Antimicrobial resistance of Gram-negative bacteria and molecular investigation of Escherichia coli genes isolated from feces of canaries (Serinus canaria, Linnaeus 1758) in Ceará, Brazil

Resistência antimicrobiana de bactérias Gram-negativas e investigação molecular de genes de Escherichia coli isoladas de fezes de canários (Serinus canaria, Linnaeus 1758) no Ceará, Brasil

Abstract

This study aimed to characterize Gram-negative bacteria isolated from domestic canaries (Serinus canaria) at an exhibition in Ceará, Brazil, by evaluating their antimicrobial resistance profiles and the presence of molecular markers associated with diarrheagenic Escherichia coli (DEC) and avian pathogenic Escherichia coli (APEC). A total of 82 pooled fecal samples were analyzed, of which 68 (82.9%) yielded Gram-negative bacterial growth, resulting in the recovery of 88 isolates belonging to nine species. The highest resistance rates were observed for amoxicillin (84.1%) and ampicillin (81.8%), and 45.5% (40/88) of the isolates exhibited a multidrug-resistant (MDR) profile. Among the seven E. coli isolates, three (42.9%) were MDR, while four (57.1%) carried at least one of the molecular markers investigated. One isolate carried all five molecular markers investigated for APEC (iutA, hlyF, iss, iroN, and ompT), whereas another was characterized as Shiga toxin-producing E. coli (STEC) based on the detection of the stx2 gene. These findings expand current knowledge of Gram-negative bacteria in exhibition canaries and demonstrate the occurrence of antimicrobial resistance, including MDR profiles, as well as E. coli isolates with molecular profiles associated with APEC and STEC, highlighting the importance of monitoring these birds from a One Health perspective.

Keywords:
avian pathogenic; Serinus canaria; zoonosis; gram negative; Escherichia coli

Resumo

Este estudo teve como objetivo caracterizar bactérias Gram-negativas isoladas de canários domésticos (Serinus canaria) em uma exposição no Ceará, Brasil, avaliando o perfil de resistência aos antimicrobianos e a presença de marcadores moleculares associados à Escherichia coli diarreiogênica (DEC) e à Escherichia coli patogênica aviária (APEC). Foram analisadas 82 amostras fecais em pool, das quais 68 (82,9%) apresentaram crescimento de bactérias Gram-negativas, resultando na recuperação de 88 isolados pertencentes a nove espécies. As maiores frequências de resistência foram observadas para amoxicilina (84,1%) e ampicilina (81,8%), e 45,5% (40/88) dos isolados apresentaram perfil de multirresistência. Entre os sete isolados de E. coli, três (42,9%) apresentaram multirresistência e quatro (57,1%) apresentaram pelo menos um dos marcadores moleculares investigados. Um isolado apresentou os cinco marcadores moleculares investigados para APEC (iutA, hlyF, iss, iroN e ompT), enquanto outro foi caracterizado como STEC pela detecção do gene stx2. Os achados ampliam o conhecimento sobre bactérias Gram-negativas de canários de exposição e evidenciam a ocorrência de resistência aos antimicrobianos, incluindo perfis de multirresistência, e de isolados de E. coli com perfis associados a APEC e STEC, reforçando a relevância do monitoramento desses animais sob a perspectiva da Saúde Única.

Palavras-chave:
patogênica aviária; Serinus canaria; zoonose; gram-negativas; Escherichia coli

1. Introduction

Among ornamental birds, passerines are widely kept as companion animals, with the canary (Serinus canaria) being particularly valued for its aesthetic and behavioral traits, which favor close interaction with humans (Cardoso et al., 2023; Hattab et al., 2023). Such close contact, however, may pose risks to both animal and public health through the transmission of microorganisms with zoonotic potential. Pet birds have therefore received increasing attention as potential reservoirs of zoonotic and antimicrobial-resistant bacteria, raising concerns from a One Health perspective. Although some of these bacteria are part of the normal intestinal microbiota of healthy birds, others may act as opportunistic pathogens or indicate microbial imbalance and have been associated with the emergence and spread of multidrug-resistant isolates (Horn et al., 2015; Ahmed et al., 2021).

In this context, Gram-negative bacteria frequently isolated from ornamental birds include members of the order Enterobacterales, such as Pantoea agglomerans, Hafnia alvei, Klebsiella pneumoniae, Enterobacter spp., Serratia spp., and Cronobacter spp., as well as Pseudomonas aeruginosa (Horn et al., 2015; Giacopello et al., 2015; Beleza et al., 2019). These microorganisms may act as primary or opportunistic pathogens in birds and, importantly, may also serve as reservoirs of antimicrobial resistance genes or have zoonotic potential, posing a complex challenge to global health (Horn et al., 2015; Masud et al., 2024; Melo et al., 2025; Samir et al., 2025; Mazari et al., 2026).

This issue is particularly relevant given the increasing spread of antimicrobial resistance (AMR), recognized as a major global health threat affecting humans, animals, and the environment within the One Health framework (WHO, 2023). Ornamental birds, including passerines, may contribute to this scenario by harboring and disseminating multidrug-resistant (MDR) bacteria and their genetic resistance determinants to other animals and the environment (WHO, 2023; Cocoș et al., 2025). The circulation of resistant bacteria across these compartments may be facilitated by direct contact, environmental contamination, and inappropriate antimicrobial use. Therefore, monitoring AMR in avian populations is an important component of epidemiological surveillance and may support the development of strategies aimed at limiting its spread. The detection of resistant bacteria in birds also highlights the importance of antimicrobial stewardship and measures promoting the prudent use of these drugs to reduce the dissemination of resistance (Giacopello et al., 2016; Beleza et al., 2019).

Among the Gram-negative bacteria of interest in this context, Escherichia coli is a commensal species in most hosts. However, some lineages have acquired virulence factors that enable them to cause intestinal and extraintestinal infections and are classified into different pathotypes according to their pathogenic mechanisms and genetic profiles (Croxen and Finlay, 2010). Among the pathotypes of veterinary and public health importance are avian pathogenic Escherichia coli (APEC), which causes systemic infections in birds and is associated with substantial economic losses in poultry production, and diarrheagenic E. coli (DEC), which comprises pathotypes capable of causing gastroenteritis in humans and other animals (Croxen and Finlay, 2010; Kathayat et al., 2021).

Molecular identification of these pathotypes relies on the detection of specific molecular markers. For APEC, iroN, iutA, iss, ompT, and hlyF are commonly used as molecular markers and are associated with salmochelin-mediated iron acquisition, the aerobactin system, serum complement resistance, outer membrane protease activity, and hemolysin production, respectively (Johnson et al., 2008). Among DEC, the stx1 and stx2 genes are markers of Shiga toxin-producing E. coli (STEC); eaeA is associated with intimate adherence to the intestinal epithelium in enteropathogenic E. coli (EPEC); eltB and estA encode enterotoxins produced by enterotoxigenic E. coli (ETEC); ipaH is used as a marker of enteroinvasive E. coli (EIEC); and aatA and aaiC are associated with secretion systems and virulence mechanisms of enteroaggregative E. coli (EAEC) (Taniuchi et al., 2012).

Despite growing concern regarding the spread of antimicrobial-resistant Gram-negative bacteria among companion birds, information on the occurrence of these microorganisms in canaries (Serinus canaria) kept in competitive exhibition settings in Brazil remains limited (Horn et al., 2015; Beleza et al., 2019). These events bring together birds from different breeding facilities and management conditions, increasing contact among individuals and potentially contributing to the introduction, persistence, and spread of potentially pathogenic and antimicrobial-resistant bacteria. In this context, the microbiological characterization of these birds, including antimicrobial resistance profiling and the investigation of molecular markers associated with DEC and APEC, may provide further insight into the pathogenic and epidemiological potential of circulating isolates and contribute to disease surveillance, clinical management, and antimicrobial resistance monitoring in breeding facilities and exhibition settings. This study aimed to characterize Gram-negative bacteria isolated from domestic canaries (Serinus canaria) at an exhibition in Ceará, Brazil, by evaluating their antimicrobial resistance profiles and the presence of molecular markers associated with diarrheagenic Escherichia coli (DEC) and avian pathogenic Escherichia coli (APEC).

2. Material and Methods

2.1. Sampling

This study was approved by the Ethics Committee for the Use of Animals (CEUA) of the State University of Ceará (UECE) with protocol number 0515351/2016. It was designed as an exploratory and descriptive investigation, aiming to determine the bacterial profile of pooled fecal samples obtained from canaries (Serinus canaria) bred for competition. Birds belonged to several local breeders who participated in an exhibition in Fortaleza, Ceará, in 2016. The event held 500 birds that were housed individually in cages made with stainless steel, equipped with appropriate perches, measuring 32, 22, and 21 centimeters in length, width, and height, respectively. The cage trays were covered with paper sheets, which were removed daily containing feces and feed scraps. Water and feed were provided ad libitum. Any bird that died during the event was submitted to necropsy, and samples of the liver, spleen, kidneys, and intestine were collected and individually processed for bacteriological examination.

Feces from 246 cages that were exposed during the event were analyzed. Each cage provided a sheet of paper containing canary feces. Because the birds were participating in a competitive exhibition, fecal material was collected non-invasively from the cage trays to avoid direct handling of the animals. On each sheet, excess feed was removed to prevent cross-contamination in areas where only feces were present. The papers were carefully folded to protect the fecal material from contamination during transport. Subsequently, the paper sheets were placed in sterile plastic bags and immediately transported to the Laboratory of Ornithological Studies at the Faculty of Veterinary Medicine (FAVET) of the State University of Ceará (UECE) in isothermal boxes with recyclable ice for further microbiological processing. Between the collection of the paper sheets from the trays, transport, and the start of cultivation, no more than two hours elapsed. From each paper sheet, 2g of feces were collected for microbiological evaluation. The sampling unit was defined as a pooled fecal sample composed of material collected from three cages belonging to the same breeder, with 2 g of feces collected from each paper sheet. This pooling strategy was adopted because the amount of fecal material produced by a single bird was limited, allowing sufficient material to be obtained for microbiological processing. Only areas containing visible fecal material were selected for collection, while regions containing feed residues were avoided. Thus, 82 pooled fecal samples were obtained and submitted to microbiological analysis.

2.2. Isolation and identification of Gram-negative bacteria

In the initial stage, material from each pooled fecal sample was collected with the aid of a sterile spatula, placed in 10 mL of 1% peptone water and subsequently incubated at 37 °C for 24 h under aerobic conditions. Subsequently, 1 mL aliquots were withdrawn from the pre-enrichment culture in peptone water after incubation and transferred to Brain-heart Infusion (BHI) and Selenite-Cystine (SC) broths, and a 0.1 mL aliquot from the same culture was transferred to Rappaport-Vassiliadis (RP) broth. The inoculated broths were incubated at 37 °C for 24 h under aerobic conditions. After incubation, the culture from each broth was streaked onto plates containing brilliant green agar (BGA), Salmonella-Shigella agar (SS), and MacConkey agar (MC), using a sterile inoculating loop. The plates were then incubated at 37 °C for 24 h under aerobic conditions.

From the plates corresponding to each sample, one to three representative colonies with morphologies compatible with the Gram-negative bacteria investigated in the study were selected based on differences in colony morphology, including size, shape, margin, elevation, surface, sheen, consistency, color, and mucoid appearance, and inoculated in tubes containing triple-sugar-iron agar slants (TSI), which was used as an initial biochemical screening test to assess glucose, lactose, and sucrose fermentation, gas production, and hydrogen sulfide (H₂S) production. The TSI tubes were incubated at 37 °C for 24 h under aerobic conditions. After incubation, the results were interpreted and used to guide the complementary biochemical identification. Growth from the slants was used to inoculate the following media/tests: lysine iron agar (LIA), sulfide-indole-motility (SIM) medium, Simmons citrate (CIT), methyl red (MR), Voges-Proskauer (VP), ornithine decarboxylase, urea, malonate, and carbohydrate fermentation tests (lactose, sucrose, mannitol, arabinose, rhamnose, raffinose, dulcitol, adonitol, inositol, and sorbitol) (Koneman et al., 2018). The inoculated media used for the biochemical tests were incubated at 37 °C for 24 h under aerobic conditions. The TSI results were considered in conjunction with-the complementary biochemical panel for presumptive identification, according to the biochemical profiles described by Koneman et al. (2018).

2.3. Antimicrobial susceptibility and multidrug resistance of the isolates

Isolates were submitted to antimicrobial susceptibility testing with the Kirby–Bauer disk diffusion technique and inhibition zone diameters were compared to the standards established by the Clinical and Laboratory Standards Institute (CLSI, 2023). A bacterial suspension was prepared by suspending colonies from a fresh pure culture in sterile saline solution and adjusting the turbidity to the 0.5 McFarland standard. The standardized suspension was inoculated onto Mueller–Hinton agar plates (Kasvi), and antimicrobial disks (Laborclin) containing standardized concentrations were placed on the agar surface. The plates were incubated at 37 °C for 24 h, after which the inhibition zone diameters were measured. A total of 8 antibiotics from four pharmacological classes and 4 synthetic antimicrobials from two pharmacological classes were tested. The antibiotics were: aminoglycosides (neomycin, 30 μg; streptomycin, 10 μg and gentamicin, 10 μg), beta-lactams (ampicillin, 10 μg and amoxicillin, 3 μg), phenicols (chloramphenicol, 30 μg), and tetracyclines (tetracycline, 30 μg). The synthetic antimicrobials were: sulfonamides (sulfonamide, 300 μg, and sulfamethoxazole + trimethoprim, 25 μg), and quinolones (nalidixic acid, 30 μg and enrofloxacin, 5 μg). For this test, E. coli strain ATCC 25922 was used as a control. Multidrug resistance was considered when isolates were resistant to at least three classes of antimicrobials (Magiorakos et al., 2012).

2.4. Detection of molecular markers associated with diarrheagenic Escherichia coli (DEC) and avian pathogenic E. coli (APEC)

2.4.1. DNA extraction and quantification

For molecular analysis, the E. coli isolates maintained on nutrient agar were reactivated in BHI broth, incubated at 37 °C for 24 h and streaked onto MacConkey agar to confirm purity. DNA was extracted using the boiling method described by Johnson et al. (2008) and Lima et al. (2013). Two to three colonies were collected from each plate, placed in tubes containing 1 mL of 0.5% Triton X-100, vortexed for 15 s, and boiled for 20 min at 94 °C. Tubes were then centrifuged at approximately 14,087 × g for 10 min at 4 °C. The supernatant containing DNA was subsequently recovered and quantified by spectrophotometry using a NanoDrop Spectrophotometer 2000 (Thermo Scientific, Wilmington, USA).

2.4.2. PCR amplification of virulence genes associated with DEC and APEC

For the molecular characterization of DEC, DNA samples were subjected to conventional uniplex polymerase chain reaction (PCR) assays. Nine molecular markers associated with five DEC pathotypes were investigated: stx1 (348 bp) and stx2 (584 bp) for Shiga toxin-producing Escherichia coli (STEC); eltB (508 bp) and estA (147 bp) for enterotoxigenic E. coli (ETEC); eaeA (881 bp) and bfpA for enteropathogenic E. coli (EPEC); ipaH (483 bp) for enteroinvasive E. coli (EIEC); and aatA (630 bp) and aaiC (215 bp) for enteroaggregative E. coli (EAEC) (Taniuchi et al., 2012; Beleza et al., 2019). EAEC 042, EHEC O157:H7, EIEC O124, EPEC 2348/69, and ETEC H10407 were used as positive control strains for the corresponding reactions. The isolates were also subjected to conventional uniplex PCR assays targeting the five molecular markers associated with APEC (iroN, iss, hlyF, ompT, and iutA), as described by Johnson et al. (2008). Strain 212A was used as a positive control for these reactions. A no-template control containing the complete PCR mixture, with Milli-Q water replacing the template DNA, was included as a negative control in the PCR assays.

PCR assays targeting molecular markers associated with DEC and APEC were performed using the same reaction mixture and amplification conditions. Reactions were carried out in a final volume of 20 μL using GoTaq Green Master Mix® (Promega, São Paulo, Brazil), primers at a final concentration of 0.2 μM, and template DNA in a MyCycler™ Thermal Cycler (Bio-Rad, CA, USA). For both molecular marker panels, amplification consisted of an initial step at 95 °C for 15 min, followed by 40 cycles of 95 °C for 30 s, 57 °C for 30 s, and 72 °C for 1 min, with a final extension at 72 °C for 10 min. Although there is no consensus in the scientific literature regarding the molecular criteria for defining an APEC isolate, Johnson et al. (2008) reported that isolates belonging to this pathotype and recovered from lesions in birds clinically diagnosed with colibacillosis carried, on average, four of the five markers considered predictors of APEC virulence. Therefore, in this study, isolates positive for at least four of the five molecular markers investigated (iroN, iss, hlyF, ompT, and iutA) were considered to exhibit an APEC-associated molecular profile.

2.4.3. Agarose gel electrophoresis and visualization of PCR products

The amplified products obtained from PCR assays targeting molecular markers associated with DEC and APEC were separated by electrophoresis on 2% agarose gels stained with ethidium bromide, visualized, and photographed using the ChemiDoc™ XRS System (Bio-Rad, CA, USA).

3. Results

Of the 82 pooled fecal samples analyzed, 68 (82.9%) yielded bacterial growth, resulting in the recovery of 88 Gram-negative bacterial isolates. Because more than one bacterial species could be recovered from the same sample, frequencies were expressed relative to both the total number of isolates and the total number of pooled samples analyzed. Among the positive pools, 49 (72.0%) yielded a single bacterial species, whereas 19 (28.0%) yielded two or more species. Two-species co-isolations predominated among these samples, with only one pool yielding three distinct bacterial species (Table 1).

Table 1
Occurrence and co-isolation patterns of bacterial species in pooled fecal samples.

Isolation pattern	Bacterial species isolated	No. of pooled samples
No bacterial growth	—	14
	Pantoea agglomerans	24
	Hafnia alvei	14
	Escherichia coli	4
Single-species isolation	Klebsiella pneumoniae	3
	Pseudomonas aeruginosa	1
	Serratia rubidaea	2
	Cronobacter sakazakii	1
	Total single-species isolations	49
	Pantoea agglomerans + Hafnia alvei	6
	Pantoea agglomerans + Escherichia coli	2
	Pantoea agglomerans + Klebsiella pneumoniae	2
	Pantoea agglomerans + Serratia marcescens	1
Two-species isolation	Pantoea agglomerans + Pseudomonas aeruginosa	2
	Pantoea agglomerans + Cronobacter sakazakii	1
	Hafnia alvei + Klebsiella pneumoniae	1
	Hafnia alvei + Serratia rubidaea	1
	Escherichia coli + Enterobacter cloacae	1
	Pseudomonas aeruginosa + Serratia rubidaea	1
	Total two-species isolations	18
Three-species isolation	Hafnia alvei + Klebsiella pneumoniae + Pseudomonas aeruginosa	1
	Total three-species isolations	1
Total pooled samples	—	82

• Note: A total of 82 pooled fecal samples were analyzed. Of these, 14 showed no bacterial growth, 49 yielded one bacterial species, 18 yielded two species, and one yielded three species, resulting in a total of 88 bacterial isolates.

The 88 Gram-negative bacterial isolates recovered from the pooled fecal samples comprised nine bacterial species (Table 2). Pantoea agglomerans was the most frequently isolated species, accounting for 43.2% (38/88) of all isolates and occurring in 46.3% (38/82) of the pooled fecal samples. Hafnia alvei was the second most frequent species, representing 26.1% (23/88) of the isolates and occurring in 28.0% (23/82) of the pools. All other species occurred in fewer than 10% of the pooled samples, including Klebsiella pneumoniae and Escherichia coli (8.5%; 7/82 each), Pseudomonas aeruginosa (6.1%; 5/82), Serratia rubidaea (4.9%; 4/82), Cronobacter sakazakii (2.4%; 2/82), Enterobacter cloacae (1.2%; 1/82), and Serratia marcescens (1.2%; 1/82).

Table 2
Distribution of Gram-negative bacterial isolates and occurrence of bacterial species in pooled fecal samples from canaries.

Bacterial species	Bacterial isolates n (%)	Pooled fecal samples n (%)
Pantoea agglomerans	38 (43.2)	38 (46.3)
Hafnia alvei	23 (26.1)	23 (28.0)
Klebsiella pneumoniae	7 (8.0)	7 (8.5)
Escherichia coli	7 (8.0)	7 (8.5)
Pseudomonas aeruginosa	5 (5.7)	5 (6.1)
Serratia rubidaea	4 (4.5)	4 (4.9)
Cronobacter sakazakii	2 (2.3)	2 (2.4)
Enterobacter cloacae	1 (1.1)	1 (1.2)
Serratia marcescens	1 (1.1)	1 (1.2)
Total bacterial isolates	88 (100.0)	—
Positive pooled samples	—	68 (82.9)
Negative pooled samples	—	14 (17.1)
Total pooled samples analyzed	—	82 (100.0)

• Note: Percentages of bacterial isolates were calculated considering the total number of isolates (n = 88), whereas species occurrence and sample positivity were calculated considering the total number of pooled fecal samples analyzed (n = 82). The sum of species-specific frequencies may exceed the number of positive pooled samples because more than one bacterial species could be isolated from the same pooled sample.

The antimicrobial susceptibility testing results are presented in Table 3. Overall, the highest resistance rates were observed among β-lactam antibiotics, particularly amoxicillin and ampicillin, with rates of 84.1% (74/88) and 81.8% (72/88), respectively. All Hafnia alvei, Klebsiella pneumoniae, and Pseudomonas aeruginosa isolates were resistant to amoxicillin, and a high resistance rate was also found among Pantoea agglomerans isolates (78.9%). Among quinolones, resistance to nalidixic acid was detected in 43.2% (38/88) of the isolates, including all P. aeruginosa and Serratia rubidaea isolates. In contrast, resistance to enrofloxacin was uncommon and restricted to isolates of a few bacterial species.

Table 3
Antimicrobial resistance profiles and multidrug resistance among Gram-negative bacterial isolates recovered from pooled fecal samples of canaries.

Bacterial species (n)	β-lactams		Quinolones		Tetracyclines	Phenicols	Aminoglycosides			Sulfonamides		MDR
	AMO	AMP	NAL	ENR	TET	CLO	GEN	NEO	STR	SUL	SUT	n (%)
Pantoea agglomerans (38)	30	28	15	3	5	4	1	-	11	19	7	10 (26.3)
Hafnia alvei (23)	23	IR	7	1	4	5	2	2	5	12	6	12 (52.2)
Klebsiella pneumoniae (7)	7	IR	3	-	1	1	1	-	5	5	2	6 (85.7)
Escherichia coli (7)	2	2	3	-	2	-	-	-	3	2	2	3 (42.9)
Pseudomonas aeruginosa (5)	5	5	5	3	3	2	1	1	4	4	4	5 (100.0)
Serratia rubidaea (4)	3	3	4	1	2	3	-	-	-	1	3	3 (75.0)
Cronobacter sakazakii (2)	2	2	-	-	-	-	-	-	-	1	-	-
Enterobacter cloacae (1)	1	IR	-	-	-	-	-	-	-	1	-	-
Serratia marcescens (1)	1	IR	1	-	-	-	-	-		-	-	1 (100.0)
Total (n = 88)	74	72	38	8	17	15	5	3	28	45	24	40 (45.5)

• AMO = Amoxicillin; AMP = Ampicillin; NAL = Nalidixic acid; ENR = Enrofloxacin; TET = Tetracycline; CLO = Chloramphenicol; GEN = Gentamicin; NEO = Neomycin; STR = Streptomycin; SUL = Sulfonamide; SUT = Sulfamethoxazole–trimethoprim; IR = intrinsic resistance; MDR = multidrug resistance. Values represent the number of resistant isolates for each antimicrobial; MDR values are expressed as n (%). Intrinsic resistance was not considered for MDR classification.

Among the other antimicrobial classes, sulfonamide showed the highest resistance rate, with 51.1% (45/88) of the isolates classified as resistant, whereas resistance to sulfamethoxazole–trimethoprim was detected in 27.3% (24/88). Tetracycline resistance was observed in 19.3% (17/88) of the isolates, while most isolates remained susceptible to chloramphenicol, with resistance detected in only 17.0% (15/88). Among aminoglycosides, streptomycin showed the highest resistance rate (31.8%; 28/88), in contrast to the lower rates observed for gentamicin (5.7%; 5/88) and neomycin (3.4%; 3/88).

For Escherichia coli, resistance was detected to β-lactams (amoxicillin and ampicillin), quinolones (nalidixic acid), tetracycline, sulfonamides (sulfonamide and sulfamethoxazole–trimethoprim), and the aminoglycoside streptomycin. In contrast, all isolates were susceptible to the phenicol chloramphenicol, the aminoglycosides gentamicin and neomycin, and the quinolone enrofloxacin.

Overall, 40 of the 88 bacterial isolates (45.5%) were classified as multidrug-resistant (MDR), exhibiting acquired resistance to three or more antimicrobial classes (Table 3). The highest MDR frequencies were recorded in Pseudomonas aeruginosa (100.0%; 5/5), followed by Klebsiella pneumoniae (85.7%; 6/7), Serratia rubidaea (75.0%; 3/4), Hafnia alvei (52.2%; 12/23), Escherichia coli (42.9%; 3/7), and Pantoea agglomerans (26.3%; 10/38). The single Serratia marcescens isolate was also classified as MDR. Among the E. coli isolates, three (42.9%) exhibited an MDR profile; one was resistant to three antimicrobial classes and two were resistant to four. Only one isolate, identified as P. aeruginosa, was resistant to all antimicrobial classes investigated.

PCR analysis of the seven Escherichia coli isolates revealed that four (57.1%) carried at least one of the molecular markers investigated. Among the positive isolates, one carried only the stx2 gene and was therefore characterized as Shiga toxin-producing Escherichia coli (STEC). Another isolate carried all five molecular markers investigated for APEC (iss, iroN, ompT, iutA, and hlyF) and was classified as avian pathogenic E. coli (APEC). This isolate was also resistant to nalidixic acid, tetracycline, and streptomycin. Of the two remaining positive isolates, one carried iss, ompT, and hlyF, whereas the other carried only hlyF. The remaining isolates were negative for all molecular markers investigated for DEC (stx1, stx2, eltB, estA, eaeA, bfpA, ipaH, aatA, and aaiC).

During the exhibition, only one canary died. The bird showed no apparent clinical signs upon arrival but developed motor incoordination and drooping wings within a few hours, followed by death. Necropsy revealed no significant macroscopic lesions. Microbiological examination yielded Pseudomonas aeruginosa exclusively from the liver sample, whereas no bacterial growth was detected in the spleen, kidney, or intestinal samples.

4. Discussion

Enterobacteriaceae isolated from canaries have been associated with different diseases in these birds (Kimura et al., 2021; Kaur et al., 2024). However, members of this bacterial family may also be found in apparently healthy granivorous passerines, and their occurrence has been associated with close contact between birds and humans (Horn et al., 2015; Vukoja, 2017). Consistent with the findings of the present study, previous investigations have reported several Enterobacteriaceae species in fecal samples from canaries showing no apparent signs of disease (Horn et al., 2015; Beleza et al., 2019).

Pantoea agglomerans was the most frequently isolated bacterium in the present study. Its predominance may be related to the widespread occurrence of this species in plants and seeds, which constitute a major component of the canary diet (Correia et al., 2019). Although commonly associated with environmental sources, P. agglomerans may also behave as an opportunistic pathogen, particularly under conditions that compromise host defenses. Its occurrence in birds is therefore relevant not only from a clinical perspective but also in the context of antimicrobial resistance, as isolates of this species may harbor antimicrobial resistance determinants and contribute to their persistence and dissemination in avian environments (Dutkiewicz et al., 2016; Saticioglu et al., 2018).

Among the other bacteria recovered, Hafnia alvei was the second most frequently isolated species. However, reports linking this bacterium to disease in canaries remain scarce, which may suggest a commensal or opportunistic role in these birds. In contrast, the detection of Klebsiella pneumoniae is noteworthy, as this species has previously been associated with outbreaks of yolk sac and egg infections in domestic canaries (Dutkiewicz et al., 2016), supporting its potential pathogenic relevance in this host.

Pantoea agglomerans was the most frequently isolated bacterium in this study. This species is commonly found in seeds, suggesting that dietary exposure may contribute to its occurrence in birds. Correia et al. (2019) also indicated that feed containing high concentrations of P. agglomerans may represent a potential risk to avian health. Hafnia alvei was the second most frequently isolated species; however, to the best of our knowledge, reports of disease associated with this bacterium in canaries remain scarce. Klebsiella pneumoniae, which was also recovered in the present study, has previously been associated with an outbreak of yolk sac and egg infection in domestic canaries (Nakhaee et al., 2022).

In the present study, Escherichia coli was recovered from 8.5% (7/82) of the pooled fecal samples. Comparable frequencies have been reported in studies conducted in the same region, with E. coli detected in 3.6% of cloacal swab samples and 4.5% of fecal samples collected from cage floors, respectively, from apparently healthy Belgian canaries (Horn et al., 2015; Beleza et al., 2019). The frequency of E. coli isolation among passerine birds, however, varies considerably across studies. In Italy, Giacopello et al. (2015) examined 50 fecal samples collected from cages housing canaries showing signs of illness and reported E. coli in 62.0% of the samples. Differences in the health status of the birds, environmental conditions, and sampling context may therefore contribute to the variation in E. coli isolation rates reported among studies. The clinical relevance of this bacterium in canaries is further supported by reports of E. coli-associated disease outbreaks in breeding facilities in Brazil (Kimura et al., 2021).

Salmonella was not detected in any of the samples analyzed, despite its recognized importance for both avian and public health (Lopes et al., 2016; Beleza et al., 2019). Its occurrence in captive birds may be influenced by sanitary management practices, including regular cleaning of cages, feeders, and drinkers, as well as the provision of treated water and appropriate feed management (Ssemakadde et al., 2024). Although the absence of Salmonella is noteworthy, other Gram-negative bacteria were frequently recovered in the present study, shifting attention to another relevant concern: their antimicrobial resistance profiles.

Differences in antimicrobial susceptibility were observed among the bacterial species investigated. Overall, high resistance frequencies were recorded for sulfonamides and, particularly, for the β-lactams amoxicillin and ampicillin. Some of these findings may reflect intrinsic resistance mechanisms reported for certain bacterial species, including resistance to ampicillin in Hafnia alvei, Klebsiella pneumoniae, Enterobacter cloacae, and Serratia marcescens (Kaur et al., 2024). Nevertheless, acquired resistance was also frequently observed and may be favored by factors such as inappropriate antimicrobial use (Beleza et al., 2019; Kaur et al., 2024). A similar resistance pattern was reported by Beleza et al. (2019) in canaries sampled at an exhibition in Northeastern Brazil, with high resistance rates to amoxicillin (78.7%), ampicillin (75.4%), streptomycin (45.9%), and sulfonamides (42.6%).

Multidrug resistance was observed in 45.5% (40/88) of the isolates in the present study, indicating a substantial occurrence of MDR among Gram-negative bacteria recovered from canaries. This frequency was higher than the 37.7% reported by Beleza et al. (2019) in canaries sampled at an exhibition in Northeastern Brazil, although differences in the bacterial populations recovered and antimicrobial resistance profiles should be considered when comparing these findings. Giacopello et al. (2015) also reported extensive antimicrobial resistance among Gram-negative bacteria isolated from clinically affected canaries in Italy, further demonstrating the occurrence of resistant bacteria in canary breeding environments. Among the Escherichia coli isolates in the present study, 42.9% (3/7) exhibited an MDR profile, reinforcing the relevance of antimicrobial resistance surveillance in these birds.

In the molecular analysis of E. coli strains, one was positive for the stx2 gene, which characterized it as a Shiga-Toxin-producing E. coli (STEC), a strain known for its potent toxins associated with severe intestinal disease in humans (Beleza et al., 2019). The stx2 gene was also detected in E. coli isolated from three out of 50 samples of canary feces (6%) (Gholami-Ahangaran and Zia-Jahromi, 2014). Another relevant finding was the strain of avian pathogenic E. coli (APEC), which was resistant to nalidixic acid, tetracycline, and streptomycin. In Brazil, researchers investigated the presence of APEC genes in E. coli isolates from canaries with respiratory disease, confirming the circulation of multidrug-resistant APEC strains in these birds (Kimura et al., 2021). The authors found no positive sample for the tested pathotype, demonstrating that the E. coli was probably not associated with the observed respiratory disease. On the other hand, another study investigated 21 canaries with colibacillosis from breeding sites in the State of Paraná and found avian pathogenic E. coli (APEC) genes in all strains, all of which were resistant to ampicillin, tetracycline, ciprofloxacin, enrofloxacin, norfloxacin, and nalidixic acid (Kimura et al., 2021).

The relevance of avian pathogenic Escherichia coli (APEC) in canaries is related to its ability to cause localized or systemic infections in birds (Kimura et al., 2021). Nevertheless, E. coli carrying APEC-associated molecular markers may also occur in the intestinal microbiota of apparently healthy birds and be shed in feces, potentially contributing to environmental contamination and bacterial dissemination. Although APEC- and STEC-associated molecular profiles were detected in only one isolate each in the present study, their occurrence is noteworthy because of their potential implications for avian and public health. These findings reinforce the importance of preventive measures aimed at limiting bacterial dissemination among birds and reducing potential exposure of humans in close contact with these animals (Johar et al., 2021; Silva et al., 2023).

Regarding the canary that died during the exhibition, Pseudomonas aeruginosa was isolated exclusively from the liver, whereas no bacterial growth was detected in the spleen, kidneys, or intestine. The bird did not exhibit clinical signs commonly associated with pseudomonosis, such as respiratory signs or fetid diarrhea, and necropsy revealed no significant macroscopic lesions, including airsacculitis (Abd El-Ghany, 2021). Therefore, although P. aeruginosa was recovered from the liver, its role in the death of the bird could not be established based on bacterial isolation alone. The recovery of this microorganism is nevertheless noteworthy because P. aeruginosa can exhibit multidrug-resistant profiles and has been associated with disease in canaries and other avian species (Abd El-Ghany, 2021).

An outbreak of upper respiratory tract infection associated with Pseudomonas aeruginosa was reported, causing a mortality rate of 32% (6/19) in birds of the Kori Bustard (Ardeotis kori) species maintained in aviaries in the United States. The bacteria were resistant to multiple antimicrobials, including amoxicillin, amoxicillin + clavulanic acid, chloramphenicol, tetracycline, and enrofloxacin (Bailey et al., 2000). This bacterial species has often been associated with cases of resistance to many of the commonly used antimicrobials and has already been described in a study involving canaries showing multidrug resistance (Giacopello et al., 2015; Abd El-Ghany, 2021), in which the isolate was resistant to six of the tested antimicrobials (neomycin, gentamicin, clavulanic acid, amoxicillin, sulfamethoxazole+trimethoprim and tetracycline).

This study has limitations related to the sample size and the fact that the study area was restricted to a single exhibition event. However, the proposed design was intentionally exploratory, aiming to document, for the first time in Brazil, the presence of Escherichia coli isolates of the APEC and STEC pathotypes carrying the stx2 gene in exhibition canaries. Future studies with longitudinal or cross-sectional designs and broader sampling coverage will be necessary to provide a more robust population assessment and epidemiological inference in contexts similar to the present research. It is also recommended that subsequent studies include serotyping and comprehensive characterization of the identified pathotypes, in order to deepen the understanding of the genetic diversity and pathogenic potential of the isolated isolates.

Another important consideration is that bacterial identification in this study was based on phenotypic and biochemical tests traditionally used in microbiological screening. These tests provide a presumptive identification based on colony morphology, growth characteristics, and biochemical profiles, whereas definitive identification at the genus or species level may require complementary methods, such as molecular assays or sequencing (Perry, 2019; Chauhan and Jindal, 2020). This limitation may have affected the identification of some isolates, particularly Hafnia alvei, which may be phenotypically difficult to distinguish from Escherichia albertii because of similarities in their biochemical profiles. Although molecular methods were not used for this differentiation in the present study, their inclusion in future investigations would allow more accurate discrimination between species with similar phenotypic characteristics.

5. Conclusion

In conclusion, this study characterized Gram-negative bacteria isolated from exhibition canaries and demonstrated the occurrence of antimicrobial-resistant bacteria in this population. The most frequently isolated species were Pantoea agglomerans, Hafnia alvei, and Klebsiella pneumoniae, with high resistance rates observed mainly to amoxicillin and ampicillin. Molecular analysis of Escherichia coli isolates identified one isolate carrying the stx2 gene and another exhibiting the molecular profile associated with avian pathogenic E. coli (APEC). Additionally, Pseudomonas aeruginosa was isolated from the liver of a canary that died during the exhibition. These findings expand the current knowledge of Gram-negative bacteria, antimicrobial resistance, and molecular markers associated with pathogenic E. coli in exhibition canaries in Brazil.

Acknowledgements

The authors would like to thank the Laboratory of Ornithological Studies, College of Veterinary Medicine (LABEO-FAVET) of the State University of Ceará (UECE) and Laboratory of Molecular Toxicology (LTM) of the Federal University of Ceará (UFC).

Data Availability Statement

All data generated and analyzed during this study are included in this article.

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*

e-mail: [e-mail]

Editor:

Takako Matsumura Tundisi

Publication Dates

• Publication in this collection
  25 Sept 2026
• Date of issue
  2026

History

• Received
  12 Aug 2026
• Accepted
  20 Aug 2026