Diversity of Escherichia coli strains involved in vertebral osteomyelitis and arthritis in broilers in Brazil

Abstract

Background: Locomotor disorders and infections by Escherichia coli represent major concerns to the poultry industry worldwide. Avian pathogenic E. coli (APEC) is associated with extraintestinal infections leading to respiratory or systemic disease known as colibacillosis. The most common lesions seen in cases of colibacillosis are perihepatitis, airsacculitis, pericarditis, peritonitis/salpingitis and arthritis. These diseases are responsible for significant economic losses in the poultry industry worldwide. E. coli has been recently isolated from vertebral osteomyelitis cases in Brazil and there are no data on molecular and phenotypic characteristics of E. coli strains isolated from lesions in the locomotor system of broilers. This raised the question whether specific E. coli strains could be responsible for bone lesions in broilers. The aim of this study was to assess these characteristics of E. coli strains isolated from broilers presenting vertebral osteomyelitis and arthritis in Brazil.

Results: Fifteen E. coli strains from bone lesions were submitted to APEC diagnosis and setting of ECOR phylogenic group, O serogroup, flagella type, virulence genes content, genetic patterns by Pulsed Field Gel Electrophoresis (PFGE) and Multilocus Sequence Typing (MLST). In addition, bacterial isolates were further characterized through a lethality test, serum resistance test and antibiotic resistance profile. E. coli strains harbored different genetic pattern as assessed by PFGE, regardless of flock origin and lesion site. The strains belonged to seven sequence types (STs) previously described (ST117, ST101, ST131, ST 371 and ST3107) or newly described in this study (ST5766 and ST5856). ECOR group D (66.7 %) was the most frequently detected. The strains belonged to diverse serogroups (O88, O25, O12, and O45), some of worldwide importance. The antibiotic resistance profile confirmed strains' diversity and revealed a high proportion of multidrug-resistant strains (73 %), mainly to quinolones and beta-lactams, including third generation cephalosporin. The percentage of resistance to tetracycline was moderate (33 %) but always associated with multidrug resistance.

Conclusions: Our results demonstrated that vertebral osteomyelitis and arthritis in broilers can be associated with highly diverse E. coli based on molecular and phenotypic characteristics. There was no specific virulence patterns of the E. coli strains associated with vertebral osteomyelitis or arthritis. Also, E. coli strains were frequently multidrug resistant and belonged to STs commonly shared by APEC and human ExPEC strains.

Keywords: APEC; Bacterial infections; Broilers; Multidrug-resistant E. coli; Pathology; Virulence genes.

Fig. 1

Molecular and phenotypic characterization of 15 Escherichia coli strains isolated from broilers with osteomyelitis and arthritis. Black and white boxes represent positive and negative results, respectively. Flock ID, number of the flock of origin; Lesion, VO: vertebral osteomyelitis, Art: arthritis; Serotype, ns: non-serotyped; Flagella, nm: non-motile, nc: non-correspondent to any flagellar type tested; ST, Sequence type; ECOR: ECOR phylogenetic group; APEC (Johnson et al.): APEC diagnosis according to Johnson et al. (2008); APEC (Schouler et al.); APEC diagnosis according to Schouler et al. (2012); Yes: APEC strain, No: non-APEC strain; pVAGs, pattern of virulence genes described by Schouler et al. (2012), nc: non-correspondent to the described patterns; Iron acquisition, genes encoding iron acquisition system; Adhesin, genes encoding adhesins; Toxin, genes encoding toxins; Protectin, genes encoding protectins; Invasin, genes encoding invasins; Miscellaneous, genes encoding different kinds of virulence; VAGs (%), percentage of APEC-associated virulence genes; Lethality score, number of chicks that died at the fourth day post-infection with E. coli; Serum resistance, R: serum resistant strain, I: intermediate resistant strain, S: serum sensitive strain; N° resistant AB: number of antibiotics to which the strain was resistant; Antibiotic resistance profile: gentamicin, Gen; neomycin, Neo; apramycin, Apr; amoxicillin, Amx; amoxicillin + clavulanic acid, Amc; cephalotin, Cef; cefoxitin, Fox; ceftiofur, Xnl; florfenicol, Ffc; colistin, Cst; nalidixic acid, Nal; flumequine, UB; enrofloxacin, Enr; trimethoprim, Tmp; Tmp + sulfamethoxazole, TmpStx; tetracycline, Tet; pansusceptible, PanSus

Fig. 2

Clinical signs and gross pathology of vertebral osteomyelitis (a, b, c) and arthritis (d, e, f) in broilers. a Broiler showing the classical clinical sign of severe cases of vertebral osteomyelitis. b Note the enlargement of affected vertebral body (T4), c which revels caseonecrotic material and spinal cord compression on longitudinal section. d Broiler with bilateral arthritis showing ventral recumbency and retracted legs. e Suppurative exudate in articular cavity in acute arthritis, f which extended to proximal tibiotarsus causing tibial osteomyelitis

Fig. 3

Histopathology of osteomyelitis and arthritis in broilers. a Vertebral osteomyelitis showing enlargment of vertebral body (T4) by caseonecrotic material (remanescent, arrow), which compresses spinal cord (*); HE. b Caseonecrotic hererophilic and histiocytic exudate (*) in the articular space with intralesional bacterial colonies (arrow); HE. Inset: Gram-negative bacteria stained by Goodpasture. c Necrotic synovitis (arrow) associated with caseonecrotic exudate within the articular space (*); HE. d Proximal growth plate (physis) of tibiotarsus showing extensive necrosis (*) with heterophilic exudate in a case of tibial osteomyelitis; HE

Fig. 4

Percentages of antibiotic resistance of E. coli strains isolated from vertebral osteomyelitis and arthritis in broilers by antibiotic class: (a) quinolones; (b) beta-lactams; (c) cephalosporins; (d) sulfonamides; (e) tetracyclines; (f) aminoglycosides; (g) phenicols; and (h) polypeptides