11.9% in nasal/tracheal samples). All isolates were methicillin-susceptible (MSSA) and 8.8% of them were also susceptible to all tested antibiotics. The AMR phenotype/percentage/genes detected were as follows: penicillin/79.1%/blaZ; erythromycin-clindamycin-inducible/19.1%/ermA, ermT; tetracycline/11.9%/tetK; clindamycin/4.5%/lnuA and ciprofloxacin/4.5%. Twenty-one different spa types, including 2 new ones (t7778-ST15-CC15 and t18009-ST26-CC25), were detected and ascribed to 11 clonal complexes (CCs). MSSA-CC398 (8.2%), MSSA-CC15 (7.1%) and MSSA-ST291 (5.9%) were the most prevalent lineages in storks. Moreover, tst-positive (MSSA-CC22-t223 and MSSA-CC30-t1654), eta-positive (MSSA-CC9-t209) and etb-positive strains (MSSA-CC45-t015) were detected in four storks. The 18.5% of storks harboured distinct MSSA strains (with different lineages and/or AMR genes). Nestlings of storks foraging in landfills (10 CCs) had more diverse S. aureus strains than those of parents foraging in natural habitats (3 CCs). Low level of AMR was demonstrated among S. aureus strains. The predominance of MSSA-CC398 (an emergent clade) and toxigenic MSSA strains in stork nestlings highlight the need for continuous surveillance of S. aureus in wild birds.

Bagaza virus and Plasmodium spp. coinfection in red-legged partridges (Alectoris rufa), in Southern Spain 2019.

Flaviviruses such as West Nile (WNV), Usutu (USUV) and Bagaza (BAGV) virus and avian malaria parasites are vector borne pathogens that circulate naturally between avian and mosquito hosts. WNV and USUV and potentially also BAGV constitute zoonoses. Temporal and spatial cocirculation and coinfection with Plasmodium spp., and West Nile virus has been documented in birds and mosquito vectors, and fatally USUV-infected passerines coinfected with Plasmodium spp. had more severe lesions. Also, WNV, USUV and BAGV have been found to cocirculate. Yet little is known about the interaction of BAGV and malaria parasites during consecutive or coinfections of avian hosts. Here we report mortality of free-living red-legged partridges in a hunting estate in Southern Spain that were coinfected with BAGV and Plasmodium spp. The outbreak occurred in the area where BAGV first emerged in Europe in 2010 and where cocirculation of BAGV, USUV and WNV was confirmed in 2011 and 2013. Partridges were found dead in early October 2019. Birds had mottled locally pale pectoral muscles, enlarged, congestive greenish-black tinged livers and enlarged kidneys. Microscopically congestion and predominantly mononuclear inflammatory infiltrates were evident and Plasmodium phanerozoites were present in the liver, spleen, kidneys, muscle and skin. Molecular testing and sequencing detected Plasmodium spp. and BAGV in different tissues of the partridges, and immunohistochemistry confirmed the presence and colocalization of both pathogens in the liver and spleen. Due to the importance of the red-legged partridge in the ecosystem of the Iberian Peninsula and as driver of regional economy such mortalities are of concern. Such outbreaks may reflect climate change related shifts in host, vector and pathogen ecology and interactions that could emerge similarly for other pathogens.