Abstract
Antimicrobial resistance (AMR) is a major global health challenge and its circulation in wildlife is garnering increasing attention. Seabirds, such as the yellow-legged gull (Larus michahellis), which inhabit the interface between marine and anthropogenic environments and exhibit synanthropic habits, are valuable targets for investigating AMR. Therefore, this study focused on the dissemination of bacteria resistant to the critically important antimicrobials (CIA) cefepime (CEP) and enrofloxacin (ENR), by sampling the cloacal content of 15 L. michahellis nestlings. Twenty-seven strains resistant to CEP and/or ENR were isolated. Enterobacter hormaechei subsp. hoffmannii was the most represented species, and all those isolates shared the same multidrug resistance profile. Ten resistant strains belonged to the genus Enterococcus, while three strains belonging to the Bacillus cereus complex were CEP resistant. No horizontal transfer of CEP or ENR resistance was observed. High rates of resistance to trimethoprim/sulphametoxazole, streptomycin, tetracycline, and chloramphenicol were detected. The study evidences the wide presence of CIA-resistant bacteria in L. michahellis, indicating that yellow-legged gulls may contribute to the environmental dissemination of clinically relevant resistances. This highlights the potential bidirectional circulation of resistant bacteria between anthropogenic sources and natural ecosystems, thereby increasing the likelihood of exposure to humans, domestic animals, and wildlife.
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Data availability
The datasets generated during the current study are available from the corresponding author on reasonable request. The nucleotide sequences of the partial 16 S rRNA gene analyzed for species identification have been submitted to NCBI GenBank with the accession numbers PX390185-PX390198.
References
Patel, J. et al. Measuring the global response to antimicrobial resistance, 2020-21: a systematic governance analysis of 114 countries. Lancet Infect. Dis. 23, 706–718. https://doi.org/10.1016/S1473-3099(22)00796-4 (2023).
Food and Agriculture Organization of the United Nations, World Health Organization, World Organisation for Animal Health. Second joint FAO/OIE/WHO expert workshop on non-human antimicrobial usage and antimicrobial resistance: Management options (World Health Organization, 2004). https://openknowledge.fao.org/server/api/core/bitstreams/95194054-3c82-479e-8ee4-74fe3312dcf9/content
World Health Organization. WHO list of medically important antimicrobials (World Health Organization, 2024). https://cdn.who.int/media/docs/default-source/gcp/who-mia-list-2024-lv.pdf
Grabowski, Ł. et al. Enrofloxacin-The ruthless killer of eukaryotic cells or the last hope in the fight against bacterial infections? Int. J. Mol. Sci. 23, 3648. https://doi.org/10.3390/ijms23073648 (2022).
Catry, B. et al. Use of colistin-containing products within the European Union and European Economic Area (EU/EEA): development of resistance in animals and possible impact on human and animal health. Int. J. Antimicrob. Agents. 6, 297–306. https://doi.org/10.1016/j.ijantimicag.2015.06.005 (2015).
Singleton, D. A. et al. A randomised controlled trial to reduce highest priority critically important antimicrobial prescription in companion animals. Nat. Commun. 12, 1593. https://doi.org/10.1038/s41467-021-21864-3 (2021).
Marco-Fuertes, A., Marin, C., Lorenzo-Rebenaque, L., Vega, S. & Montoro-Dasi, L. Antimicrobial resistance in companion animals: A new challenge for the One Health approach in the European Union. Vet. Sci. 9, 208. https://doi.org/10.3390/vetsci9050208 (2022).
Gehring, R., Mochel, J. P. & Schmerold, I. Understanding the background and clinical significance of the WHO, WOAH, and EMA classifications of antimicrobials to mitigate antimicrobial resistance. Front. Vet. Sci. 10, 1153048. https://doi.org/10.3389/fvets.2023.1153048 (2023).
Feldman, S. F. et al. A nationwide population-based study of Escherichia coli bloodstream infections: incidence, antimicrobial resistance and mortality. Clin. Microbiol. Infect. 28 879.e1-879.e7 (2022).
Choudhury, D. et al. Clonal and resistance profiles of fluoroquinolone-resistant uropathogenic Escherichia coli in countries with different practices of antibiotic prescription. Front. Microbiol. 15, 1446818. https://doi.org/10.3389/fmicb.2024 (2024).
Golli, A. L. et al. Prevalence of multidrug-resistant pathogens causing bloodstream infections in an intensive care unit. Infect. Drug Resist. 5, 5981–5992. https://doi.org/10.2147/IDR.S383285 (2022).
Kumar, N. R., Balraj, T. A., Kempegowda, S. N. & Prashant, A. Multidrug-resistant sepsis: A critical healthcare challenge. Antibiotics 13, 46. https://doi.org/10.3390/antibiotics13010046 (2024).
Huemer, M., Mairpady Shambat, S., Brugger, S. D. & Zinkernagel, A. S. Antibiotic resistance and persistence-Implications for human health and treatment perspectives. EMBO Rep. 21, e51034. https://doi.org/10.15252/embr.202051034 (2020).
Mancuso, G., Midiri, A., Gerace, E. & Biondo, C. Bacterial antibiotic resistance: The most critical pathogens. Pathogens 10, 1310. https://doi.org/10.3390/pathogens10101310 (2021).
GBD 2021 Antimicrobial Resistance Collaborators. Global burden of bacterial antimicrobial resistance 1990–2021: a systematic analysis with forecasts to 2050. Lancet 404, 1199–1226. https://doi.org/10.1016/S0140-6736(24)01867-1 (2024).
Samreen, Ahmad, I., Malak, H. A. & Abulreesh, H. H. Environmental antimicrobial resistance and its drivers: a potential threat to public health. J. Glob Antimicrob. Resist. 27, 101–111. https://doi.org/10.1016/j.jgar.2021.08.001 (2021).
Navarro, J. et al. Feathered detectives: Real-time GPS tracking of scavenging gulls pinpoints illegal waste dumping. PLoS One. 11, e0159974. https://doi.org/10.1371/journal.pone.015997 (2016).
Zorrozua, N. et al. Evaluating the dependence of opportunistic yellow-legged gulls (Larus michahellis) on marine habitat and fishing discards. Ibis 166, 112–128. https://doi.org/10.1111/ibi.13227 (2024).
Ramírez, F. et al. Humans shape the year-round distribution and habitat use of an opportunistic scavenger. Ecol. Evol. 10, 4716–4725. https://doi.org/10.1002/ece3.622 (2020).
Navarro, J. et al. Pathogen transmission risk by opportunistic gulls moving across human landscapes. Sci. Rep. 9 https://doi.org/10.1038/s41598-019-46326-1 (2019).
Jacoby, G. A. et al. qnrB, another plasmid-mediated gene for quinolone resistance. Antimicrob. Agents Chemother. 50, 1178–1182. https://doi.org/10.1128/AAC.50.4.1178-1182.2006 (2006).
Manzulli, V. et al. Discrimination of Bacillus cereus group members by MALDI-TOF mass spectrometry. Microorganisms 9, 1202. https://doi.org/10.3390/microorganisms9061202 (2021).
Palmeira, J. D. et al. Emergence and spread of cephalosporinases in wildlife: A review. Animals 11, 1765. https://doi.org/10.3390/ani11061765 (2021).
Adachi, F., Yamamoto, A., Takakura, K. & Kawahara, R. Occurrence of fluoroquinolones and fluoroquinolone-resistance genes in the aquatic environment. Sci. Total Environ. 444, 508–514. https://doi.org/10.1016/j.scitotenv.2012.11.077 (2013).
Lai, O. et al. Observed prevalence and characterization of fluoroquinolone-resistant and multidrug-resistant bacteria in loggerhead sea turtles (Caretta caretta) from the Adriatic Sea. Antibiotics 14, 252. https://doi.org/10.3390/antibiotics14030252 (2025).
Champagnon, J., Carré, H. & Gili, L. Effects of research disturbance on nest survival in a mixed colony of waterbirds. PeerJ 7, e7844. https://doi.org/10.7717/peerj.7844 (2019).
Sethuvel, D. P. M. et al. β-lactam resistance in ESKAPE pathogens mediated through modifications in penicillin-binding proteins: An overview. Infect. Dis. Ther. 12, 829–841. https://doi.org/10.1007/s40121-023-00771-8 (2023).
Timmler, S. B. et al. CroR Regulates expression of pbp4(5) to promote cephalosporin resistance in Enterococcus faecalis. mBio 13, e0111922. https://doi.org/10.1128/mbio.01119-22 (2022).
Pallares, R. et al. Cephalosporins as risk factor for nosocomial Enterococcus faecalis bacteremia. A matched case-control study. Arch. Intern. Med. 153, 1581–1586 (1993).
Siesing, P. C., Alva-Jørgensen, J. P., Brodersen, J., Arpi, M. & Jensen, P. E. Rising incidence of Enterococcus species in microbiological specimens from orthopedic patients correlates to increased use of cefuroxime: a study concentrating on tissue samples. Acta Orthop. 84, 319–322. https://doi.org/10.3109/17453674.2013.792028 (2013).
Sangiorgio, G., Calvo, M., Migliorisi, G., Campanile, F. & Stefani, S. The impact of Enterococcus spp. in the immunocompromised host: A comprehensive review. Pathogens 13, 409. https://doi.org/10.3390/pathogens13050409 (2024).
Ferguson, D. M. et al. Virulence genes among Enterococcus faecalis and Enterococcus faecium isolated from coastal beaches and human nonhuman sources in Southern California and Puerto Rico. J. Pathog. 2016, 3437214. https://doi.org/10.1155/2016/3437214 (2016).
Kwit, R. et al. Prevalence of Enterococcus spp. and the whole-genome characteristics of Enterococcus faecium and Enterococcus faecalis strains isolated from free-living birds in Poland. Pathogens 12, 836. https://doi.org/10.3390/pathogens12060836 (2023).
Manzulli, V. et al. Evaluation of in vitro antimicrobial susceptibility of Bacillus anthracis strains isolated during anthrax outbreaks in Italy from 1984 to 2017. J. Vet. Sci. 20, 58–62. https://doi.org/10.4142/jvs.2019.20.1.58 (2019).
Turnbull, P. C. et al. MICs of selected antibiotics for Bacillus anthracis, Bacillus cereus, Bacillus thuringiensis, and Bacillus mycoides from a range of clinical and environmental sources as determined by the Etest. J. Clin. Microbiol. 42, 3626–3634. https://doi.org/10.1128/JCM.42.8.3626-3634.2004 (2004).
Chen, Y., Succi, J., Tenover, F. C. & Koehler, T. M. Beta-lactamase genes of the penicillin-susceptible Bacillus anthracis Sterne strain. J. Bacteriol. 185, 823–830. https://doi.org/10.1128/JB.185.3.823-830.2003 (2003).
Intra, J. et al. Antimicrobial resistance patterns of Enterobacter cloacae and Klebsiella aerogenes strains isolated from clinical specimens: A twenty-year surveillance study. Antibiotics 12, 775. https://doi.org/10.3390/antibiotics12040775 (2023).
Chang, C. Y., Huang, P. H. & Lu, P. L. The resistance mechanisms and clinical impact of resistance to the third generation cephalosporins in species of Enterobacter cloacae complex in Taiwan. Antibiotics 11, 1153. https://doi.org/10.3390/antibiotics11091153 (2022).
Matsuo, A. et al. Molecular epidemiology and β-lactam resistance mechanisms of Enterobacter cloacae complex isolates obtained from bloodstream infections, Kyoto, Japan. Microbiol. Spectr. 13, e0248524. https://doi.org/10.1128/spectrum.02485-24 (2025).
Lautenbach, E. et al. Longitudinal trends in fluoroquinolone resistance among Enterobacteriaceae isolates from inpatients and outpatients, 1989–2000: differences in the emergence and epidemiology of resistance across organisms. Clin. Infect. Dis. 38, 655–662. https://doi.org/10.1086/381549 (2004).
Pallares-Vega, R. et al. Temperature and nutrient limitations decrease transfer of conjugative IncP-1 plasmid pKJK5 to wild Escherichia coli strains. Front. Microbiol. 12, 656250. https://doi.org/10.3389/fmicb.2021.656250 (2021).
Hooper, D. C. & Jacoby, G. A. Mechanisms of drug resistance: quinolone resistance. Ann. N Y Acad. Sci. 1354, 12–31. https://doi.org/10.1111/nyas.12830 (2015).
Partridge, S. R., Kwong, S. M., Firth, N. & Jensen, S. O. Mobile genetic elements associated with antimicrobial resistance. Clin. Microbiol. Rev. 31, e00088–e00017 (2018). 10.1128/CMR.00088 – 17.
Prats, G. et al. Antibiotic resistance trends in enteropathogenic bacteria isolated in 1985–1987 and 1995–1998 in Barcelona. Antimicrob. Agents Chemother. 44, 1140–1145 (2000).
Pugliese, N., Maimone, F., Scrascia, M., Materu, S. F. & Pazzani, C. SXT-related integrating conjugative element and IncC plasmids in Vibrio cholerae O1 strains in Eastern Africa. J. Antimicrob. Chemother. 63, 438–442. https://doi.org/10.1093/jac/dkn542 (2009).
Marinho, C. M., Santos, T., Gonçalves, A., Poeta, P. & Igrejas, G. A decade-long commitment to antimicrobial resistance surveillance in Portugal. Front. Microbiol. 7, 1650. https://doi.org/10.3389/fmicb.2016.01650 (2016).
Ramos, R., Ramirez, F., Sanpera, C., Jover, L. & Ruiz, X. Feeding ecology of yellow-legged gulls Larus michahellis in the western Mediterranean: A comparative assessment using conventional and isotopic methods. Mar. Ecol. Prog Ser. 377, 289–297. https://doi.org/10.3354/meps07792 (2009).
Manzulli, V. et al. Psychrobacter raelei sp. nov., isolated from a dog with peritonitis. Int. J. Syst. Evol. Microbiol. 74 https://doi.org/10.1099/ijsem.0.006353 (2024).
Galkiewicz, J. P. & Kellogg, C. A. Cross-kingdom amplification using bacteria-specific primers: complications for studies of coral microbial ecology. Appl. Environ. Microbiol. 74, 7828–7831. https://doi.org/10.1128/AEM.01303-08 (2008).
Huang, X. & Madan, A. CAP3: A DNA sequence assembly program. Genome Res. 9, 868–877. https://doi.org/10.1101/gr.9.9.868 (1999).
Chalita, M. et al. EzBioCloud: a genome-driven database and platform for microbiome identification and discovery. Int. J. Syst. Evol. Microbiol. 74, 006421. https://doi.org/10.1099/ijsem.0.006421 (2024).
Guastalegname, M. et al. An outbreak of human systemic anthrax, including one case of anthrax meningitis, occurred in Calabria Region (Italy): A description of a successful One Health approach. Life 12, 909. https://doi.org/10.3390/life12060909 (2022).
Clinical and Laboratory Standard Institute. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically 7th edn (CLSI, 2006).
Clinical and Laboratory Standard Institute. Performance standards for antimicrobial disk susceptibility tests 9th edn (CLSI, 2006).
Clinical and Laboratory Standard Institute. Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals 7th edn (CLSI, 2024).
Clinical and Laboratory Standard Institute. Performance standards for antimicrobial susceptibility testing 35th edn (CLSI, 2025).
The European Committee on Antimicrobial Susceptibility Testing. Breakpoint tables for interpretation of MICs and zone diameters, version 15.0 (2025). http://www.eucast.org/clinical_breakpoints
Magiorakos, A. P. et al. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin. Microbiol. Infect. 18, 268–281. https://doi.org/10.1111/j.1469-0691.2011.03570.x (2012).
Islam, M. S. et al. Detection of blaTEM, blaCTX–M, blaCMY, and blaSHV genes among extended-spectrum beta-lactamase-producing Escherichia coli isolated from migratory birds travelling to Bangladesh. Microb Ecol. 83, 942–950. https://doi.org/10.1007/s00248-021-01803-x (2022).
Kanokudom, S. et al. Rapid detection of extended spectrum β-lactamase producing Escherichia coli isolated from fresh pork meat and pig cecum samples using multiplex recombinase polymerase amplification and lateral flow strip analysis. PLoS One. 16, e0248536. https://doi.org/10.1371/journal.pone.0248536 (2021).
Ye, J. et al. Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinform. 13, 134. https://doi.org/10.1186/1471-2105-13-134 (2012).
Nishihara, Y. et al. Differences in β-lactamase activity and carbapenem resistance among the Bacillus cereus group. Antimicrob. Agents Chemother. 70, e0130225. https://doi.org/10.1128/aac.01302-25 (2026).
Clopper, C. J. & Pearson, E. S. The Use of confidence or fiducial limits illustrated in the case of the binomial. Biometrika 26, 404–413 (1934). ;.2307/2331986.
R Core Team. R: A language and environment for statistical computing (R Foundation for Statistical Computing, 2026).
Acknowledgements
We are grateful to Antonella Bove and Diana Romito for their technical assistance.
Funding
This study has been funded by the project “CALLIOPE - CasA dell’InnovaziOne Per il one hEalth” (funders: Italian Ministry of Enterprises and Made in Italy; Italy’s Development and Cohesion Fund), and by the Ricerca Corrente IZSPB 01/2023 “Antibiotico resistenza, il ruolo dell’avifauna selvatica (ARAS)” (funder: Italian Ministry of Health). The Open Access Fees have been covered by the “Fondo per la Qualità e l’Internazionalizzazione della Ricerca” according to the D.R.2924/2024 (funder: University of Bari).
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Conceptualization: NP, AC; Data curation: NP, OL, VM, SC, GC; funding acquisition: AC, AP; investigation – sampling procedures: RS, CL, AC; investigation – isolation and cultural procedures: NP, CI, RS, RL, MP, DS, VG; investigation – MALDI/TOF identification: VM, DG; investigation – 16 S rRNA gene identification: NP, SC, AB, LC, AP; methodology: NP, VM, LC, AP; project administration: AC; resources: DG, AP, AC; supervision: NP, DG, AP, AC; validation: OL, EC; visualization: NP, VG; writing – original draft: NP, OL, AC; writing – review and editing: DG, EC, AP.
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The study has been approved by the Italian Institute for Environmental Protection and Research (ISPRA), with the authorization number 25336/2024, prot. 0028044. All procedures complied with the Italian and European regulations for animal welfare in scientific studies. No animals were harmed during this study, and all procedures involving animals were conducted by expert veterinarian personnel.
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Pugliese, N., Intermite, C., Samarelli, R. et al. Commensal and opportunistic bacteria resistant to fourth-generation cephalosporins or fluoroquinolones isolated from yellow-legged gulls (Larus michahellis) settled in Taranto, Southern Italy. Sci Rep (2026). https://doi.org/10.1038/s41598-026-49997-9
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DOI: https://doi.org/10.1038/s41598-026-49997-9


