title: Recommendation for a Standardised Method of Broth Microdilution Susceptibility Testing for Porcine Bordetella bronchiseptica.
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 10(5) cfu/ml was proposed.
authors.affiliation: Institute for Food Quality and Food Safety, University of Veterinary Medicine Hannover, Foundation, Bischofsholer Damm 15, D-30173, Hannover, Germany.
authors.forename: Sandra
authors.initials: S
authors.lastname: Prüller
authors.affiliation: Department of Biometry, Epidemiology and Information Processing, WHO Collaborating Centre for Research and Training in Veterinary Public Health, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany.
authors.forename: Cornelia
authors.initials: C
authors.lastname: Frömke
authors.affiliation: Federal Office of Consumer Protection and Food Safety, Berlin, Germany.
authors.forename: Heike
authors.initials: H
authors.lastname: Kaspar
authors.affiliation: Institute for Food Quality and Food Safety, University of Veterinary Medicine Hannover, Foundation, Bischofsholer Damm 15, D-30173, Hannover, Germany.
authors.forename: Günter
authors.initials: G
authors.lastname: Klein
authors.affiliation: Federal Office of Consumer Protection and Food Safety, Berlin, Germany.
authors.forename: Lothar
authors.initials: L
authors.lastname: Kreienbrock
authors.affiliation: Institute for Food Quality and Food Safety, University of Veterinary Medicine Hannover, Foundation, Bischofsholer Damm 15, D-30173, Hannover, Germany.
authors.forename: Corinna
authors.initials: C
authors.lastname: Kehrenberg
mesh_terms: D000818:Animals; D000890:Anti-Infective Agents / Q000494:pharmacology; D001885:Bordetella Infections / Q000662:veterinary*; D016950:Bordetella bronchiseptica / Q000187:drug effects / Q000302:isolation & purification*; D004198:Disease Susceptibility; D008826:Microbial Sensitivity Tests; D015203:Reproducibility of Results; D013552:Swine / Q000382:microbiology*; D013553:Swine Diseases
chemical_list: D000890:Anti-Infective Agents