Prevalence and Genetic Profiles of Staphylococcus aureus Isolated from Milk in Mastitic Dairy Cows Based on Conventional and Multiplex PCR Assays in Khon Kaen Province, Thailand

Abstract

The objective of this study was to investigate the prevalence and genetic profiles of Staphylococcus aureus isolated from mastitic milk in dairy cows by conventional methods and multiplex polymerase chain reaction (m-PCR). Four hundred and thirty-five quarter milk samples were recruited from 14 dairy herds in Khon Kaen Province, Thailand, and determined as subclinical cases by California Mastitis test (CMT). Seventy-Six S. aureus isolates were then bacteriologically identified according to culture and biochemical tests. The prevalence of S. aureus, coagulase negative staphylococci (CNS) and other bacteria were 17.5%, 32.4% and 12.4%, respectively. Genetic profiles of S. aureus were carried out by m-PCR using 3 specific primers targeting
16s rDNA gene (420 bp) for detection of the Staphylococcus spp., gene encoding coagulase (coa: 159, 240 bp) and factors essential for methicillin-resistance (femA: 344 bp) for detecting species level of S. aureus. Genetic profiles were assigned to 6 different patterns based on specific genes detection out of the 76 S. aureus isolates. All of the isolates yielded an amplicon with a size of approximately 420 bp of the 16s-rDNA. Amplification of the coa gene yielded two distinct products of 159-bp and 240-bp amplicons in 28 (36.8%) and 29 (38.1%) isolates, respectively. The fem A gene (344 bp) was highly expressed in 69 isolates (92.1%).On the basis of genetic variation, DNA band patterns were divided into 6 patterns (pattern I-VI) and recognized
as follows: pattern I (420-344-240-159 bp), pattern II (420-344 bp, pattern III (800-600-420-344-240 bp), pattern IV (800-600-420- 344 bp), pattern V (600-500-420-240 bp) and pattern VI (800-600-420-240-159 bp), accounted for 25% (19/76), 43.4% (33/76), 5.26% (4/76), 10.52% (8/76), 3.94% (3/76) and 5.25% (4/76), respectively. The results of this study showed that the prevalence
of S. aureus is relatively high and distributed in several farms. m-PCR assay with 3 specific genes can be successfully applied to assess the genetic characteristics of S. aureus isolates from different herds. Furthermore, the genetic diversity of specific genes indicates the same or different clonal distribution of in several dairy herds in the nearby area.