Enteric colonization of captive exotic psittacines by Gram negative bacteria: Sanitary risks and antimicrobial resistance profileABSTRACT
Gram-negative are considered part of the intestinal microbiota of mammals, birds and humans. These microorganisms are capable of surviving in the environment for long periods and are recognized as important extraintestinal pathogens, associated with nosocomial infections. Since the past times, psittacine have been birds that have caused great admiration in the human population and the number of specimens in homes, as unconventional pets, has increased considerably. The aim of this study was to identify Gram-negative intestinal colonization and evaluate antimicrobial susceptibility in exotic psittacines from a commercial aviary in São Paulo, Brazil. During routine health examinations of birds from an exotic psittacine commercial aviary, cloacal swabs were collected from 70 birds, from nine different species. Laboratory assays were performed to identify Gram-negative species and tests for resistance and sensitivity to antibiotics. A total of 12 distinct species of Gram-negative bacteria were identified in the group of birds examined, namely: Escherichia coli, Kluyvera ascorbata, Acinetobacter baumannii, Klebsiella pneumoniae, Enterobacter asburiae, Stenotrophomonas maltophilia, Proteus mirabilis, Raoultella planticola, Serratia marcescens, Alcaligenes faecalis, Leclercia ascorbata and Salmonella spp. Among the 12 species identified, Escherichia coli was the most prevalent, detected in 45% (9/20) of the sampled exotic psittacines, representing 12.8% of the total birds examined. The antibiogram results indicated greater bacterial sensitivity to amoxicillin, ceftiofur, enrofloxacin and gentamicin, while higher resistance was observed to tetracycline. These findings highlight the importance of continued investigation into the intestinal microbiota of captive exotic birds, particularly regarding antimicrobial resistance patterns, to support better management and treatment strategies.
Keywords:
antibiogram; exotic birds; enterobactérias; bacterial culture
RESUMO
Gram-negativos são considerados parte da microbiota intestinal de mamíferos, aves e humanos. Esses microrganismos são capazes de sobreviver no ambiente por longos períodos e são reconhecidos como importantes patógenos intraintestinais, associados a infecções nosocomiais. Desde tempos remotos, os psitacídeos têm sido aves que têm causado grande admiração na população humana, e o número de espécimes em domicílios, como animais de estimação não convencionais, tem aumentado consideravelmente. O objetivo deste estudo foi identificar a colonização intestinal por Gram-negativos e avaliar a suscetibilidade antimicrobiana em psitacídeos exóticos de um aviário comercial em São Paulo, Brasil. Durante os exames de rotina de saúde de aves de um aviário comercial de psitacídeos exóticos, foram coletados swabs cloacais de 70 aves, de nove espécies diferentes. Ensaios laboratoriais foram realizados para identificar espécies Gram-negativas e testes de resistência e sensibilidade a antibióticos. Um total de 12 espécies distintas de bactérias Gram-negativas foram identificadas no grupo de aves examinadas, a saber: Escherichia coli, Kluyvera ascorbata, Acinetobacter baumannii, Klebsiella pneumoniae, Enterobacter asburiae, Stenotrophomonas maltophilia, Proteus mirabilis, Raoultella planticola, Serratia marcescens, Alcaligenes faecalis, Leclercia ascorbata e Salmonella spp. Dentre as 12 espécies identificadas, Escherichia coli foi a mais prevalente, detectada em 45% (9/20) dos psitacídeos exóticos amostrados, representando 12,8% do total de aves examinadas. Os resultados do antibiograma indicaram maior sensibilidade bacteriana a amoxicilina, ao ceftiofur, ao enrofloxacino e à gentamicina, enquanto maior resistência foi observada à tetraciclina. Esses achados ressaltam a importância da investigação contínua da microbiota intestinal de aves exóticas mantidas sob cuidados humanos, particularmente em relação aos padrões de resistência antimicrobiana, para subsidiar melhores estratégias de manejo e tratamento.
Palavras-chave:
antibiograma; aves exóticas; enterobactérias; cultura bacteriana
INTRODUCTION
Psittaciformes is a group of birds that includes macaws, parrots, parakeets, cockatoos, cockatiels, lorises, and other species. It comprises around 350 species distributed between the Tropics of Cancer and Capricorn, with South America and Oceania harboring the greatest biodiversity of psittacine species (Grespan and Raso, 2014). These birds are popular and adored for their sociable characteristics, intelligence, ability to repeat sounds and the exuberant colors of their feathers, making them one of the most popular groups of “pet” animals (Forshaw and Knight, 2010).
In Brazil, the commercial breeding of this group of birds has grown considerably, being the state of São Paulo having the largest number of commercial aviaries, possibly because it has the highest national commercial demand for psittacines. The intestinal microbiota of psittacines is colonized mainly by Gram-positive bacteria, however, keeping wild birds under human care can modify their intestinal microbiota, favoring the presence of Gram-negative bacteria in the gastrointestinal tract (Saidenberg et al., 2012; Mattes et al., 2005). The high concentration of bacteria of this group of microorganisms can result in opportunistic infections in wild animals, in addition to the potential zoonotic risk and threat of serious nosocomial infections (Hidasi et al., 2013).
The main genera of enteric bacteria are Escherichia, Shigella, Salmonella, Klebsiella, Proteus, Morganella, Yersinia, Enterobacter, Citrobacter and Serratia. These genera are associated with opportunistic diseases, while others constitute the commensal flora of humans and animals (Marques et al., 2021). These microorganisms are widespread in soil and water and can be naturally present in animals. They develop a huge variety of resistance patterns, divided as natural or acquired resistance to antibiotics. Gene acquirement, through horizontal transfer among species and genera or by mutation, can complicate or nullify the effect of infection treatments (Losch et al., 2008; Silva et al., 2010).
The high rate of health complications associated with Gram-negative bacterial infections is associated with numerous factors, including physical-chemical properties of the strains, production of toxins, presence of capsules, adhesion factors, indiscriminate use of antibiotics, among others, transforming this group of bacteria into a global health problem (Canton and Morosini, 2011, Biagioni et al., 2023).
Infection by Gram-negative pathogens in birds can cause clinical manifestations such as prostration, enteritis, hepatitis, hepato and splenomegaly, airsacculitis, polyserositis and impairment of the cardiocirculatory and reproductive systems, as well as decreased egg-laying, unfertilized eggs and consequently low birth rates of chicks (Knöbl et al., 2017; Allegretti, 2009).
The aim of this study was to identify intestinal colonization by Gram-negative bacteria and assess antimicrobial sensitivity in a collection of exotic psittacines from a commercial aviary in the countryside of the state of São Paulo, Brazil.
MATERIAL AND METHODS
During routine examinations of exotic psittacine birds kept in a commercial aviary, cloacal swabs were collected from 70 adult animals. The birds were housed in suspended cages and kept in pairs or groups of individuals. Using physical restraint and with the aid of leather gloves and nets, nine different bird species were handled, including 23 Eastern rosellas (Platycercus eximius), 21 Rose-ring parakeets (Alexandrinus krameri), 10 Grey parrots (Psittacus erithacus), five Australian ringneck (Barnardius zonarius), three red-rumped parrots (Psephotus haematonotus), a pair of Princess parrot (Polytelis alexandrae), a pair of Plum-headed parakeets (Psittacula cyanocephala), a pair of Regent parakeets (Polytelis anthopeplus) and a pair of Australian king-parrots (Alisterus scapularis). The research was authorized by the Animal Use Ethics Committee of the São Paulo State University, under protocol number CEUA 0241/2018 of Unesp/Botucatu. The samples were collected with sterile swabs and transported under refrigeration to the Laboratory of Avian of the School of Veterinary Medicine and Animal Science (FMVZ) of the University of São Paulo (USP).
The swabs were placed in 0.1% peptone water and BHI broth (brain heart infusion - Difco, Sparks, MD, USA) and incubated at 37°C for 18 hours. In a second step, aliquots removed from the tubes in peptone water were inoculated in sodium tetrathionate broth (Difco, Sparks, MD, USA), in a proportion of 1:10, and incubated at 37°C for 24 hours. After the enrichment, the cultures were streaked onto selective media: MacConkey Agar (Difco, Sparks, MD, USA), Xylose Lysine Deoxycholate Agar (XLD - Difco, Sparks, MD, USA) and Salmonella Chromagar™ Orientation (Difco, Sparks, MD, USA) and subsequently incubated at 37°C for 24-48 hours.
The colonies were identified by Matrix Associated Laser Desorption-Ionization-Time of Flight mass spectrometry (MALDI- TOF MS). The ribosomal proteins were extracted as described by Hijazin et al. (2012) and the protein spectra were captured using the FlexControl™software and MTB_autoX method by a - Microflex™ mass spectrometer (Bruker Daltonics, Inc., Billerica, MA, USA). The identification was performed by BioTyper™ 3.0 (Bruker Daltonics, Inc., Billerica, MA, USA) with species-level identification defined by log score values ≥2.0, and genus-level identification by scores between 1.7 and <2.0.
The antimicrobial susceptibility test was carried out according to CLSI guidelines (CLSI, 2020). The strains were seeded onto Mueller-Hinton agar (Difco®, Detroit, MI) and incubated at 37ºC for 18 hours. The colonies were suspended in saline until they reached the concentration of 0.5 MacFarland standard (1.5x108 CFU/mL). A sterile swab with the suspension was inoculated onto Mueller-Hinton plates, and the antimicrobial disks (DME®, São Paulo, Br) were added: amikacin (AK-30µg), amoxicillin (AMO-10μg); ceftiofur (CTF-30μg); enrofloxacin (ENO-5μg); gentamycin (GEN-10μg); sulfametoxazol/trimetoprim (SUT-25µg) and tetracycline (TET-30 μg). The inhibition zones were measured after incubating the plate at 37ºC for 18 hours. The E. coli ATCC 25922 strain was used as the internal control and results were interpreted according to CLSI breakpoints for Enterobacteriaceae (CLSI, 2020).
RESULTS AND DISCUSSION
The research presented results for the presence of enterobacteria in exotic psittacines kept under human care. Bacteriological culture showed positive results in 28.5% (20/70) of birds, in five species of psittacines. Among Eastern rosella 47.8% (11/23) tested positive. The isolated bacteria from these bird species includes Escherichia coli, Klebsiella pneumoniae, Proteus miriabilis, Raoutella planticola, Serratia marcescens, Leclercia ascorbate and Enterobacter spp. Half of all individuals of grey parrots (50%, 5/10) presented: E. coli, Salmonella spp. and Enterobacter spp. Among rose-ring-necked parakeets, only 9.5% (2/21), were positive for Gram-negative, resulting in cultures of E. coli and Acinetobacter baumanii. Finally, one specimen of Princess parrot 50% (1/2) and one Regent parakeet 50% (1/2) presented positive cultures for E. coli and Klebsiella ascobarta and in the other bird species K. pneumoniae and Enterobacter asburiae, respectively (Table 1). In total, 12 distinct species of Gram-negative bacteria were identified in the birds examined, namely: Escherichia coli, Kluyvera ascorbata, Acinetobacter baummanii, Klebsiella pneumoniae, Enterobacter asburiae, Stenothrophomonas maltophilia, Proteus mirabilis, Raoultella planticola, Serratia marcescens, Alcaligenes faecalis, Leclercia ascorbata and Salmonella spp. (Table 2).
Scientific name, common name and number of specimens of exotic psittacines examined and numbers of birds positive for Gram-negative bacteria
Next, antimicrobial sensitivity tests were conducted for common antibiotics used in wild bird medicine, including amikacin, amoxicillin, ceftiofur, enrofloxacin, gentamicin, sulfametoxazol/trimetoprim and tetracyclines. The results of the antibiograms indicated greater bacterial susceptibility to the following antibiotics: amikacin, amoxicillin, ceftiofur, enrofloxacin, gentamicin and sulfametoxazol/trimetoprim. In contrast, the strains showed higher resistance to the tetracycline. The result of the present study indicated that the bacteria E. coli is the most common in psittacines kept under human care, supporting results reported by other authors (Marques et al., 2024, 2021; Hidasi et al., 2013; Lopes et al., 2016; Marietto et al., 2010; Biagioni et al., 2023; Maciel et al., 2019; 2012, Davies et al., 2021). Regarding the antimicrobial resistance, quinolone has been consistently more effective in eliminating bacteria, while tetracycline has lower bactericidal action, corroborating the results found in the present study (Trindade and Figueira, 2018, Biagioni et al., 2023, Machado et al., 2016; Pereira et al., 2020).
The psittacines studied had a low egg-laying rate combined with a low hatching rate, especially in the bird species with higher positivity for the bacteria studied, most likely associated with the presence of microorganisms. Another relevant aspect is that the birds did not show any apparent clinical signs. The partnership between laboratories, universities and private aviaries in the passive and active research of Gram-negative bacteria should be part of the disease prevention program, especially because of the ease of obtaining samples and the low cost in relation to the benefit of laboratory results. Hygiene and correct handling of wild birds kept under human care, as well as the quality and good origin of the water and food, are directly related to the occurrence of Gram-negative bacteria.
CONCLUSION
This study indicated a high frequency of occurrence of Gram-negative bacteria species in the digestive system of exotic psittacines. The most frequent specie was E. coli, detected in 45% (9/20) of animals with the presence of Gram-negative bacteria, corresponding to 12.8% of the total of exotic psittacines researched. Among the antibiotics tested for resistance and susceptibility, the most effective antibiotic was enrofloxacin, and the least effective was tetracycline. Further studies on the intestinal microbiota are needed to determine the prevalence of Gram-negative bacteria in psittacine birds under kept human care.
ACKNOWLEDGMENTS
To the veterinarians Natalia Todesco, Carol Sanches, Paolla Nicole, Marina Camargo and Julia Vial and Marcelo Nanini.
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