First study on capsular serotypes and virulence factors of Pasteurella multocida isolates from Phan Rang sheep in Vietnam

Abstract

Background and aim: Pasteurella multocida is considered as a main factor mediating pneumonic pasteurellosis in ruminants, including sheep. It is also a current threat to Phan Rang sheep in Vietnam. This study aimed to characterize P. multocida isolated from Phan Rang sheep, their antibiotic resistance profile, and the prevalence of some virulence-associated genes of these strains.

Materials and methods: Bacteria were isolated on brain heart infusion, 10% sheep blood agar plates, and screened by biochemical tests. The polymerase chain reaction technique was used with specific primers to identify P. multocida, the presence of virulence-associated genes, and serotypes of isolates. Antimicrobial susceptibility and biofilm formation of isolates were examined using the disk diffusion method and crystal violet-based method, respectively.

Results: A total of 41 P. multocida strains were isolated from 485 samples from clinically sick and healthy sheep. Of the isolates, 58.53% were serotype A, 9.75% were serotype B, and 31.71% were serotype D. Healthy animals were infected with serotype D only. All 15 virulence genes were identified in all strains isolated from clinically sick sheep, while strains isolated from healthy sheep carried 11/15 virulence genes tested. Among virulence-associated genes exbB, exbD, tonB, ompA, oma87, fimA, hgbA, and nanB were detected in over 90% of isolates, whereas hgbB, nanH, tbpA and pfhA were less frequent. Interestingly, pmHAS and tadD were highly prevalent in capsular type A strains, whereas the toxA gene was detected in capsular type D strains only. All of the isolated strains were fully susceptible to enrofloxacin, ciprofloxacin, neomycin, and ofloxacin. About 92.68% were susceptible to chloramphenicol and 90.24% to amikacin, but there was high resistance to erythromycin, tetracycline, and amoxicillin. Our results reveal that 53.65% of 41 isolates could produce biofilm, whereas 46.34% could not.

Conclusion: Pasteurella multocida from Phan Rang sheep possess many virulence genes and resistance to several common antibiotics such as erythromycin, tetracycline, and amoxicillin. The results are an important warning regarding antibiotic resistance of P. multocida.

Keywords: Pasteurella multocida; Phan Rang sheep; antimicrobial resistance; biofilm formation; capsular type.

Figure-1

Pasteurella multocida-specific polymerase chain reaction (PCR) assay by kmt1 primers and multiplex PCR for capsular typing of P. multocida isolates. (a) Verification of P. multocida by PCR. Lane M, 1 kb DNA ladder marker (ThermoScientific, USA); lanes 1–10: detection of P. multocida from isolates (1–10); lane P: Positive control (633 bp), lane N: Negative control. (b) Capsular identification in isolated P. multocida by multiplex PCR assay. Lane M, 1 kb DNA ladder marker (ThermoScientific, USA); lane N, negative control; lanes 1–10 isolates (1–10); A positive control of capA (1044 bp); B, positive control of capB (760 bp); and D, positive control of capD (657 bp).

Figure-2

Polymerase chain reaction assay checking the prevalence of virulence genes of Pasteurella multocida isolated from Phan Rang sheep. Lane M, 1 kb DNA ladder marker (ThermoScientific, USA); lanes 1–10: Samples, lane N: Negative control, lane P: Positive control. (a) Detection of toxA gene; (b) detection of tadD gene; (c) detection of ompA gene; (d) detection of pmHAS gene.

Figure-3

Biofilm formation of 41 Pasteurella multocida strains. The ability of isolates to form biofilm was measured at 540 nm using crystal violet after 24 h of growth in 96-well plates. Error bars indicate standard deviation from three independent experiments. The orange line indicates control measurement.

Figure-S1

Antibiotic resistance profile of Pasteurella multocida isolates: Resistant (R) in red, intermediate (I) in yellow, and susceptible (S) in green color.