Antimicrobial multiresistance in microorganisms of the Enterobacterales order isolated from exotic passerines from farms in the Metropolitan Region of FortalezaAbstract
The aim of this study was to assess the degree of antimicrobial resistance in strains of the Enterobacterales order isolated from exotic passerine birds originated from breeding facilities in the metropolitan region of Fortaleza. For this purpose, 104 strains of ten different bacterial species were used. To perform the antibiogram, ten antibiotics from eight pharmacological classes were employed. Among the main results, a high resistance rate was found for some antimicrobials, notably ampicillin (91.35%) and sulfamethoxazole-trimethoprim (47.12%). Additionally, relevant rates of resistance were also found for meropenem, a controlled-use antibiotic. Concerning the bacterial species, Escherichia coli showed resistance to nine out of the ten tested antibiotics, and out of the 24 strains used, eight showed resistance to at least three antibiotics, classified as multidrug-resistant. Thus, the results presented reinforce the need for surveillance regarding multidrug-resistant strains because, besides causing harm to animal health, they could also become a public health problem due to the zoonotic potential that some strains possess.
Keywords:
antibiotics; enterobacteria;
Escherichia coli
; zoonosis; public health
Resumo
O objetivo deste trabalho foi avaliar o grau de resistência a antimicrobianos em cepas da ordem Enterobacterales, isoladas de passeriformes exóticos oriundos de criadouros da região metropolitana de Fortaleza. Para tanto, foram utilizadas 104 cepas de dez espécies bacterianas diferentes. Para realizar o antibiograma, foram utilizados dez antibióticos de oito classes farmacológicas. Dentre os principais resultados, estão a alta taxa resistência encontrada para alguns antimicrobianos, com destaque para ampicilina (91,35%) e sulfazotrim (47,12%). Além disso, também foram encontradas taxas relevantes de resistência para o meropenem, antibiótico de uso controlado. Em relação as espécies bacterianas, a Escherichia coli apresentou resistência a nove dos dez antibióticos testados e, das 24 cepas utilizadas, oito apresentaram resistência a pelo menos três antibióticos, sendo classificadas como multirresistentes. Assim, os resultados apresentados reforçam a necessidade de vigilância no tocante as cepas multirresistentes, pois além de trazer prejuízos a saúde animal também podem vir a se tornar problema de saúde pública, dado o potencial zoonótico que algumas cepas possuem.
Palavras-chave:
antibióticos; enterobactérias;
Escherichia coli
; zoonose; saúde pública
1. Introduction
Antimicrobial resistance (AMR) in bacteria is one of the main global public health concerns, affecting both humans and animals (WHO, 2023). This phenomenon is particularly alarming in veterinary medicine, where the indiscriminate use of antimicrobials in ornamental birds can lead to the development of multidrug-resistant strains (O'Neill, 2016). In the Brazilian context, this issue is even more relevant, as the country has approximately 41 million songbirds and ornamental birds, making it the third largest in pet population, reflecting the growth of this practice (ABINPET, 2022). The increasing number of companion birds, combined with the improper use of antimicrobials, may favor the emergence of antibiotic-resistant microorganisms, representing a significant concern for both animal and human health (Van et al., 2019).
Among the bacteria most frequently isolated from captive birds are those belonging to the order Enterobacterales, such as Escherichia coli, Klebsiella spp., Enterobacter spp., and Salmonella spp., which pose a potential risk due to their role in zoonotic infections (Adeolu et al., 2016; Martinez-Rodriguez et al., 2023). The presence of multidrug-resistant strains of these microorganisms in captive birds is worrisome, given their relevance to public health (Kozdruń et al., 2015). Transmission can occur through direct contact with birds or indirectly via contaminated surfaces, water, and soil in bird habitats (Van et al., 2019).
Although previous studies have primarily focused on commercial birds (Rabello et al., 2020; Nhung et al., 2019), research on ornamental passerines remains limited, particularly in the Northeast region of Brazil, and more specifically in the state of Ceará. There is a notable lack of data on the antimicrobial resistance profiles of bacterial isolates from exotic passerines kept in captivity (Gaio et al., 2019). In this context, the objective of this study was to perform antimicrobial susceptibility testing and evaluate the degree of resistance observed in bacterial strains of the order Enterobacterales isolated from exotic passerines housed in farms located in the metropolitan region of Fortaleza, Brazil.
2. Material and Methods
A total of 104 strains from the Enterobacteriaceae family were used in this study, with the following bacterial species: Pantoea agglomerans (35), Escherichia coli (24), Hafnia alvei (11), Klebsiella pneumoniae (10), Serratia rubidaea (8), Enterobacter gergoviae (6), Cronobacter sakazakii (4), Serratia marcescens (4), Enterobacter cloacae (1), and Serratia liquefaciens (1). The strains were isolated by Nogueira (2020) from 120 exotic passerines of three species: Gouldian Finch (Erythrura gouldiae), Zebra Finch (Taeniopygia guttata), and Munia (Lonchura domestica), which belonged to breeding facilities located in the metropolitan region of Fortaleza, Brazil. Collection took place between August and October 2019, with a single cloacal swab collected individually from each bird. After isolation and identification, the strains were stored on nutrient agar and added to the bacterial collection of the Laboratory of Ornithological Studies at the Faculty of Veterinary Medicine of the State University of Ceará. For the antimicrobial sensitivity test, the strains were reactivated in BHI broth, plated on MacConkey agar, and incubated for 24 hours at 37 °C.
The strains were subjected, following the recommendations of the Clinical and Laboratory Standards Institute (CLSI, 2017), to the Kirby-Bauer disk diffusion technique. Müller-Hinton agar was used as the culture medium, samples were seeded across the plate surface, and antibiotic disks were then distributed. The diameter of the inhibition zones was measured after 24 hours of incubation at 37 °C. Ten antibiotics from eight pharmacological classes were evaluated: aminoglycosides (gentamicin 10 µg and tobramycin 10 µg), fluoroquinolones (ciprofloxacin 5 µg and nalidixic acid 30 µg), cephalosporins (ceftriaxone 30 µg), penicillins (ampicillin 10 µg), penicillins associated with beta-lactamase inhibitors (amoxicillin with clavulanic acid 30 µg), tetracyclines (tetracycline 30 µg), folate pathway inhibitors (sulfamethoxazole 23.75 µg - trimethoprim 1.25 µg), and carbapenems (meropenem 10 µg). Strains resistant to at least three classes of antibiotics were considered multidrug-resistant (Magiorakos et al., 2012)
3. Results
Among the tested antimicrobials, ampicillin (89.4%) was the antibiotic to which the strains most commonly showed resistance, followed by sulfamethoxazole (48.5%), nalidixic acid (41.7%), and amoxicillin with clavulanic acid (33.2%). Regarding bacterial species, Pantoea agglomerans showed resistance to all ten antibiotics, notably ampicillin (91.4%) and sulfamethoxazole (62.86%). Escherichia coli exhibited resistance to nine out of the ten antibiotics tested, with ampicillin (79.16%) being the antibiotic to which the strains showed the highest resistance (Table 1). Out of the 104 strains used in the study, only 15 (14.42%) showed no resistance to any class of antimicrobials, while 53 (50.96%) were resistant to at least 3 classes of antibiotics, characterizing them as multidrug-resistant. In this context, eight (5.8%) strains stood out as they showed resistance to six classes of antibiotics e apenas uma (0,9%) foi resistente a todos as classes testadas. Out of the 24 (23.04%) Escherichia coli strains, thirteen (54.2%) exhibited multidrug resistance and only three strains did not show resistance to any of the antibiotics. The highest number of cases of multidrug resistance for this bacterial species was presented in only one sample (4.1%), involving new classes of antibiotics (Table 2).
Frequency of enterobacteria resistant to antibiotics tested from samples isolated from exotic passerines from breeding sites in the Metropolitan Region of Fortaleza – CE, Brazil.
Multidrug-resistant Enterobacteriaceae isolated from exotic passerines breeding sites in the Metropolitan Region of Fortaleza – CE. Brazil.
4. Discussion
As observed in this study, ampicillin has also been reported as one of the antibiotics with the highest resistance rates in microorganisms of the order Enterobacterales isolated from passerines. Horn et al. (2015) analyzed strains of these microorganisms obtained from cloacal swabs of canaries (Serinus canaria) and found that ampicillin showed the second-highest resistance rate (54.1%) among the various antibiotics tested. Furthermore, the authors highlighted the importance of this antimicrobial in therapeutic protocols for treating birds and the concern regarding the resistance that bacterial strains can develop.
Among the bacterial species investigated in this study, a high resistance rate to ampicillin (79.1%) was found in Escherichia coli strains, corroborating the findings of Braconaro et al. (2015), who observed ampicillin resistance in 100% of E. coli strains isolated from trafficked passerines. Similarly, Gaio et al. (2019) reported a 98% resistance rate to ampicillin in E. coli strains isolated from native wild passerines received by the Wild Animal Screening Center (CETAS). Horn et al. (2015), in their study with strains isolated from Belgian canaries, reported similar results regarding resistance to sulfamethoxazole (in combination with trimethoprim), with a resistance rate of 44%, compared to the 47.12% rate found in the present study.
The high resistance rates observed in this study may be influenced by several factors, including the environment to which these birds are exposed. Although this study did not directly analyze environmental factors, previous research, such as those by Machado et al. (2016) and Gaio et al. (2019), suggests that antimicrobial resistance is generally more prevalent in captive birds than in free-living birds. This may be related to increased contact between individuals, facilitating the spread of resistant strains, as well as the selective pressure caused by the indiscriminate use of antimicrobials in captive environments. Additionally, the study by Iroha et al. (2015) emphasizes that improper management and excessive use of antimicrobials in captive environments significantly contribute to the emergence of resistant strains.
Regarding meropenem, a critical antimicrobial often used as a last resort in human medicine, a resistance rate of 25% was found. This finding is concerning, as previous studies have reported low or no resistance to this antimicrobial in bacteria isolated from wild or captive birds (Iroha et al., 2015; Sousa et al., 2019; Ong et al., 2020; Foti et al., 2020). The resistance observed in this study may reflect the emergence of carbapenem-resistant strains, which is a growing global concern. However, it is important to note that Antimicrobial Sensitivity Testing (AST) can occasionally produce false-positive results, particularly for carbapenems such as meropenem (Ong et al., 2020; CLSI, 2020).
Regarding multidrug resistance, high rates were observed, exceeding those reported in several other studies involving passerines. Beleza et al. (2019), using a methodology similar to that employed in this study, exclusively analyzed Belgian canaries (Serinus canaria) and identified a multidrug resistance rate of 37.7% for total bacteria. These authors emphasized the need for greater control over the use of antimicrobials to prevent future therapeutic complications in bird management in Brazil. On the other hand, studies with cloacal swab samples from passerines originating from wildlife trafficking found lower multidrug resistance rates (15.6%) for bacteria of the order Enterobacterales (Teixeira et al., 2024). This result may be attributed to the fact that many birds are newly captured from the wild and, therefore, have not been exposed to antimicrobial treatments, as is more commonly the case with birds from domestic breeders.
5. Conclusion
In this study, antimicrobial resistance to various classes of antibiotics was observed, with ampicillin, sulfamethoxazole, nalidixic acid, amoxicillin with clavulanic acid, and meropenem standing out. Furthermore, the issue of multidrug resistance exhibited by almost half of the strains used in the study reinforces the concern and caution that should be taken when establishing therapeutic protocols using antimicrobials, highlighting the challenge of treating infections in these birds. Therefore, the importance of constant surveillance regarding the issue of antimicrobial resistance in exotic passerine breeding is evident, as these strains can not only cause sanitary losses to the flock but also have zoonotic potential, thus directly impacting public health.
Acknowledgements
We would to thank Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) most sincerely for promoting this research. Furthermore, we would also like to thank the collaboration of exotic passerine breeders in the city of Fortaleza for giving us this opportunity.
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