Recommendation for a Standardised Method of Broth Microdilution Susceptibility Testing for Porcine Bordetella bronchiseptica.

Sandra Prüller,Cornelia Frömke,Heike Kaspar,Corinna Kehrenberg,Lothar Kreienbrock,Günter Klein,Daniela Flavia Hozbor

doi:10.1371/journal.pone.0123883

Abstract

The objective was to establish and standardise a broth microdilution susceptibility testing method for porcine Bordetella (B.) bronchiseptica. B. bronchiseptica isolates from different geographical regions and farms were genotyped by macrorestriction analysis and subsequent pulsed-field gel electrophoresis. One reference and one type strain plus two field isolates of B. bronchiseptica were chosen to analyse growth curves in four different media: cation-adjusted Mueller-Hinton broth (CAMHB) with and without 2% lysed horse blood, Brain-Heart-Infusion (BHI), and Caso broth. The growth rate of each test strain in each medium was determined by culture enumeration and the suitability of CAMHB was confirmed by comparative statistical analysis. Thereafter, reference and type strain and eight epidemiologically unrelated field isolates of B. bronchiseptica were used to test the suitability of a broth microdilution susceptibility testing method following CLSI-approved performance standards given in document VET01-A4. Susceptibility tests, using 20 antimicrobial agents, were performed in five replicates, and data were collected after 20 and 24 hours incubation and statistically analysed. Due to the low growth rate of B. bronchiseptica, an incubation time of 24 hours resulted in significantly more homogeneous minimum inhibitory concentrations after five replications compared to a 20-hour incubation. An interlaboratory comparison trial including susceptibility testing of 24 antimicrobial agents revealed a high mean level of reproducibility (97.9%) of the modified method. Hence, in a harmonization for broth microdilution susceptibility testing of B. bronchiseptica, an incubation time of 24 hours in CAMHB medium with an incubation temperature of 35°C and an inoculum concentration of approximately 5 x 105 cfu/ml was proposed.

Highlights

Bordetella bronchiseptica, a strict aerobic, gram-negative coccobacillus belongs to the genus Bordetella within the alcaligenaceae family [1]
Growth curves of four isolates cultured in different media showed a difference in the results between optical density (OD600) and culture enumeration
While the mean value of three replications of optical density measurement showed a beneficial growth in cation-adjusted Mueller-Hinton broth (CAMHB) + 2% lysed horse blood, the mean value of three replications of cfu/mL revealed an almost consistent growth in the four different media (Fig B–I in S1 File)

Summary

Introduction

Bordetella bronchiseptica, a strict aerobic, gram-negative coccobacillus belongs to the genus Bordetella within the alcaligenaceae family [1] This pathogen is known to cause respiratory. The pathogen can lead to symptoms ranging from mild bronchitis to severe bronchopneumonia It is involved in atrophic rhinitis, associated with toxogenic strains of Pasteurella multocida, which can lead to severe distorsion and atrophy of the turbinate bones in the nasal terminus [3,4,5]. In dogs it causes tracheobronchitis and is one of the main pathogens of kennel cough [6]. Respiratory tract infections due to B. bronchiseptica are known in rabbits and guinea pigs [8]

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Conclusion