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Use of MALDI-TOF for identification and surveillance of gram-negative bacteria in captive wild psittacines

Uso do MALDI-TOF para identificação e monitoramento de bactérias Gram-negativas em psitacídeos cativos

Y. M. Davies L. S. Franco F. B. Barbosa C. L. Vanin V. T. M. Gomes L. Z. Moreno M. R. F. Barbosa M. I. Z. Sato A. M. Moreno T. Knöbl About the authors

Abstract

Microbiological studies of the sanitary and health status of psittacine birds that will be reintroduced is important in evaluating whether these animals act as carriers of pathogenic agents to other animals and humans. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a faster and more accurate method to identify bacteria than conventional microbiology methods. The aim of this study was to evaluate the health status of psittacines housed in captivity, by assessment of Gram-negative bacteria from fecal microbiota through MALDI- TOF MS identification. The results indicate high frequency of Gram-negative bacteria in feces (96.5%), especially from the Enterobacteriaceae family (88.7%). The most prevalent bacteria were Escherichia coli (39.0%), Proteus vulgaris (12.2%), Klebsiella spp. (12.1%) and Raoultella ornithinolytica (8.7%). Proteus hauseri, Citrobacter spp., Morganella morgannii, Providencia rettgeri, Enterobacter spp. and Escherichia hermannii were isolated with lower frequency. . All these agents are potentially pathogenic for parrots and can cause systemic infections in other animals and humans. These findings reinforce that MALDI- TOF MS proved to be a rapid and accurate method of identification of the microorganism and evaluation of the health status of psittacines, providing relevant data to assist decision-making regarding the sanitary protocols in wildlife centers, and possible future reintroduction of wild birds.

Keywords:
parrots; psittacine birds; MALDI -TOF MS; enterobacteria; public health

Resumo

Estudos microbiológicos da sanidade de psitacídeos que serão reintroduzidos são importantes para avaliar se esses animais atuam como portadores de agentes patogênicos para outros animais e humanos. A espectrometria de massa por ionização/dessorção de matriz assistida por laser/tempo de vôo (MALDI-TOF MS) é um método mais rápido e preciso para identificar bactérias na comparação com métodos convencionais de microbiologia. O objetivo deste estudo foi avaliar o estado de saúde de psitacídeos cativos, identificando bactérias Gram-negativas da microbiota fecal por MALDI -TOF MS. Os resultados indicaram alta frequência de bactérias Gram-negativas nas fezes (96,5%), principalmente da família Enterobacteriaceae (88,7%). As mais prevalentes foram Escherichia coli (39,0%), Proteus vulgaris (12,2%), Klebsiella spp. (12,1%) e Raoultella ornithinolytica (8,7%). Proteus hauseri, Citrobacter spp., Morganella morgannii, Providencia rettgeri, Enterobacter spp. e Escherichia hermannii foram isolados com menor frequência. Todos esses agentes são potencialmente patogênicos para os papagaios e podem causar infecções sistêmicas em outros animais e seres humanos. Esses achados reforçam que o MALDI- TOF MS é um método rápido e preciso de identificação do microrganismo e avaliação do estado de saúde dos psitacídeos, fornecendo dados relevantes para auxiliar na tomada de decisões sobre os protocolos sanitários em centros de triagem de animais selvagens e sobre a possibilidade de reintrodução futura.

Palavras-chave:
aves; psitacídeos; MALDI- TOF MS; enterobactérias; saúde pública

1. Introduction

Brazil presents the greatest diversity of parrots in the world, but 25 of the 87 species recognized in Brazilian territories are critically endangered, under threat of extinction or vulnerable (Birdlife International, 2017BIRDLIFE INTERNATIONAL, 2017 [viewed 10 December 2019]. State of the world’s bids: taking the pulse of the planet [online]. Availabe from: https://www.birdlife.org/sowb2018
https://www.birdlife.org/sowb2018...
). The ex situ maintenance and breeding of birds recovered from wildlife trade, followed by rehabilitation and release, represent an alternative for preservation of some species. However, when breeding in captivity, the intestinal and respiratory microbiota of birds may be modified, especially under poor sanitary management (Mattes et al., 2005MATTES, B.R., CONSIGLIO, S.A.S., ALMEIDA, B.Z., GUIDO, M.C., ORSI, R.B., SILVA, R.M., COSTA, A., FERREIRA, A.J.P. and KNÖBL, T., 2005. Influência da biossegurança na colonização intestinal por Escherichia coli em psitacídeos. Arquivos do Instituto Biológico, vol. 72, pp. 13-16.).

The gastrointestinal microbiome of psittacine free-living birds has been poorly understood, but the most of reports shows that the normal psittacine bird microbiota is composed by Gram-positive facultative bacteria, that is, aerobic and anaerobic, and producers of lactic acid (Gerlach, 1994GERLACH, H., 1994. Bacteria. In: B.W. RITCHIE, G.J. HARRISON and L.R. HARRISON, eds. Avian Medicine: principles and application. Lake Worth, Fla: Wingers Pub, pp. 1384.; Xenoulis et al., 2010XENOULIS, P.G., GRAY, P.L., BRIGHTSMITH, D., PALCULICT, B., HOPPES, S., STEINER, J.M., TIZARD, I. and SUCHODOLSKI, J.S., 2010. Molecular characterization of the cloacal microbiota of wild and captive parrots. Veterinary Microbiology, vol. 146, no. 3-4, pp. 320-325. http://dx.doi.org/10.1016/j.vetmic.2010.05.024. PMid:20646876.
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; Allegretti et al., 2014ALLEGRETTI, L., REVOLLEDO, L., ASTOLFI-FERREIRA, C.S., CHACÓN, J.L., MARTINS, L.M., SEIXAS, G.H.F. and FERREIRA, A.J.P., 2014. Isolation and molecular identification of lactic acid bacteria and Bifidobacterium spp. from faeces of the blue-fronted Amazon parrot in Brazil. Beneficial Microbes, vol. 5, no. 4, pp. 497-503. http://dx.doi.org/10.3920/BM2013.0082. PMid:25062609.
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; Saidenberg et al, 2015SAIDENBERG, A.B.S., GILARDI, J.D., MUNN, C.A. and KNÖBL, T., 2015. Evaluation of fecal gram stains and prevalence of endoparasites in free-living Macaws. Atas de Saúde Ambiental, vol. 3, pp. 3-11.). However, captive birds usually have a greater diversity of intestinal microbial community, including Gram negative bacteria (Hidasi et al., 2013HIDASI, H.W., HIDASI-NETO, J., MORAES, D.M.C., LINHARES, G.F.C., JAYME, V.S. and ANDRADE, M.A., 2013. Enterobacterial detection and Escherichia coli antimicrobial resistance in parrots seized from the illegal wildlife trade. Journal of Zoo and Wildlife Medicine, vol. 44, no. 1, pp. 1-7. http://dx.doi.org/10.1638/1042-7260-44.1.1. PMid:23505696.
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; Sanches et al., 2017SANCHES, L.A., GOMES, M., TEIXEIRA, R.H.F., CUNHA, M.P.V., OLIVEIRA, M.G.X., VIEIRA, M.A.M., GOMES, T.A.T. and KNOBL, T., 2017. Captive wild birds as reservoirs of enteropathogenic E. coli (EPEC) and Shiga-toxin producing E. coli (STEC). Brazilian Journal of Microbiology, vol. 48, no. 4, pp. 760-763. http://dx.doi.org/10.1016/j.bjm.2017.03.003. PMid:28619663.
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), probably due the influence of anthropogenic action, modified diet and use of antimicrobials (Mattes et al., 2005MATTES, B.R., CONSIGLIO, S.A.S., ALMEIDA, B.Z., GUIDO, M.C., ORSI, R.B., SILVA, R.M., COSTA, A., FERREIRA, A.J.P. and KNÖBL, T., 2005. Influência da biossegurança na colonização intestinal por Escherichia coli em psitacídeos. Arquivos do Instituto Biológico, vol. 72, pp. 13-16.; Davies et al., 2016aDAVIES, Y.M., CUNHA, M.P., OLIVEIRA, M.G., OLIVEIRA, M.C., PHILADELPHO, N., ROMERO, D.C., MILANELO, L., GUIMARÃES, M.B., FERREIRA, A.J.P., MORENO, A.M., SÁ, L.R. and KNÖBL, T., 2016a. Virulence and antimicrobial resistance of Klebsiella pneumoniae isolated from passerine and psittacine birds. Avian Pathology, vol. 45, no. 2, pp. 194-201.; Clavijo and Flórez, 2018CLAVIJO, V. and FLÓREZ, M.J.V., 2018. The gastrointestinal microbiome and its association with the control of pathogens in broiler chicken production: a review. Poultry Science, vol. 97, no. 3, pp. 1006-1021. http://dx.doi.org/10.3382/ps/pex359. PMid:29253263.
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; Pereira et al., 2019PEREIRA, R., BORTOLUZZI, C., DURRER, A., FAGUNDES, N.S., PEDROSO, A.A., RAFAEL, J.M., PERIM, J.E.L., ZAVARIZE, K.C., NAPTY, G.S., ANDREOTE, F.D., COSTA, D.P. and MENTEN, J.F.M., 2019. Performance and intestinal microbiota of chickens receiving probiotic in the feed and submitted to antibiotic therapy. Journal of Animal Physiology and Animal Nutrition, vol. 103, no. 1, pp. 72-86. http://dx.doi.org/10.1111/jpn.13004. PMid:30485573.
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).

The high prevalence of Gram-negative opportunistic bacteria, such as Enterobacter spp., Escherichia coli, Klebsiella spp., Citrobacter spp. and Aeromonas spp., increase the health hazard, because these microorganisms can become pathogenic in stressful and adverse situations (Rupley, 1999RUPLEY, A.E., 1999. Manual de clínica aviária. São Paulo: Ed. Roca. 582 p.; Sandmeier and Coutteel, 2006SANDMEIER, P. and COUTTEEL, P., 2006. Management of canaries, finches and mynahs. In: G.J. HARRISON and LIGHTFOOT, T. L., eds. Clinical avian medicine. Florida: Editor Spix Publishing, vol. 2, p.896-897.; Davies et al., 2016aDAVIES, Y.M., CUNHA, M.P., OLIVEIRA, M.G., OLIVEIRA, M.C., PHILADELPHO, N., ROMERO, D.C., MILANELO, L., GUIMARÃES, M.B., FERREIRA, A.J.P., MORENO, A.M., SÁ, L.R. and KNÖBL, T., 2016a. Virulence and antimicrobial resistance of Klebsiella pneumoniae isolated from passerine and psittacine birds. Avian Pathology, vol. 45, no. 2, pp. 194-201.). Therefore, a microbiological study of the sanitary and health status of animals that will be reintroduced is important in evaluating whether these animals act as carriers of pathogenic agents to other animals and humans (Braconaro et al., 2015BRACONARO, P., SAIDENBERG, A.B.S., BENITES, N.R., ZUNIGA, E., DA SILVA, A.M., SANCHES, T.C., ZWARG, T., BRANDÃO, P.E. and MELVILLE, P.A., 2015. Detection of bacteria and fungi and assessment of the molecular aspects and resistance of Escherichia coli isolated from confiscated passerines intended for reintroduction programs. Microbial Pathogenesis, vol. 88, pp. 65-72. http://dx.doi.org/10.1016/j.micpath.2015.08.006. PMid:26279195.
http://dx.doi.org/10.1016/j.micpath.2015...
; Davies et al., 2016aDAVIES, Y.M., CUNHA, M.P., OLIVEIRA, M.G., OLIVEIRA, M.C., PHILADELPHO, N., ROMERO, D.C., MILANELO, L., GUIMARÃES, M.B., FERREIRA, A.J.P., MORENO, A.M., SÁ, L.R. and KNÖBL, T., 2016a. Virulence and antimicrobial resistance of Klebsiella pneumoniae isolated from passerine and psittacine birds. Avian Pathology, vol. 45, no. 2, pp. 194-201.; Gioia Di-Chiacchio et al., 2018GIOIA DI-CHIACCHIO, R.M., CUNHA, M.P.V., DE SÁ, L.R.M., DAVIES, Y.M., PEREIRA, C.B.P., MARTINS, F.H., MUNHOZ, D.D., ABE, C.M., FRANZOLIN, M.R., DOS SANTOS, L.F., GUTH, B.E.C., ELIAS, W.P., PIAZZA, R.M.F. and KNÖBL, T., 2018. Novel Hybrid of typical enteropathogenic Escherichia coli and Shiga-Toxin-Producing E. coli (tEPEC/STEC) emerging from pet birds. Frontiers in Microbiology, vol. 9, pp. 1-9. http://dx.doi.org/10.3389/fmicb.2018.02975. PMid:30574131.
http://dx.doi.org/10.3389/fmicb.2018.029...
), and can provide relevant data to assist zoo-sanitary protocols and the management of birds in wildlife centers (Dutra et al., 2016DUTRA, L.M., YOUNG, R.J., GALDINO, C.A. and VASCONCELLOS, A.S., 2016. Apprenhended saffron finches now how to survive predators? A careful look at reintroduction candidates. Behavioural Processes, vol. 125, pp. 6-12. http://dx.doi.org/10.1016/j.beproc.2016.01.007. PMid:26827615.
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).

Clinical microbiology has developed continuously and there has been a constant search for new techniques for rapid and accurate identification of pathogens (Cherkaoui et al., 2010CHERKAOUI, A., HIBBS, J., EMONET, S., TANGOMO, M., GIRARD, M., FRANCOIS, P. and SCHRENZEL, J., 2010. Comparison of two matrix-assisted laser desorption ionization-time of flight mass spectrometry methods with conventional phenotypic identification for routine identification of bacteria to the species level. Journal of Clinical Microbiology, vol. 48, no. 4, pp. 1169-1175. http://dx.doi.org/10.1128/JCM.01881-09. PMid:20164271.
http://dx.doi.org/10.1128/JCM.01881-09...
; Stępień-Pyśniak et al., 2017STĘPIEŃ-PYŚNIAK, D., HAUSCHILD, T., RÓŻAŃSKI, P. and MAREK, A., 2017. MALDI-TOF Mass Spectrometry as a useful tool for identification of Enterococcus spp. from wild birds and differentiation of closely related species. Journal of Microbiology and Biotechnology, vol. 27, no. 6, pp. 1128-1137. http://dx.doi.org/10.4014/jmb.1612.12036. PMid:28285496.
http://dx.doi.org/10.4014/jmb.1612.12036...
). In comparison with conventional biochemical or phenotypic tests, MALDI-TOF MS has been increasingly applied in veterinary microbiology diagnostics (Davies et al., 2018DAVIES, Y.M., XAVIER DE OLIVEIRA, M.G., PAULO VIEIRA CUNHA, M., SOARES FRANCO, L., PULECIO SANTOS, S.L., ZANOLLI MORENO, L., TÚLIO DE MOURA GOMES, V., ZANOLLI SATO, M.I., SCHIAVO NARDI, M., MICKE MORENO, A., BECKER SAIDENBERG, A., ROSE MARQUES DE SÁ, L. and KNÖBL, T., 2018. Edwardsiella tarda outbreak affecting fishes and aquatic birds in Brazil. The Veterinary Quarterly, vol. 38, no. 1, pp. 99-105. http://dx.doi.org/10.1080/01652176.2018.1540070. PMid:30668277.
http://dx.doi.org/10.1080/01652176.2018....
; Moreno et al., 2018MORENO, L.Z., MATAJIRA, C.E., POOR, A.P., MESQUITA, R.E., GOMES, V.T., SILVA, A.P.S., AMIGO, C.R., CHRIST, A.P., BARBOSA, M.R.F., SATO, M.I.Z. and MORENO, A.M., 2018. Identification through MALDI-TOF mass spectrometry and antimicrobial susceptibility profiling of bacterial pathogens isolated from sow urinary tract infection. The Veterinary Quarterly, vol. 38, no. 1, pp. 1-8. http://dx.doi.org/10.1080/01652176.2017.1397302. PMid:29090643.
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; Van Driessche et al., 2019VAN DRIESSCHE, L., BOKMA, J., DEPREZ, P., HAESEBROUCK, F., BOYEN, F. and PARDON, B., 2019. Rapid identification of respiratory bacterial pathogens from bronchoalveolar lavage fluid in cattle by MALDI-TOF MS. Scientific Reports, vol. 9, no. 1, pp. 18381. http://dx.doi.org/10.1038/s41598-019-54599-9. PMid:31804604.
http://dx.doi.org/10.1038/s41598-019-545...
; Ulrich et al., 2020ULRICH, S., GOTTSCHALK, C., STRAUBINGER, R.K., SCHWAIGER, K. and DÖRFELT, R., 2020. Acceleration of the identification of sepsis‐inducing bacteria in cultures of dog and cat blood. The Journal of Small Animal Practice, vol. 61, no. 1, pp. 42-45. http://dx.doi.org/10.1111/jsap.13056. PMid:31313312.
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).

The MALDI-TOF MS system is used successfully for precise identification of bacteria isolated from various types of specimens (Stępień-Pyśniak et al., 2017STĘPIEŃ-PYŚNIAK, D., HAUSCHILD, T., RÓŻAŃSKI, P. and MAREK, A., 2017. MALDI-TOF Mass Spectrometry as a useful tool for identification of Enterococcus spp. from wild birds and differentiation of closely related species. Journal of Microbiology and Biotechnology, vol. 27, no. 6, pp. 1128-1137. http://dx.doi.org/10.4014/jmb.1612.12036. PMid:28285496.
http://dx.doi.org/10.4014/jmb.1612.12036...
; Davies et al., 2016bDAVIES, Y.M., GUIMARÃES, M.B., MILANELO, L., OLIVEIRA, M.G.X., GOMES, V.T.D.M., AZEVEDO, N.P., CUNHA, M.P.V., MORENO, L.Z., ROMERO, D.C., CHRIST, A.P.G., SATO, M.I.Z., MORENO, A.M., FERREIRA, A.J.P., SÁ, L.R.M. and KNÖBL, T., 2016b. A survey on gram-negative bacteria in saffron finches (Sicalis flaveola) from illegal wildlife trade in Brazil. Brazilian Journal of Veterinary Research and Animal Science, vol. 53, no. 3, pp. 286. http://dx.doi.org/10.11606/issn.1678-4456.bjvras.2016.109042.
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; Cabral et al., 2020CABRAL, B.G., DAVIES, Y.M., MENÃO, M.C., SAIDENBERG, A., GOMES, V.T.M., MORENO, L.Z., SATO, M.I.Z., MORENO, A.M. and KNÖBL, T., 2020. Companion psittacine birds as reservoir of gentamicin and vancomycin-resistant Enterococcus spp. Pesquisa Veterinária Brasileira, vol. 40, no. 2, pp. 129-133. http://dx.doi.org/10.1590/1678-5150-pvb-6147.
http://dx.doi.org/10.1590/1678-5150-pvb-...
). Therefore, species identification would improve timeliness and reduce isolate identification costs in clinical bacteriology laboratories (Cherkaoui et al., 2010CHERKAOUI, A., HIBBS, J., EMONET, S., TANGOMO, M., GIRARD, M., FRANCOIS, P. and SCHRENZEL, J., 2010. Comparison of two matrix-assisted laser desorption ionization-time of flight mass spectrometry methods with conventional phenotypic identification for routine identification of bacteria to the species level. Journal of Clinical Microbiology, vol. 48, no. 4, pp. 1169-1175. http://dx.doi.org/10.1128/JCM.01881-09. PMid:20164271.
http://dx.doi.org/10.1128/JCM.01881-09...
).

The aim of this study was to evaluate the health status of psittacines housed in captivity in the state of São Paulo, Brazil by assessment of Gram-negative bacteria from fecal microbiota through MALDI- TOF MS identification, assist in the development of more effective sanitary maintenance protocols, and possible future reintroduction of wild birds.

2. Materials and Methods

The development of this project was approved by the Ethics Committee on Animal Use of the School of Veterinary Medicine and Animal Science of the University of São Paulo (USP-FMVZ), protocol number 5174111215 and has SISBIO license: 46561-2.

2.1. Animals

A total of 58 psittacines were evaluated, male and female (not sexed birds), of different ages, which were housed in captivity at the state of São Paulo, Brazil. The birds were fed with seeds and grains and drinking water ad libitum and were kept in individual enclosures. The birds have not received antibiotic treatment in the last six months prior to this study.

Among the animals collected in this period are blue-fronted Amazon parrot (Amazona aestiva) (n= 22), Mangrove parrot (Amazona amazonica) (n= 2), Galician parrot (Amazona xanthops) (n= 1), Blue macaw (Anodorhynchus hyacinthinus) (n= 2), Blue-and-yellow macaw (Ara ararauna) (n= 15), Scarlet macaw (Ara macao) (n= 12), and Red-and-green macaw (Ara chloropterus) (n= 4).

2.2. Microbiological evaluation

Stool samples were collected from individual birds. The material was identified, packed and kept refrigerated until referral to the Laboratory of Avian Medicine at the Department of Pathology of FMVZ-USP for microbiological culture and isolation. The individual samples were inoculated in 2 mL of BHI broth (brain heart infusion, Difco™) and incubated at 37°C for 24 h. The isolation was performed on MacConkey agar (Difco™) and incubated at 37°C for 24 h. Then, all different morphotypes were selected (one to three different bacteria species per bird) and maintained at -80oC in BHI with 30% of glycerol, for further identification by MALDI-TOF MS.

2.3. MALDI-TOF MS identification

For MALDI-TOF MS (Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) sample preparation, bacterial proteins were extracted using an ethanol/formic acid protocol (Kuhnert et al., 2012KUHNERT, P., BISGAARD, M., KORCZAK, B.M., SCHWENDENER, S., CHRISTENSEN, H. and FREY, J., 2012. Identification of animal Pasteurellacea by MALDI-TOF mass spectrometry. Journal of Microbiological Methods, vol. 89, no. 1, pp. 1-7. http://dx.doi.org/10.1016/j.mimet.2012.02.001. PMid:22343217.
http://dx.doi.org/10.1016/j.mimet.2012.0...
). The isolates were seeded on TSA (Tryptic Soy agar, Difco™) incubated at 37oC for 18 h. Then, one colony of each isolate was mixed in 300 µL of deionized water. Nine hundred microliters of absolute ethanol were added to each sample. The tubes were mixed by inversion and centrifuged for 3 min at 12,800 rpm. The supernatant was removed, and the samples are dried at room temperature. After, the pellet was resuspended in 35 µL of formic acid (70%) and then, 35 µL of acetonitrile were added. Samples were mixed by inversion and centrifuged at 12,800 g for 3 min. The supernatant (protein suspension) was removed.

Isolates were tested in duplicate. The protein suspension (1 µL) was transferred to a polished steel MALDI target plate (Bruker Daltonik) and allowed to dry at room temperature. The sample was overlaid with 1 µL of matrix (10 mg α-cyano-4-hydroxy-cinnamic acid mL−1 in 50% acetonitrile/ 2.5% trifluoroacetic acid), and mass spectra in the 2–20 kDa range were acquired using a Microflex™ mass spectrometer (Bruker Daltonik). For the MALDI-TOF MS analysis, the spectra were loaded into MALDI BioTyper™ 3.0 and compared with the manufacturer’s library, which resulted in the log (score) value. Standard Bruker interpretative criteria were applied; scores ≥ 2.0 were accepted for species assignment and scores ≥ 1.7 but ≤ 2.0 for genus identification.

3. Results

Table 1 shows the distribution of Gram-negative bacteria identified. A total of 115 colonies were selected and identified. Of these isolates, 102/115 (88.7%) were from Enterobacteriaceae family, and 13/115 (11.3%) were to the families Moraxellaceae (Acinetobacter johnsonii) (0.9%), Aeromonadaceae (Aeromonas spp.) (1.7%), Alcaligenaceae (Alcaligenes faecalis) (3.5%) and Pseudomonadaceae (Pseudomonas spp.) (1.7%). Among the Enterobacteriaceae, the most frequently isolated species was Escherichia coli (39.0%). Other genera of bacteria that were isolated less frequently were Proteus vulgaris (12.2%), Raoultella ornithinolytica (8.7%), Klebsiella oxytoca (7.8%), Klebsiella pneumoniae (4.3%), Citrobacter spp. (4.3%), Proteus hauseri (4.3%), Morganella morgannii (3.5%), Providencia rettgeri (1.7%), Enterobacter spp. (1.7%) and Escherichia hermannii (0.9%).

Table 1
Frequency of Gram-negative bacteria in fecal samples from captive parrots. São Paulo, Brazil.

All of the birds evaluated presented Gram negative bacteria in their fecal microbiota, except for two birds (2/58), an Amazona aestiva and an Ara ararauna. One to three different bacterial species per bird were isolated: 18.9% (11/58) of birds presented a single Gram-negative pathogen, while 81.1% (45/58) presented a mix culture, composed by two (31/58) and three (14/58) bacteria species. Escherichia coli stood out with the highest frequency (10/11), among single isolates. The mixed culture were further grouped in 24 distinct profiles, with predominance of Escherichia coli. The higher frequency mixed profiles were composed by Escherichia coli associated with Proteus spp. (7/56) and Escherichia coli associated with Raoultella ornithinolytica (4/56). These data were showed on Table 2.

Table 2
Profiles of Gram-negative bacteria isolated from captive parrots. São Paulo, Brazil.

4. Discussion

Routine identification of Gram-negative bacterial isolates from various sources is based on biochemical methods, and is most often confirmed by PCR using primers and conditions for different bacteria species. However, the use of species-specific PCR for identification is laborious and requires a large number of suitable species primers (Cunha et al., 2016CUNHA, M.P., GUIMARÃES, M., DAVIES, Y., MILANELO, L. and KNÖBL, T., 2016. Bactérias gram-negativas em cardeais (Paroaria coronata e Paroaria dominicana) apreendidos do tráfico de animais silvestres. Brazilian Journal of Veterinary Research and Animal Science, vol. 53, no. 1, pp. 107-111. http://dx.doi.org/10.11606/issn.1678-4456.v53i1p107-111.
http://dx.doi.org/10.11606/issn.1678-445...
; Stępień-Pyśniak et al., 2017STĘPIEŃ-PYŚNIAK, D., HAUSCHILD, T., RÓŻAŃSKI, P. and MAREK, A., 2017. MALDI-TOF Mass Spectrometry as a useful tool for identification of Enterococcus spp. from wild birds and differentiation of closely related species. Journal of Microbiology and Biotechnology, vol. 27, no. 6, pp. 1128-1137. http://dx.doi.org/10.4014/jmb.1612.12036. PMid:28285496.
http://dx.doi.org/10.4014/jmb.1612.12036...
).

This study used matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), based on the protein profile of microorganisms, which is a distinctive and unique molecular fingerprint of a species (Kwak et al., 2015KWAK, H.L., HAN, S.K., PARK, S., PARK, S.H., SHIM, J.Y., OH, M., RICKE, S.C. and KIM, H., 2015. Development of a rapid and accurate identification method for Citrobacter species isolated from pork products using a matrix-assisted laser-desorption ionization time-offlight mass spectrometry (MALDI-TOF MS). Journal of Microbiology and Biotechnology, vol. 25, no. 9, pp. 1537-1541. http://dx.doi.org/10.4014/jmb.1503.03071. PMid:26017224.
http://dx.doi.org/10.4014/jmb.1503.03071...
; Santos et al., 2015SANTOS, T., CAPELO, J.L., SANTOS, H.M., OLIVEIRA, I., MARINHO, C., GONÇALVES, A., ARAÚJO, J. E., POETA, P., and IGREJAS, G., 2015. Use of MALDI-TOF mass spectrometry fingerprinting to characterize Enterococcus spp. and Escherichia coli isolates. Journal of Proteomics, vol. 127, Part B, pp. 321-331.; Davies et al., 2016bDAVIES, Y.M., GUIMARÃES, M.B., MILANELO, L., OLIVEIRA, M.G.X., GOMES, V.T.D.M., AZEVEDO, N.P., CUNHA, M.P.V., MORENO, L.Z., ROMERO, D.C., CHRIST, A.P.G., SATO, M.I.Z., MORENO, A.M., FERREIRA, A.J.P., SÁ, L.R.M. and KNÖBL, T., 2016b. A survey on gram-negative bacteria in saffron finches (Sicalis flaveola) from illegal wildlife trade in Brazil. Brazilian Journal of Veterinary Research and Animal Science, vol. 53, no. 3, pp. 286. http://dx.doi.org/10.11606/issn.1678-4456.bjvras.2016.109042.
http://dx.doi.org/10.11606/issn.1678-445...
; Stępień-Pyśniak et al., 2017STĘPIEŃ-PYŚNIAK, D., HAUSCHILD, T., RÓŻAŃSKI, P. and MAREK, A., 2017. MALDI-TOF Mass Spectrometry as a useful tool for identification of Enterococcus spp. from wild birds and differentiation of closely related species. Journal of Microbiology and Biotechnology, vol. 27, no. 6, pp. 1128-1137. http://dx.doi.org/10.4014/jmb.1612.12036. PMid:28285496.
http://dx.doi.org/10.4014/jmb.1612.12036...
; Davies et al., 2018DAVIES, Y.M., XAVIER DE OLIVEIRA, M.G., PAULO VIEIRA CUNHA, M., SOARES FRANCO, L., PULECIO SANTOS, S.L., ZANOLLI MORENO, L., TÚLIO DE MOURA GOMES, V., ZANOLLI SATO, M.I., SCHIAVO NARDI, M., MICKE MORENO, A., BECKER SAIDENBERG, A., ROSE MARQUES DE SÁ, L. and KNÖBL, T., 2018. Edwardsiella tarda outbreak affecting fishes and aquatic birds in Brazil. The Veterinary Quarterly, vol. 38, no. 1, pp. 99-105. http://dx.doi.org/10.1080/01652176.2018.1540070. PMid:30668277.
http://dx.doi.org/10.1080/01652176.2018....
). Here, MALDI-TOF MS was used as a rapid and accurate method for identification of 115 isolates collected from fecal samples of captive psittacine birds.

In terms of cost, Cherkaoui et al. (2010)CHERKAOUI, A., HIBBS, J., EMONET, S., TANGOMO, M., GIRARD, M., FRANCOIS, P. and SCHRENZEL, J., 2010. Comparison of two matrix-assisted laser desorption ionization-time of flight mass spectrometry methods with conventional phenotypic identification for routine identification of bacteria to the species level. Journal of Clinical Microbiology, vol. 48, no. 4, pp. 1169-1175. http://dx.doi.org/10.1128/JCM.01881-09. PMid:20164271.
http://dx.doi.org/10.1128/JCM.01881-09...
reported that using MALDI-TOF MS identification, the laboratory would have saved approximately US$5 per isolate in marginal costs and reduced average turnaround time by more than an 8-h shift, with no loss in accuracy. Seng et al. (2009)SENG, P., DRANCOURT, M., GOURIET, F., LA SCOLA, B., FOURNIER, P.E., ROLAIN, J.M. and RAOULT, D., 2009. Ongoing revolution in bacteriology: routine identification of bacteria by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Clinical Infectious Diseases, vol. 49, no. 4, pp. 543-551. http://dx.doi.org/10.1086/600885. PMid:19583519.
http://dx.doi.org/10.1086/600885...
found similar results comparing phenotypic methods (EU 4.66-6.06), MALDI-TOF MS (EU 1.43) and other automated identification system (bioMerieux Vitek system) (EU 5.9–8.23), also the mean time required for MALDI-TOF mass spectrometry identification of 1 isolate was 6 min. Despite the advantages associated with lower cost per analysis and faster diagnosis, the high initial investment to acquire the equipment (more than 200 thousand dollars) can be an impediment for the most of veterinary laboratories. According to the results of this study, 96.5% of the psittacine birds presented colonization by Gram-negative bacteria (Tables 1 and 2). In asymptomatic birds, Gram-negative bacteria should be considered an indication of need for improvement of health management, thereby minimizing the risk of developing opportunistic infections (Boseret et al., 2013BOSERET, G., LOSSON, B., MAINIL, J.G., THIRY, E. and SAEGERMAN, C., 2013. Zoonoses in pet birds: review and perspectives. Veterinary Research, vol. 44, no. 36, pp. 1-17. http://dx.doi.org/10.1186/1297-9716-44-36. PMid:23687940.
http://dx.doi.org/10.1186/1297-9716-44-3...
). The main issue is not only related to the pathogenicity of certain strains, but the possibility of transmission to other animals and humans (Saidenberg et al., 2012aSAIDENBERG, A.B., GUEDES, N.M.R., SEIXAS, G.H.F., ALLAGAYER, M.C., ASSIS, E.P., SILVEIRA, L.F., MELVILLE, P.A. and BENITES, N.R., 2012a. A survey for Escherichia coli in asymptomatic free-ranging parrots. International Scholarly Research Network Veterinary Science, vol. 2012, pp. 984813. PMid:23738135., Saidenberg et al. 2012bSAIDENBERG, A.B., TEIXEIRA, R.H.F., GUEDES, N.M.R., ALLGAYER, M.C., MELVILLE, P.A. and BENITES, N.R., 2012b. Molecular detection of enteropathogenic Escherichia coli in asymptomatic captive psittacines. Pesquisa Veterinária Brasileira, vol. 32, no. 9, pp. 922-926. http://dx.doi.org/10.1590/S0100-736X2012000900017.
http://dx.doi.org/10.1590/S0100-736X2012...
; Gioia Di-Chiacchio et al., 2018GIOIA DI-CHIACCHIO, R.M., CUNHA, M.P.V., DE SÁ, L.R.M., DAVIES, Y.M., PEREIRA, C.B.P., MARTINS, F.H., MUNHOZ, D.D., ABE, C.M., FRANZOLIN, M.R., DOS SANTOS, L.F., GUTH, B.E.C., ELIAS, W.P., PIAZZA, R.M.F. and KNÖBL, T., 2018. Novel Hybrid of typical enteropathogenic Escherichia coli and Shiga-Toxin-Producing E. coli (tEPEC/STEC) emerging from pet birds. Frontiers in Microbiology, vol. 9, pp. 1-9. http://dx.doi.org/10.3389/fmicb.2018.02975. PMid:30574131.
http://dx.doi.org/10.3389/fmicb.2018.029...
).

In this study, MALDI-TOF MS revealed 13/115 (11.3%) of the isolates belonged to the families Moraxellaceae (Acinetobacter johnsonii), Aeromonadaceae (Aeromonas spp.), Alcaligenaceae (Alcaligenes faecalis) and Pseudomonadaceae (Pseudomonas spp.). Acinetobacter spp. are widely distributed in the environment (soil) and constitute one of the predominant organisms in water. It has evolved as one of the most important nosocomial pathogens in the past decade (Özvatan et al., 2016ÖZVATAN, T., AKALIN, H., SINIRTAŞ, M., OCAKOĞLU, G., YILMAZ, E., HEPER, Y., KELEBEK, N., İŞÇIMEN, R. and KAHVECI, F., 2016. Nosocomial Acinetobacter pneumonia: treatment and prognostic factors in 356 cases. Respirology (Carlton, Vic.), vol. 21, no. 2, pp. 363-369. http://dx.doi.org/10.1111/resp12698. PMid:26635315.
http://dx.doi.org/10.1111/resp12698...
). There are reports of infection by Acinetobacter spp. associated with mycobacteriosis in falcons (Muller et al., 2010MULLER, M.G., GEORGE, A.R. and WALOCHNIK, J., 2010. Acinetobacter baumammii in Localised Cutaneous Mycobacteriosis in Falcons. Veterinary Medicine International, vol. 2010, pp. 1-7. http://dx.doi.org/10.4061/2010/321797. PMid:20871867.
http://dx.doi.org/10.4061/2010/321797...
), asymptomatic saffron finch (Sicalis flaveola) seized from illegal wildlife trade (Davies et al., 2016bDAVIES, Y.M., GUIMARÃES, M.B., MILANELO, L., OLIVEIRA, M.G.X., GOMES, V.T.D.M., AZEVEDO, N.P., CUNHA, M.P.V., MORENO, L.Z., ROMERO, D.C., CHRIST, A.P.G., SATO, M.I.Z., MORENO, A.M., FERREIRA, A.J.P., SÁ, L.R.M. and KNÖBL, T., 2016b. A survey on gram-negative bacteria in saffron finches (Sicalis flaveola) from illegal wildlife trade in Brazil. Brazilian Journal of Veterinary Research and Animal Science, vol. 53, no. 3, pp. 286. http://dx.doi.org/10.11606/issn.1678-4456.bjvras.2016.109042.
http://dx.doi.org/10.11606/issn.1678-445...
), in captive grey parrot (Klotz et al., 2018KLOTZ, P., JACOBMEYER, L., STAMM, I., LEIDNER, U., PFEIFER, Y., SEMMLER, T., PRENGER-BERNINGHOFF, E. and EWERS, C., 2018. Carbapenem-resistant Acinetobacter baumannii ST294 harbouring the OXA-72 carbapenemase from a captive grey parrot. The Journal of Antimicrobial Chemotherapy, vol. 73, no. 4, pp. 1098-1100. http://dx.doi.org/10.1093/jac/dkx490. PMid:29309610.
http://dx.doi.org/10.1093/jac/dkx490...
), and in canaries infected by Macrorhabdus ornithogaster (Robino et al., 2019ROBINO, P., FERROCINO, I., ROSSI, G., DOGLIERO, A., ALESSANDRIA, V., GROSSO, L., GALOSI, L., TRAMUTA, C., COCOLIN, L. and NEBBIA, P., 2019. Changes in gut bacterial communities in canaries infected by Macrorhabdus ornithogaster. Avian Pathology, vol. 48, no. 2, pp. 111-120. http://dx.doi.org/10.1080/03079457.2018.1553294. PMid:30499334.
http://dx.doi.org/10.1080/03079457.2018....
).

Aeromonas spp. are frequently associated with aquatic environments and may lead gastroenteritis and septicemia in humans, after immunosuppression (Panigrahy et al., 1981PANIGRAHY, B., MATHEWSON, J.J., HALL, C.F. and GRUMBLES, L.C., 1981. Unsual disease conditions in pet and aviary birds. Journal of the American Veterinary Medical Association, vol. 178, no. 4, pp. 394-395. PMid:7240000.). It has been described in wild waterfowl birds that can act as carriers of pathogenic Aeromonas species in their intestines (Laviad-Shitrit et al., 2018LAVIAD-SHITRIT, S., IZHAKI, I., ARAKAWA, E. and HALPERN, M., 2018. Wild waterfowl as potential vectors of Vibrio cholerae and Aeromonas species. Tropical Medicine & International Health, vol. 23, no. 7, pp. 758-764. http://dx.doi.org/10.1111/tmi.13069. PMid:29733476.
http://dx.doi.org/10.1111/tmi.13069...
), in ostriches (Struthio camelus) (França et al., 2009FRANÇA, M., WALKER, R.L., KOKKA, R. and SHIVAPRASAD, H.L., 2009. Aeromonas Species Associated with Necrotizing Enteritis and Septicemia in an Adult Male Ostrich (Struthio camelus). Avian Diseases, vol. 53, no. 2, pp. 310-316. http://dx.doi.org/10.1637/8458-082508-Case.1. PMid:19630242.
http://dx.doi.org/10.1637/8458-082508-Ca...
), and asymptomatic saffron finches (Sicalis flaveola) seized from illegal wildlife trade (Davies et al., 2016bDAVIES, Y.M., GUIMARÃES, M.B., MILANELO, L., OLIVEIRA, M.G.X., GOMES, V.T.D.M., AZEVEDO, N.P., CUNHA, M.P.V., MORENO, L.Z., ROMERO, D.C., CHRIST, A.P.G., SATO, M.I.Z., MORENO, A.M., FERREIRA, A.J.P., SÁ, L.R.M. and KNÖBL, T., 2016b. A survey on gram-negative bacteria in saffron finches (Sicalis flaveola) from illegal wildlife trade in Brazil. Brazilian Journal of Veterinary Research and Animal Science, vol. 53, no. 3, pp. 286. http://dx.doi.org/10.11606/issn.1678-4456.bjvras.2016.109042.
http://dx.doi.org/10.11606/issn.1678-445...
).

Pseudomonas spp. is an opportunistic pathogen present in environmental, but virulent or antibiotic resistant strains may be associated with high morbidity and mortality in humans and animals (Giacopello et al., 2015GIACOPELLO, C., FOTI, M., FISICHELLA, V. and LO PICCOLO, F., 2015. Antibiotic-Resistance patterns of Gram-negative bacterial isolates from breeder canaries (Serinus canaria domestica) with clinical disease. Journal of Exotic Pet Medicine, vol. 24, no. 1, pp. 84-91. http://dx.doi.org/10.1053/j.jepm.2014.12.009.
http://dx.doi.org/10.1053/j.jepm.2014.12...
). This pathogen has also been described in cloacal swabs of asymptomatic wild birds (Brittingham et al., 1988BRITTINGHAM, M.C., TEMPLE, S.A. and DUNCAN, R.M., 1988. A survey of the prevalence of selected bacteria on wild birds. Journal of Wildlife Diseases, vol. 24, no. 2, pp. 299-307. http://dx.doi.org/10.7589/0090-3558-24.2.299. PMid:3286907.
http://dx.doi.org/10.7589/0090-3558-24.2...
) and passerine birds seized from illegal wildlife trade (Davies et al., 2016bDAVIES, Y.M., GUIMARÃES, M.B., MILANELO, L., OLIVEIRA, M.G.X., GOMES, V.T.D.M., AZEVEDO, N.P., CUNHA, M.P.V., MORENO, L.Z., ROMERO, D.C., CHRIST, A.P.G., SATO, M.I.Z., MORENO, A.M., FERREIRA, A.J.P., SÁ, L.R.M. and KNÖBL, T., 2016b. A survey on gram-negative bacteria in saffron finches (Sicalis flaveola) from illegal wildlife trade in Brazil. Brazilian Journal of Veterinary Research and Animal Science, vol. 53, no. 3, pp. 286. http://dx.doi.org/10.11606/issn.1678-4456.bjvras.2016.109042.
http://dx.doi.org/10.11606/issn.1678-445...
; Cunha et al., 2016CUNHA, M.P., GUIMARÃES, M., DAVIES, Y., MILANELO, L. and KNÖBL, T., 2016. Bactérias gram-negativas em cardeais (Paroaria coronata e Paroaria dominicana) apreendidos do tráfico de animais silvestres. Brazilian Journal of Veterinary Research and Animal Science, vol. 53, no. 1, pp. 107-111. http://dx.doi.org/10.11606/issn.1678-4456.v53i1p107-111.
http://dx.doi.org/10.11606/issn.1678-445...
).

According to Saidenberg et al. (2015)SAIDENBERG, A.B.S., GILARDI, J.D., MUNN, C.A. and KNÖBL, T., 2015. Evaluation of fecal gram stains and prevalence of endoparasites in free-living Macaws. Atas de Saúde Ambiental, vol. 3, pp. 3-11., bacteria from the Enterobacteriaceae family are not part of the intestinal microbiota of free-living psittacine birds. However, studies with isolation of E. coli from different sites (cloaca, oropharynx and feces), involving different species of birds, whether free-living or captive, symptomatic or asymptomatic, resulted in the identification of different enterobacteria present in the digestive system (Xenoulis et al., 2010XENOULIS, P.G., GRAY, P.L., BRIGHTSMITH, D., PALCULICT, B., HOPPES, S., STEINER, J.M., TIZARD, I. and SUCHODOLSKI, J.S., 2010. Molecular characterization of the cloacal microbiota of wild and captive parrots. Veterinary Microbiology, vol. 146, no. 3-4, pp. 320-325. http://dx.doi.org/10.1016/j.vetmic.2010.05.024. PMid:20646876.
http://dx.doi.org/10.1016/j.vetmic.2010....
; Davies et al., 2016bDAVIES, Y.M., GUIMARÃES, M.B., MILANELO, L., OLIVEIRA, M.G.X., GOMES, V.T.D.M., AZEVEDO, N.P., CUNHA, M.P.V., MORENO, L.Z., ROMERO, D.C., CHRIST, A.P.G., SATO, M.I.Z., MORENO, A.M., FERREIRA, A.J.P., SÁ, L.R.M. and KNÖBL, T., 2016b. A survey on gram-negative bacteria in saffron finches (Sicalis flaveola) from illegal wildlife trade in Brazil. Brazilian Journal of Veterinary Research and Animal Science, vol. 53, no. 3, pp. 286. http://dx.doi.org/10.11606/issn.1678-4456.bjvras.2016.109042.
http://dx.doi.org/10.11606/issn.1678-445...
; Cunha et al., 2016CUNHA, M.P., GUIMARÃES, M., DAVIES, Y., MILANELO, L. and KNÖBL, T., 2016. Bactérias gram-negativas em cardeais (Paroaria coronata e Paroaria dominicana) apreendidos do tráfico de animais silvestres. Brazilian Journal of Veterinary Research and Animal Science, vol. 53, no. 1, pp. 107-111. http://dx.doi.org/10.11606/issn.1678-4456.v53i1p107-111.
http://dx.doi.org/10.11606/issn.1678-445...
; Sanches et al., 2017SANCHES, L.A., GOMES, M., TEIXEIRA, R.H.F., CUNHA, M.P.V., OLIVEIRA, M.G.X., VIEIRA, M.A.M., GOMES, T.A.T. and KNOBL, T., 2017. Captive wild birds as reservoirs of enteropathogenic E. coli (EPEC) and Shiga-toxin producing E. coli (STEC). Brazilian Journal of Microbiology, vol. 48, no. 4, pp. 760-763. http://dx.doi.org/10.1016/j.bjm.2017.03.003. PMid:28619663.
http://dx.doi.org/10.1016/j.bjm.2017.03....
; Vaz et al., 2017VAZ, F.F., SERAFINI, P.P., LOCATELLI-DITTRICH, R., MEURER, R., DURIGON, E.L., DE ARAÚJO, J., THOMAZELLI, L.M., OMETTO, T., SIPINSKI, E.A.B., SEZERBAN, R.M., ABBUD, M.C. and RASO, T.F., 2017. Survey of pathogens in threatened wild red-tailed Amazon parrot (Amazona brasiliensis) nestlings in Rasa Island, Brazil. Brazilian Journal of Microbiology, vol. 48, no. 4, pp. 747-753. http://dx.doi.org/10.1016/j.bjm.2017.03.004. PMid:28629971.
http://dx.doi.org/10.1016/j.bjm.2017.03....
), suggesting that, kept in captivity with adequate sanitary management (Mattes et al., 2005MATTES, B.R., CONSIGLIO, S.A.S., ALMEIDA, B.Z., GUIDO, M.C., ORSI, R.B., SILVA, R.M., COSTA, A., FERREIRA, A.J.P. and KNÖBL, T., 2005. Influência da biossegurança na colonização intestinal por Escherichia coli em psitacídeos. Arquivos do Instituto Biológico, vol. 72, pp. 13-16.), they could act as asymptomatic carriers (Loiko et al., 2007LOIKO, M.R., ABILHEIRA, F.S., GUEDES, N.R., PASSOS, D.T., WEIMER, T.A., OLIVEIRA, S.J. and ALLYGAYER, M.C., 2007. Identificação da microbiota da orofaringe e cloaca em filhotes de arara-azul-grande (Anodorhynchus hyacinthinus) de vida livre do Pantanal-MS. Revista de Iniciação Científica da ULBRA, vol. 6, no. 6, pp. 29-35.; Sanches et al., 2017SANCHES, L.A., GOMES, M., TEIXEIRA, R.H.F., CUNHA, M.P.V., OLIVEIRA, M.G.X., VIEIRA, M.A.M., GOMES, T.A.T. and KNOBL, T., 2017. Captive wild birds as reservoirs of enteropathogenic E. coli (EPEC) and Shiga-toxin producing E. coli (STEC). Brazilian Journal of Microbiology, vol. 48, no. 4, pp. 760-763. http://dx.doi.org/10.1016/j.bjm.2017.03.003. PMid:28619663.
http://dx.doi.org/10.1016/j.bjm.2017.03....
). This group of bacteria can colonize most tissues of birds, and is often considered a secondary pathogen, that is, it will only cause infections in adverse situations, such as immunosuppression or previous treatment with the use of antibiotics (Gerlach, 1994GERLACH, H., 1994. Bacteria. In: B.W. RITCHIE, G.J. HARRISON and L.R. HARRISON, eds. Avian Medicine: principles and application. Lake Worth, Fla: Wingers Pub, pp. 1384.; Davies et al., 2016aDAVIES, Y.M., CUNHA, M.P., OLIVEIRA, M.G., OLIVEIRA, M.C., PHILADELPHO, N., ROMERO, D.C., MILANELO, L., GUIMARÃES, M.B., FERREIRA, A.J.P., MORENO, A.M., SÁ, L.R. and KNÖBL, T., 2016a. Virulence and antimicrobial resistance of Klebsiella pneumoniae isolated from passerine and psittacine birds. Avian Pathology, vol. 45, no. 2, pp. 194-201.). However, in some cases they can act as primary pathogens, since there are substantial differences in the virulence of enterobacteria and in the host's immune response (Gioia Di-Chiacchio et al., 2018GIOIA DI-CHIACCHIO, R.M., CUNHA, M.P.V., DE SÁ, L.R.M., DAVIES, Y.M., PEREIRA, C.B.P., MARTINS, F.H., MUNHOZ, D.D., ABE, C.M., FRANZOLIN, M.R., DOS SANTOS, L.F., GUTH, B.E.C., ELIAS, W.P., PIAZZA, R.M.F. and KNÖBL, T., 2018. Novel Hybrid of typical enteropathogenic Escherichia coli and Shiga-Toxin-Producing E. coli (tEPEC/STEC) emerging from pet birds. Frontiers in Microbiology, vol. 9, pp. 1-9. http://dx.doi.org/10.3389/fmicb.2018.02975. PMid:30574131.
http://dx.doi.org/10.3389/fmicb.2018.029...
).

The data obtained in this study showed a predominance of some species of enterobacteria: Escherichia coli (39.0%), Proteus vulgaris (12.2%), Raoultella ornithinolytica (8.7%), Klebsiella oxytoca (7.8%), Klebsiella pneumoniae (4.3%), Citrobacter spp. (4.3%) and Proteus hauseri (4.3%) (Table 1). The identification of other genera such as Morganella morgannii (3.5%), Providencia rettgeri (1.7%), Enterobacter spp. (1.7%) and Escherichia hermannii (0.9%) were less frequently found.

These data are consistent with the results obtained by Davies et al. (2016b)DAVIES, Y.M., GUIMARÃES, M.B., MILANELO, L., OLIVEIRA, M.G.X., GOMES, V.T.D.M., AZEVEDO, N.P., CUNHA, M.P.V., MORENO, L.Z., ROMERO, D.C., CHRIST, A.P.G., SATO, M.I.Z., MORENO, A.M., FERREIRA, A.J.P., SÁ, L.R.M. and KNÖBL, T., 2016b. A survey on gram-negative bacteria in saffron finches (Sicalis flaveola) from illegal wildlife trade in Brazil. Brazilian Journal of Veterinary Research and Animal Science, vol. 53, no. 3, pp. 286. http://dx.doi.org/10.11606/issn.1678-4456.bjvras.2016.109042.
http://dx.doi.org/10.11606/issn.1678-445...
and Cunha et al. (2016)CUNHA, M.P., GUIMARÃES, M., DAVIES, Y., MILANELO, L. and KNÖBL, T., 2016. Bactérias gram-negativas em cardeais (Paroaria coronata e Paroaria dominicana) apreendidos do tráfico de animais silvestres. Brazilian Journal of Veterinary Research and Animal Science, vol. 53, no. 1, pp. 107-111. http://dx.doi.org/10.11606/issn.1678-4456.v53i1p107-111.
http://dx.doi.org/10.11606/issn.1678-445...
, who identified Klebsiella spp., E. coli, Proteus spp., and Citrobacter spp. in stool samples from several species of clinically healthy passerines. On the other hand, in parrot studies, Davies et al. (2016a)DAVIES, Y.M., CUNHA, M.P., OLIVEIRA, M.G., OLIVEIRA, M.C., PHILADELPHO, N., ROMERO, D.C., MILANELO, L., GUIMARÃES, M.B., FERREIRA, A.J.P., MORENO, A.M., SÁ, L.R. and KNÖBL, T., 2016a. Virulence and antimicrobial resistance of Klebsiella pneumoniae isolated from passerine and psittacine birds. Avian Pathology, vol. 45, no. 2, pp. 194-201. and Vaz et al. (2017)VAZ, F.F., SERAFINI, P.P., LOCATELLI-DITTRICH, R., MEURER, R., DURIGON, E.L., DE ARAÚJO, J., THOMAZELLI, L.M., OMETTO, T., SIPINSKI, E.A.B., SEZERBAN, R.M., ABBUD, M.C. and RASO, T.F., 2017. Survey of pathogens in threatened wild red-tailed Amazon parrot (Amazona brasiliensis) nestlings in Rasa Island, Brazil. Brazilian Journal of Microbiology, vol. 48, no. 4, pp. 747-753. http://dx.doi.org/10.1016/j.bjm.2017.03.004. PMid:28629971.
http://dx.doi.org/10.1016/j.bjm.2017.03....
reported the presence of Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, Citrobacter freundii and E. coli associated with clinical manifestations.

Due to the opportunistic nature of the agent, isolation only of feces, cloaca or oropharynx is not enough to associate the presence of bacteria with the occurrence of disease. A more accurate assessment of the risk can be obtained with the aid of molecular techniques to search for genes related to bacterial virulence factors, such as the production of adhesins and toxins (Hidasi et al., 2013HIDASI, H.W., HIDASI-NETO, J., MORAES, D.M.C., LINHARES, G.F.C., JAYME, V.S. and ANDRADE, M.A., 2013. Enterobacterial detection and Escherichia coli antimicrobial resistance in parrots seized from the illegal wildlife trade. Journal of Zoo and Wildlife Medicine, vol. 44, no. 1, pp. 1-7. http://dx.doi.org/10.1638/1042-7260-44.1.1. PMid:23505696.
http://dx.doi.org/10.1638/1042-7260-44.1...
; Gioia-Di Chiacchio et al., 2018). Also, the pathogenesis of infections by enterobacteria varies depends on the presence of virulence factors (Broberg et al., 2014BROBERG, C.A., PALACIOS, M. and MILLER, V.L., 2014. Klebsiella: a long way to go towards understanding this enigmatic jet-setter. F1000prime Reports, vol. 6, no. August, pp. 64. http://dx.doi.org/10.12703/P6-64. PMid:25165563.
http://dx.doi.org/10.12703/P6-64...
), and can be aggravated due to resistance to antibiotics (El Fertas-Aissani et al., 2013EL FERTAS-AISSANI, R., MESSAI, Y., ALOUACHE, S. and BAKOUR, R., 2013. Virulence profiles and antibiotic susceptibility patterns of Klebsiella pneumoniae strains isolated from different clinical specimens. Pathologie Biologie, vol. 61, no. 5, pp. 209-216. http://dx.doi.org/10.1016/j.patbio.2012.10.004. PMid:23218835.
http://dx.doi.org/10.1016/j.patbio.2012....
).

These findings reinforce the accurate microorganism identification and evaluation of the health status of psittacines kept as companion pets, or those that will be reintroduced in the wild, providing relevant data to assist decision-making regarding the sanitary protocols and destination of birds (Dutra et al., 2016DUTRA, L.M., YOUNG, R.J., GALDINO, C.A. and VASCONCELLOS, A.S., 2016. Apprenhended saffron finches now how to survive predators? A careful look at reintroduction candidates. Behavioural Processes, vol. 125, pp. 6-12. http://dx.doi.org/10.1016/j.beproc.2016.01.007. PMid:26827615.
http://dx.doi.org/10.1016/j.beproc.2016....
). The automated identification system MALDI- TOF MS proved to be a rapid and accurate method of identification of the bacterial isolates, and will serve as an important microbiological screening tool. Supplemental extraction strategies, as well as expanded databases including other bacterial groups of clinical importance, identifiers of resistance to antimicrobials, and genotype markers, will soon enhance the utility of MALDI-TOF MS (Cherkaoui et al., 2010CHERKAOUI, A., HIBBS, J., EMONET, S., TANGOMO, M., GIRARD, M., FRANCOIS, P. and SCHRENZEL, J., 2010. Comparison of two matrix-assisted laser desorption ionization-time of flight mass spectrometry methods with conventional phenotypic identification for routine identification of bacteria to the species level. Journal of Clinical Microbiology, vol. 48, no. 4, pp. 1169-1175. http://dx.doi.org/10.1128/JCM.01881-09. PMid:20164271.
http://dx.doi.org/10.1128/JCM.01881-09...
).

5. Conclusion

Although the parrots analyzed were apparently in healthy clinical condition, the frequency of isolation of Gram-negative bacteria, especially enterobacteria, was high. All these agents are potentially pathogenic for parrots and can cause systemic infections in other animals and humans.

In summary, MALDI-TOF MS-based identification provides less expensive and faster bacterial species identification than conventional phenotypic identification methods. This is especially relevant for routine clinical microbiology, since most results can be reported earlier. Further studies would be necessary to determine the virulence and resistance of these strains and the possible risks regarding public health and biodiversity conservation.

Acknowledgements

CAPES and CNPq research grants are gratefully. This study was financed in part by The Coordenação de Aperfeiçoamento de Pessoal de Nível Superior Brasil (CAPES – Finance Code 001). T.K. is a CNPq fellow (306577/2017-8).

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Publication Dates

  • Publication in this collection
    29 Mar 2021
  • Date of issue
    2022

History

  • Received
    30 Jan 2020
  • Accepted
    08 Aug 2020
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