Molecular and Phenotypic Characterization of Aerococcus viridans Associated with Subclinical Bovine Mastitis

Abstract

Aerococcus viridans is a wide spread bacterium in the environment and clinically this organism is associated with different diseases in animals and humans. However, the geno- and phenotypic characterization of A. viridans associated with bovine mastitis has not yet been reported. The objectives of this study were to investigate the genetic and phenotypic diversity of A. viridans isolates using three different molecular methods including 16S rRNA gene sequencing, pulsed-field gel electrophoresis and random amplified polymorphic DNA (RAPD) along with biochemical tests, including antimicrobial susceptibility test. In total, 60 A. viridans strains were cultured from dairy herds presenting with subclinical mastitis. The results of biochemical tests revealed that most of the isolates (75.0%) were accurately identified by API Rapid 20 Strep system and the majority of A. viridans strains (96.7%) were found to be catalase negative, while two (3.3%) isolates were weakly positive. All isolates were resistant to trimethoprim-sulfamethoxazole, followed by streptomycin (96.7%), tetracycline (65.0%) and clindamycin (56.7%) by minimum inhibition concentration-determining broth microdilution technique. As compared to the sequence of 16S rRNA gene, both PFGE and RAPD showed their capacities to discriminate the intra-species diversity of A. viridans. Furthermore, most of the isolates obtained from the same herd or region belonged to the same major RAPD group, which indicated that RAPD is an appropriate assay for tracking the origins of isolates and epidemiological studies of A. viridans. This is a novel approach to use three molecular techniques and to compare their efficiency regarding the genetic diversity of A. viridans. The data suggest that A. viridans associated with subclinical mastitis has a considerable phenotypic and genotypic diversity.

Fig 1. Dendrogram resulting from a computer-assisted analysis of the PFGE profiles of A. viridans isolates recovered from bovine subclinical mastitis.

The Dice coefficient and a tolerance of 1.5% were used for calculating the similarities and clustering among the profiles.

Fig 2. Representatives of RAPD fingerprints identified among A. viridans isolates from subclinical mastitis.

Lanes 1, 9, 17 molecular size markers (in base pairs; DNA ladder ranging from 100 to 5,000 bp); Lane 2 A.V. 39 (RAPD group 9, BJ-S, 2012); Lane 3 A.V. 38 (RAPD group 9, BJ-S, 2012); Lane 4 A.V. 31 (RAPD group 6, BJ-H, 2010); Lane 5 A.V. 45 (RAPD group 6, BJ-T, 2012); Lane 6 A.V. 29 (RAPD group 6, BJ-H, 2010); Lane 7 A.V. 51 (RAPD group 8, TJ-X, 2013); Lane 8 A.V. 48 (RAPD group 8, TJ-X, 2013); Lane 10 A.V. 30 (RAPD group 6, BJ-H, 2010); Lane 11 A.V. 24 (RAPD group 7, HB-S, 2010); Lane 12 A.V.43 (RAPD group 14, BJ-T, 2012); Lane 13 A.V. 28 (RAPD group 6, BJ-H, 2010); Lane 14 A.V. 41 (RAPD group 13, BJ-T, 2012); Lane 15 A.V. 33 (RAPD group 6, BJ-H, 2010); Lane 16 A.V. 53 (RAPD group 6, TJ-X, 2013).

Fig 3. Dendrogram resulting from a computer-assisted analysis of the RAPD profiles of A. viridans isolates recovered from bovine subclinical mastitis.

The Dice coefficient and a tolerance of 1.5% were used for calculating the similarities and clustering among the profiles. NA, PFGE method was not applicable for the characterization of this isolate according to the results of PFGE in our study.