Characterization of multidrug-resistant avian pathogenic Escherichia coli: an outbreak in canaries

Abstract

The canary (Serinus canaria) is appreciated for its beautiful song, colors, and docile temperament and drives a lucrative business. However, diseases caused by avian pathogenic Escherichia coli (APEC) compromise the health of canaries, and the inadequate antimicrobial treatment can lead to the emergence of resistant strains. This study aimed to characterize 21 isolates of E. coli obtained from canaries infected with colibacillosis during an outbreak in northern Paraná State, Brazil. APEC and diarrheagenic E. coli (DEC) virulence genes were screened for by polymerase chain reaction (PCR). All isolates were positive for the hlyF, iss, and ompT genes, which are characteristic of APEC. The iroN gene was found in 95.2% of isolates, and none had the iutA gene. The ipaH gene, characteristic of enteroinvasive E. coli (EIEC), was found in 71.4% of isolates, all belonging to the phylogenetic group B1. High genetic similarity (>95%) was found using enterobacterial repetitive intergenic consensus PCR (ERIC-PCR). The isolates belonged to serotypes O117:H4 (71.4%) and O1:H20 (23.8%). This is the first report of a clonal colibacillosis outbreak in canaries caused by APEC. All isolates were resistant to ampicillin, nalidixic acid, ciprofloxacin, enrofloxacin, norfloxacin, and tetracycline. The high rate of multidrug resistance in our study shows the importance of avoiding the inadequate antibiotic treatment. We suggest that further studies should be conducted to contribute to the understanding of colibacillosis in canaries since the health of animals is linked to human and environmental health, as defined by the concept of One Health.

Introduction

The canary (Serinus canaria) is a songbird in the Fringillidae family, known for its docile temperament [1]. This bird can be kept as a pet and used in singing competitions. Canaries are highly valued for their beautiful song and appearance, which includes a variety of colors [2]. In addition, the popularity of this bird as a pet is increasing. In the USA, canaries are the fourth most popular pet, after dogs, cats, and fish [3]. Thus, canary breeding has become a lucrative business [2, 4].

Escherichia coli is a Gram-negative bacterium that colonizes the intestinal lumen of birds and mammals. However, pathogenic strains can cause disease in humans and other animals, including birds [5]. Colibacillosis is caused by avian pathogenic E. coli (APEC) and has the potential to compromise the health of birds. This disease can manifest in different ways, and symptoms may include prostration, conjunctivitis, airsacculitis, cellulitis, swollen head syndrome, peritonitis, salpingitis, osteomyelitis, and septicemia [5].

Inadequate antimicrobial treatment in animals can lead to the emergence of resistant strains and, consequently, therapeutic failure [6, 7]. There are currently few studies on the genetic profile and susceptibility of E. coli isolated from canaries to antimicrobials. Therefore, the objective of the present study was to characterize E. coli isolated from canaries with colibacillosis. We hope our results will contribute to a better understanding of the etiological agent causing colibacillosis and inform measures to prevent the spread of antimicrobial resistance, since, according to the concept of One Health, multidrug-resistant bacteria affect not only veterinary medicine but also the environment and human medicine.

Material and methods

Bacterial isolates

From January to February 2015, 21 samples were collected from different adult birds belonging to a canary breeder in northern Paraná State, Brazil. According to the local veterinarian, all canaries had the same clinical symptoms (facial edema), with lesions in the infraorbital sinus, and there were no other significant macroscopic lesions. The birds received therapeutic doses of antibiotics (tetracyclines, sulfonamides, enrofloxacin, and gentamicin); however, there were no clinical signs of improvement in the canaries. The birds were sacrificed to identify the causative agent of the disease, and during the necropsy, samples were collected directly from the yellowish caseous exudate using sterile cotton swabs (ABSORVE®). The samples were submitted to diagnose for aspergillosis, pasteurellosis, and staphylococcosis using conventional microbiological methods. For the diagnosis of chlamydiosis and mycoplasmosis, molecular tests such as PCR were performed according to the recommendations of the American Association of Avian Pathologists [8].

After the microbiological tests were conducted, E. coli was the microorganism isolated from the collected samples. E. coli isolates were stored in brain heart infusion (BHI) growth medium (Difco^TM) containing 20% glycerol (Sigma®) at −80°C for subsequent extraction of bacterial DNA. For this, each isolate was inoculated in 1.5 mL of nutrient broth (Difco^TM) and kept under agitation for 24 h at 37°C. The isolates were cultured on MacConkey agar (Difco^TM) plates and incubated for 24 h. Subsequently, colonies were suspended in a microtube with 200 μL of sterile water and boiled for 10 min at 100°C. Finally, samples were centrifuged at 10,000×g for 5 min, and the supernatant containing DNA was stored.

Antibiotic susceptibility testing

Antibiotic susceptibility was determined by the disk diffusion method, according to the Clinical and Laboratory Standards Institute [9, 10] guidelines. The following antibiotics were used: ciprofloxacin and enrofloxacin (5 μg); ampicillin, norfloxacin, and gentamicin (10 μg); cefazolin, nalidixic acid, cefotaxime, cefoxitin, ceftazidime, tetracycline, and chloramphenicol (30 μg); amoxicillin clavulanic acid (30 μg); and trimethoprim-sulfamethoxazole (1.25/23.75 μg) (Oxoid Ltd., Basingstoke, Hants, UK). Enrofloxacin is a drug used in clinical veterinary medicine, and the interpretation of the results was based on breakpoints for poultry, on CLSI VET08 (2018). The E. coli ATCC 25922 strain was used as a quality control.

Polymerase chain reaction

In this study, genes that encode the most prevalent virulence factors in diarrheagenic E. coli (DEC) (eae, ehxA, stx1, stx2, elt, ipaH) and APEC (hlyF, iss, ompT, iroN, iutA) were detected as previously described [11–13]. In addition, we also evaluated the papG (fimbria P), ibeA (invasion of brain endothelium), fyuA (yersiniabactin receptor), traT (increased serum survival), fimH (mannose-specific adhesin subunit of type 1 fimbriae), hlyA (hemolysin A), and cnf1 (cytotoxic necrotizing factor 1) genes, which are also virulence factor genes [14].

Analyses of the papC (fimbria P) and cnf2 (cytotoxic necrotizing factor 2) genes were also performed [15, 16]. In addition, the isolates were classified into phylogenetic groups (A, B1, B2, C, D, E, and F) based on the presence or absence of genes chuA, yjaA, arpA, and DNA fragment TspE4.C2 [17].

The PCR reactions contained 1.25 U of Taq DNA polymerase (Invitrogen) in 1X PCR buffer (Invitrogen), 0.2 mM of each dNTP, 2.5 mM of MgCl₂, and 1 μM of each primer. The following strains were used as positive and negative controls: E. coli E2348/69 (O127:H6, eae+, bfpA+, localized adherence), E. coli EDL 933 (O157:H7, eae+, stx1+, stx2+, ehxA+), EIEC EDL 1284 (ipaH+), ETEC H10407 (elt+), E. coli J96, and E. coli K12 HB101 (negative control).

PCR products were stained with GelRed® (Biotium) and subjected to electrophoresis on a 2% agarose gel. A 1 kb Plus DNA ladder (Invitrogen) was used as the molecular marker. After electrophoresis, the DNA bands were visualized on a UV transilluminator, and images were captured using an Image Capture System (LPixImageHE, Loccus Biotecnologia).

Plasmid-mediated quinolone resistance genes (PMQR)

Qualitative PCR assays were used to assess the presence of PMQR genes (qnrA, qnrB, and qnrS) [18] and also other additional PMQR determinants, qnrD, aac(6’)-Ib-cr, qepA, oqxA, and oqxB [19–21]. PCR analysis was performed as described in the previous section.

Enterobacterial repetitive intergenic consensus

The clonality of the isolates was investigated using the method previously described [22], with some modifications. DNA was extracted using a PureLink™ Genomic DNA Mini Kit (Invitrogen). Amplification was performed in a thermocycler with initial denaturation at 95°C for 7 min, followed by 35 cycles at 94°C for 1 min, 52°C for 1 min, 65°C for 8 min, and a final extension step at 65°C for 16 min. PCR analysis was performed as previously described. The captured images were analyzed with BioNumerics software version 7.6 (Applied Maths, Sint-Martens-Latem, Belgium). The dendrogram was generated based on the Dice similarity coefficient with a 2% position tolerance and the unweighted pair group method with arithmetic mean (UPGMA).

Multilocus sequence typing

Multilocus sequence typing (MLST) was performed according to Achtman’s scheme (http://enterobase.warwick.ac.uk/species/ecoli/allele_st_search). The characterization of bacterial species was based on the sequencing of fragments of internal sequences from seven housekeeping genes (adk, fumC, gyrB, icd, mdh, purA, and recA). For sequencing, the amplicons were purified with a column-based kit (Pure Link Quick PCR Purification Kit, Invitrogen, Germany). The purified product was sequenced using Sanger methodology in the Soil Biotechnology Laboratory of the Brazilian Agricultural Research Corporation (Embrapa). The sequence type (ST) allele was assigned using Achtman’s scheme (http://enterobase.warwick.ac.uk/species/ecoli/allele_st_search).

Serotyping

The E. coli isolates were serotyped using agglutination assays as per a previously described method [23]. Following the procedure, 96-well microtiter plates and rabbit serum (SERUNAM) against 188 somatic antigens (O) and 53 flagellar antigens (H) for E. coli were obtained. Briefly, serological typing of the O antigen of the E. coli isolates was performed using cultures boiled with fluent steam for 1 h. Fifty microliters of each of the 188 O antisera diluted 1:100 using an automatic dispenser (Dynatech Laboratories, Quick Spense Controller) were distributed in three 96-well U-bottom microplates. Subsequently, 50 μL of the prepared O antigen was added, and the plates were incubated at 50°C overnight. Finally, the serogroup was determined using specific O antisera. Serotyping of flagellar antigen (H) was conducted as in the case of somatic antigens. Fifty microliters of each of the 53 H antisera (1:100) were distributed in a 96-well U-bottom microplate, and 50 μL of the H antigen of each isolate was added and incubated at 50°C for 2 h. The H antigen was determined using specific H antisera.

Invasion of HeLa cells

Considering the high genetic similarity between the ERIC types, the presence of the ipaH gene and the sensitivity to the antibiotics cefazolin, gentamicin, trimethoprim-sulfamethoxazole, and amoxicillin-clavulanic acid, three isolates (C8, C65, and C67) were selected for the invasion assay in HeLa cells [24]. The strain EIEC O152 was used as a positive control, and E. coli K12 HB101 was used as a negative control. Bacterial invasion in HeLa cells was detected by the intensity of fluorescence emitted by the hydrolysis of the MUG substrate (4-methylumbelliferyl-ß-D glucuronide), which releases a fluorogenic molecule (4-methylumbelliferone). Duncan’s multiple range test was performed using R version 3.6.0. Differences were considered statistically significant when p < 0.05 (confidence level of 95%, α = 0.05).

Results

Identification of the etiologic agent of the infection

A canary breeder in northern Paraná State, Brazil, had a disease outbreak involving 21 canaries. To identify the causative agent, a differential diagnosis was established by conventional microbiological methods and PCR. The results were negative for aspergillosis, chlamydiosis, pasteurellosis, staphylococcosis, and mycoplasmosis. E. coli was detected after the analyses, and it was concluded that the canaries had colibacillosis.

Antibiotic sensitivity

All E. coli isolates were resistant to quinolones and fluoroquinolones (ciprofloxacin, enrofloxacin, norfloxacin, and nalidixic acid), ampicillin, and tetracycline (Table 1). The resistance-profile to the first-generation cephalosporin (cefazolin) was of 71.4%. Some isolates were resistant to chloramphenicol (90.5%), trimethoprim-sulfamethoxazole (76.2%), gentamicin (61.9%), and amoxicillin/clavulanic acid (4.8%) (Table 1). However, all isolates were sensitive to cefoxitin (cephamycin) and third-generation cephalosporins (cefotaxime and ceftazidime). Therefore, the isolates were not producers of extended-spectrum β-lactamase (ESBL).

Table 1.

Antimicrobial resistance and genetic profile of Escherichia coli isolates (n=21) isolated from canaries between January and February 2015

Isolates	Genetic profile	Antimicrobial resistance profile
C8	iroN/ompT/hlyF/iss/traT/fimH	AMP, NAL, CIP, NOR, ENR, TET, CHL
C11	iroN/ompT/hlyF/iss/ipaH/traT/fimH	AMP, NAL, CIP, NOR, ENR, TET, CFZ, CHL, GEN, STX
C17	iroN/ompT/hlyF/iss/ipaH/traT/fimH	AMP, NAL, CIP, NOR, ENR, TET, CFZ, CHL, GEN, STX
C18	iroN/ompT/hlyF/iss/ipaH/traT/fimH/qnrS	AMP, NAL, CIP, NOR, ENR, TET, CFZ, CHL, GEN, STX
C19	iroN/ompT/hlyF/iss/ipaH/traT/fimH/qnrS	AMP, NAL, CIP, NOR, ENR, TET, CFZ, CHL, GEN, STX
C62	iroN/ompT/hlyF/iss/traT/fimH	AMP, NAL, CIP, NOR, ENR, TET, CFZ, CHL
C63	iroN/ompT/hlyF/iss/traT/fimH	AMP, NAL, CIP, NOR, ENR, TET, CFZ
C64	iroN/ompT/hlyF/iss/traT/fimH	AMP, NAL, CIP, NOR, ENR, TET, CFZ, CHL
C65	iroN/ompT/hlyF/iss/ipaH/traT/fimH	AMP, NAL, CIP, NOR, ENR, TET, CHL, STX
C66	iroN/ompT/hlyF/iss/ipaH/traT/fimH	AMP, NAL, CIP, NOR, ENR, TET, CHL, STX
C67	iroN/ompT/hlyF/iss/ipaH/traT/fimH	AMP, NAL, CIP, NOR, ENR, TET, CFZ, CHL, STX
C68	iroN/ompT/hlyF/iss/ipaH/traT/fimH/qnrB	AMP, NAL, CIP, NOR, ENR, TET, CFZ, CHL, GEN, STX
C69	iroN/ompT/hlyF/iss/traT/fimH	AMP, NAL, CIP, NOR, ENR, TET, CFZ
C70	iroN/ompT/hlyF/iss/ipaH/traT/fimH/qnrS	AMP, NAL, CIP, NOR, ENR, TET, CHL, GEN, STX
C71	iroN/ompT/hlyF/iss/ipaH/traT/fimH	AMP, NAL, CIP, NOR, ENR, TET, CHL, GEN, STX
C72	iroN/ompT/hlyF/iss/ipaH/traT/fimH	AMP, NAL, CIP, NOR, ENR, TET, CFZ, CHL, GEN, STX
C73	iroN/ompT/hlyF/iss/ipaH/traT/fimH/qnrS	AMP, NAL, CIP, NOR, ENR, TET, CFZ, CHL, GEN, STX
C74	iroN/ompT/hlyF/iss/ipaH/traT/fimH	AMP, NAL, CIP, NOR, ENR, TET, CHL, GEN, STX
C75	ompT/hlyF/iss/ipaH/traT/fimH/qnrS/qnrB	AMP, NAL, CIP, NOR, ENR, TET, AMC, CFZ, CHL, GEN, STX
C76	iroN/ompT/hlyF/iss/ipaH/traT/fimH	AMP, NAL, CIP, NOR, ENR, TET, CFZ, CHL, GEN, STX
C77	iroN/ompT/hlyF/iss/traT/fimH	AMP, NAL, CIP, NOR, ENR, TET, CFZ, CHL, GEN, STX

Abbreviations: AMC amoxicillin-clavulanic acid, AMP ampicillin, CFZ cefazolin, CHL chloramphenicol, CIP ciprofloxacin, ENR enrofloxacin, GEN gentamicin, NAL nalidixic acid, NOR norfloxacin, SXT trimethoprim-sulfamethoxazole, TET tetracycline

Virulence factors, phylogenetic classification, and resistance factor genes

In this study, all E. coli isolates were positive for the virulence factor genes ompT, iss, hlyF, traT, and fimH, and 95% of the isolates were positive for the iroN gene (Table 1). In addition, other virulence factor genes were detected as follows: 15 of 21 isolates (71.4%) were identified as having the ipaH (Table 1), and none of the isolates had the iutA, eae, ehxA, stx1, stx2, elt, papC, papG, ibeA, fyuA, hlyA, cnf1, nor cnf2 genes. The PMQR determinants were positive for the qnrS gene in only five isolates (23.8%), and two isolates were positive for the qnrB gene (9.5%) (Table 1). None of the isolates were positive for the qepA, oqxA, oqxB, nor aac(6´)Ib genes. In addition, all isolates belonged to phylogenetic group B1 (Fig. 1).

Fig. 1.

Dendrogram obtained by ERIC-PCR of Escherichia coli (n = 21) isolated from canaries. Each strain is identified by an ID, phylogenetic group (PG), and serotype. The strains were grouped into three ERIC types (ET1, ET2, and ET3), with 95% genetic similarity between the clusters. ^a ND, not determined

ERIC-PCR, serotyping, and MLST

The genotyping profiles of E. coli according to ERIC-PCR fingerprinting are shown in Fig. 1. Our isolates were classified into three ERIC-types, and the degree of genetic similarity among isolates was above 95% (Fig. 1). The isolates belonged to two distinct serotypes (O117: H4 and O1: H20), the most prevalent being O117:H4 (71.4%), and five of them had the O1:H20 serotype (23.8%). The serotype of one isolate (4.8%) could not be determined among the surveyed serotypes. In addition, the sequence types (STs) of two isolates of different ERIC types, C8 (O1:H20) and C74 (O117:H4), were the same (ST224).

Invasion of HeLa cells

The fluorescence intensity of the bacterial invasion in HeLa cells was compared with that of the positive and negative controls using Duncan’s multiple range test. The positive (EIEC O152) and negative (E. coli K12 HB101) controls were classified as “strongly invasive” and “non-invasive,” respectively. The isolates chosen (C8, C65, and C67) belonged to different serotypes, O1:H20 (C8) and O117:H4 (C65 and C67), and presented the same invasion profile. The fluorescence of the isolates was similar to that of the positive control, and based on Duncan’s multiple range test, it was classified as “moderately invasive.”

Discussion and conclusions

E. coli was the etiologic agent of an outbreak involving 21 canaries with facial edema from a canary breeder in northern Paraná State, Brazil. E. coli was isolated from the lesions of each bird, and it was concluded that the canaries had colibacillosis.

According to data from ERIC-PCR, which showed a genetic similarity rate above 95% in ERIC-types (ET1, ET2, and ET3), we can infer that the E. coli isolates were clonal. This may explain the homogeneity found between the results of serotyping and the phylogenetic group (Fig. 1). Our results show that all isolates belonged to phylogenetic group B1, and, although other authors have reported a predominance of APEC of the phylogenetic group B2, in our region, we have found APEC of the phylogenetic group B1 [13, 25]. Barbieri et al. (2012) showed that E. coli belonging to phylogenetic group B1 was the etiological agent of colisepticemia that caused the death of peacocks [26], demonstrating its pathogenicity in other bird species, although this phylogenetic group is associated with commensal strains.

Considering the presented clonality, two isolates (C8 and C74) belonging to two different ERIC types (ET1 and ET2) were chosen to determine the ST. Both had the same ST (ST224); however, one isolate (C8) belonged to serotype O1:H20 and the other (C74) to the O117:H4 serotype. Although there are a limited number of publications on ST224 in birds, several reports have shown the same ST in food-producing animals [27–30], cats, and dogs [31, 32]. Furthermore, ST224 has also been identified in a fatal cat infection, demonstrating its pathogenicity in pet animals [33].

Despite the lack of consensus in the scientific literature on which are the ideal genetic markers for APEC, there are reports in the literature that associate the presence of five genes carried by plasmids (iutA, hlyF, iss, iroN, and ompT) with highly pathogenic strains, and the presence of three of these genes in strains isolated from a characteristic colibacillosis lesion is considered APEC [13]. In our study, all isolates had putative hemolysin (hlyF), serum resistance (iss), and outer membrane protein (ompT) genes. The salmochelin (iroN) gene was present in 95% of the isolates, and despite the high prevalence of the aerobactin siderophore system in ExPEC [34], none of the isolates had the iutA gene. Therefore, the isolates in this study can be characterized as APEC, as they had four or more virulence genes [13]. To the best of our knowledge, this is the first report of a clonal outbreak in canaries caused by APEC.

Interestingly, in our study, there was a high prevalence of another virulence gene (ipaH) in 71.4% of isolates. This gene is present in enteroinvasive E. coli (EIEC) [35], and in the literature review, no data were found on the presence of the ipaH gene in other bird species, except in poultry [36, 37]. Moreover, previous studies have reported the occurrence of STEC (Shiga toxin-producing E. coli) and EPEC (enteropathogenic E. coli) in a wide range of bird species [38–40]. The high incidence of this virulence gene in our study, and the fact that some isolates were positive for the invasion assay of HeLa cells, indicate that the ipaH gene was being expressed. Nevertheless, further studies are needed to confirm this.

In contrast to what has been reported in previous studies, it is common for E. coli to produce extended-spectrum β-lactamases (ESBL) in animals farmed for food [34, 41, 42]. Other studies have reported the same profile in different bird species [43–45]. However, all the E. coli isolates in this study were non-ESBL-producing, as evidenced by their sensitivity to cephamycin and third-generation cephalosporins.

In this study, all isolates of E. coli were resistant to tetracycline, ampicillin, and quinolones and fluoroquinolones, and because of their resistance to three or more classes of antibiotics, they were classified as multidrug resistant [46]. Previous studies have reported the presence of multidrug-resistant bacteria in birds [47–50], and some of these antibiotics are drugs used to treat various infectious diseases in humans, such as β-lactams, fluoroquinolones, aminoglycosides, and sulfamethoxazole/trimethoprim [51]. This is concerning, because the canary breeder reported using medications such as tetracyclines, sulfonamides, enrofloxacin, and gentamicin, without the knowledge of the etiologic agent. Thus, we emphasize the importance of avoiding the inadequate antibiotic treatment in canaries, which can lead to the emergence of multidrug-resistant strains [6, 7].

Although the E. coli isolates showed high resistance to quinolones and fluoroquinolones, plasmid-mediated quinolone resistance genes were not found to a large extent. Only 23.8% (qnrS) and 9.5% (qnrB) of the isolates were positive for these genes. It has been reported in the literature that chromosomal mutations in the genes that encode gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE), increased fluoroquinolone minimal inhibitory concentrations (MIC) [52]. We believe that chromosomal mutations in quinolone resistance genes, which have not been investigated, may be involved in the high rate of resistance to this class of drug.

The results of this study indicate that inadequate antibiotic treatment may lead to multidrug resistance to currently available drugs. In addition, E. coli can express virulence genes for diarrheagenic E. coli, even though it is not common to find in these in APEC. Therefore, we emphasize the importance of adequate antimicrobial treatment and recommend constant monitoring of the use of antibiotics in canaries. We also recommend further studies be conducted to increase our understanding of colibacillosis in canaries, since the health of animals is interconnected with human and environmental health, as defined by the concept of One Health.

Author contribution

Conceptualization: Benito Guimarães de Brito, Kelly Cristina Tagliari de Brito, and Renata Katsuko Takayama Kobayashi

Methodology, formal analysis, and investigation: Angela Hitomi Kimura, Vanessa Lumi Koga, Luís Eduardo de Souza Gazal, and Armando Navarro-Ocaña

Writing—original draft preparation: Angela Hitomi Kimura and Renata Katsuko Takayama Kobayashi

Writing—review and editing: Angela Hitomi Kimura, Luís Eduardo de Souza Gazal, Gerson Nakazato, Benito Guimarães de Brito, Kelly Cristina Tagliari de Brito, and Renata Katsuko Takayama Kobayashi

Funding acquisition: Renata Katsuko Takayama Kobayashi

Supervision: Renata Katsuko Takayama Kobayashi

Funding

This study was supported in part by the National Council for Scientific and Technological Development (CNPq) (433656/2018-2) and a scholarship from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brasil—Finance Code 001 to A.H.K.

Declarations

Conflict of interest

The authors declare no competing interests.