This study aimed to characterize Escherichia coli isolates from cloacal samples of white stork nestlings, with a special focus on extended-spectrum β-lactamases (ESBLs)-producing E. coli isolates and their plasmid content. 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 bla_ESBL-carrying plasmids were completely sequenced. 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 β-lactam-phenicol-sulfonamide resistance (bla_TEM/cmlA/floR/sul1/sul2/sul3). Moreover, four white stork nestlings carried ESBL-producing E. coli (4.5%) with the following characteristics: bla_SHV-12/ST38-D, bla_SHV-12/ST58-B1, bla_CTX-M-1/ST162-B1, and bla_CTX-M-32/ST155-B1. Whole-genome sequencing followed by Southern blot hybridizations on S1-PFGE gels in ESBL-positive isolates proved that the bla_CTX-M-1 gene and one of the bla_SHV-12 genes were carried by IncI1/pST3 plasmids, while the second bla_SHV-12 gene and the bla_CTX-M-32 gene were located on IncF plasmids. The two bla_SHV-12 genes and the two bla_CTX-M genes had similar but non-identical close genetic environments, as all four genes were flanked by a variety of insertion sequences. 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.

Background/Objectives: In 2022, an outbreak of H5N1 highly pathogenic avian influenza (HPAI) killed 60% of the largest breeding colony of Dalmatian pelicans (DPs) in the world at Mikri Prespa Lake (Greece), prompting a multidisciplinary study on HPAI and other pathogens. This study determines the antimicrobial resistance rates of cloacal enterococci and Escherichia coli in DPs. Methods: Fifty-two blood and cloacal swab samples were collected from 31 nestlings (20 DP/11 great white pelicans) hatched after the H5N1 outbreak at the Prespa colony and 21 subadult/adult DPs captured at a spring migration stopover. The swabs were inoculated in non-selective and chromogenic-selective media. Identification was performed using MALDI-TOF, and antimicrobial susceptibility was tested. The genetic content was characterized using PCR and sequencing, and the clonality of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli isolates was characterized using Multilocus Sequence Typing. Results: Twenty-eight non-repetitive E. coli and 45 enterococci isolates were recovered in non-selective media; most of them were susceptible to all antibiotics tested (85.7% E. coli/91.1% enterococci). Three of the fifty-two samples (6%, all adults) contained ESBL-E. coli isolates (detected in chromogenic ESBL plates), all carrying the bla_CTX-M-15 gene and belonging to the lineage ST69. Conclusions: Despite the susceptibility of most fecal E. coli and enterococci isolates to all antibiotics tested, the finding that E. coli of lineage ST69 carry bla_CTX-M-15 is of concern. This high-risk clone needs further investigation to elucidate its primary sources and address the growing threat of antimicrobial resistance from an integrated “One Health” perspective. Furthermore, it is imperative to study the potential impacts of ESBL-E. coli on the endangered DP further.

Antimicrobial resistance (AMR) represents a threat to human, animal, and environmental health. This study evaluated the potential role of birds as AMR dispersers in white storks as a model species investigating dispersal between locations connected by their movements. Throughout a year, 346 samples were collected from two landfills (225 fecal samples) and two wetlands (93 fecal/28 water samples) based on satellite tracking data that confirmed continuous stork movements between foraging (landfill) and nighttime roosting (wetlands) locations. Samples were seeded in selective media with antibiotics for the isolation of antibiotic-resistant bacteria. A total of 313 isolates from 35 different bacterial species were obtained, with an AMR prevalence of 43.1% in fecal samples from landfills, 7.5% in fecal samples from wetlands, and 21.4% in water from wetlands. Multidrug resistant bacteria were only found in fecal samples (19.6% landfills/4.3% wetlands) and extended-spectrum beta-lactamase producing-bacteria were found exclusively in fecal samples from landfills with a marked presence during the fall migration period. Our study reveals marked differences in AMR prevalence and resistance phenotypes between study locations and fecal and water samples throughout the year, thus not supporting a clear role of storks as AMR dispersers. Nevertheless, similar changes in AMR phenotype prevalences during fall migration in stork faecal samples from one of the tested landfills and its paired wetland, and the significant increase in ESBL-producing Enterobacterales prevalence matching the arrival of migratory white storks in fall, underline the need for more in-depth genome-based studies to elucidate the role of white storks as dispersers or sentinels of AMR.

In 2022, an avian influenza outbreak caused a massive mortality of Dalmatian pelicans (Pelecanus crispus, hereafter DP) in their world's largest breeding colony located at Mikri Prespa Lake (Greece), while great white pelicans (Pelecanus onocrotalus, hereafter GWP) were not affected. The aim of this study was to describe bacterial diversity and antimicrobial resistance carriage from choanal clefts of DPs and GWPs, to elucidate differences between the two species of pelicans and age groups. Fifty-two choanal swab samples were collected from 31 nestlings (N) (20 DP/11 GWP) late in the 2022 breeding season, and 21 adults (A) DPs (DP-A) in early 2023. Samples were seeded in general and chromogenic selective media. Isolates were identified by MALDI-TOF mass spectrometry and antimicrobial susceptibility was evaluated by disk diffusion method for staphylococci/mammaliicocci, enterococci and Enterobacterales. A total of 248 non-repetitive isolates (46A/202N) were recovered, representing 39 bacterial species from 18 genera. A very high prevalence of S. aureus (80.8 % of samples tested) was detected in both age groups (85.7 %A/77.4 %N), with nestlings also showing greater staphylococcal/mammaliicoccal diversity. Most isolates recovered were susceptible to all the antibiotics tested, and none S. aureus was methicillin-resistant. Nevertheless, one multi-drug resistant (MDR) CTX-M-15-producing E. coli-ST69 isolate was found in one DP-A. This study revealed a high rate of antimicrobial susceptibility and staphylococcal/mammaliicoccal diversity in choanal samples of pelicans, with the occasional detection of MDR isolates of concern, like the extended-spectrum beta-lactamase-producing E. coli strain.

In 2022, an outbreak of H5N1 highly pathogenic avian influenza (HPAI) killed 60% of the largest breeding colony of Dalmatian pelican (DP) in the world, at Mikri Prespa Lake (Greece), prompting a multidisciplinary study on HPAI and other pathogens. This study determines the antimicrobial resistance rates of cloacal enterococci and Escherichia coli in DPs. Fifty-two blood and cloacal swab samples were collected from 31 nestlings (20 DP/11 great white pelicans) hatched after the H5N1 outbreak at the Prespa colony and 21 subadult/adult DPs captured at a spring migration stopover. Swabs were inoculated in non-selective and chromogenic selective media. Identification was performed by MALDI-TOF, and antimicrobial susceptibility was tested. The genetic content was characterized by PCR-sequencing and the clonality of extended-spectrum-betalactamase (ESBL)-producing-E. coli isolates by Multi-Locus-Sequence-Typing. Twenty-eight non-repetitive E. coli and 45 enterococci isolates were recovered in non-selective media; most of them were susceptible to all antibiotics tested (85.7% E. coli/91.1% enterococci). Three of 52 samples (6%, all adults) contained ESBL-E. coli isolates (detected in chromogenic ESBL plates), all carrying the bla_CTX-M-15 gene and belonging to the lineage ST69. Despite the susceptibility of most fecal E. coli and enterococci isolates to all antibiotics tested, the identification of E. coli of lineage ST69 carrying blaCTX-M-15 is of concern. This high-risk clone needs further investigation to elucidate its primary sources and address the growing threat of antimicrobial resistance from an integrated 'One Health' perspective. Furthermore, it is imperative to further study the potential impacts of ESBL-E. coli on the endangered DP.

Antimicrobial resistance (AMR) represents a serious problem affecting human, animal and ecosystem health. This study evaluated the potential role of white storks as AMR dispersers between two locations frequently used for foraging and resting. Through a whole year, 346 samples (318 feces/ 28 water) were collected in two landfills (225 fecal samples) and two wetlands (93 fecal/ 28 water samples) with a previous tracking that confirmed continuous storks movements between foraging (landfill) and nighttime roosting (wetlands) locations. All samples were seeded in selective media with antibiotics for the isolation of antibiotic resistant bacteria. A total of 313 isolates from 35 different bacterial species were obtained, with an AMR prevalence of 43.1% in fecal samples from landfills, 7.5% in fecal samples from wetlands and 21.4% in water from wetlands. Multidrug resistant bacteria were only found in fecal samples (19.6% landfills/ 4.3% wetlands) and Extended-spectrum beta-lactamase producing-bacteria was found exclusively in fecal samples from landfills with a marked presence during the fall migration stork’s migration period. Despite subtle circumstantial evidence of dispersion between one landfill and its paired wetland and a possible link with migratory movements, the marked differences in prevalence and resistance phenotypes found in each location did not allow establishing a clear dispersal activity by storks.