Prevalence and Characterization of Staphylococcus aureus Cultured From Raw Milk Taken From Dairy Cows With Mastitis in Beijing, China

Abstract

The colonization of dairy herds and subsequent contamination of raw milk by Staphylococcus aureus (S. aureus), especially those expressing a multi-drug resistance (MDR), biofilm and toxins producing ability, remains an important issue for both the dairy producer and public health. In this study, we investigated the prevalence, antimicrobial resistance, virulence, and genetic diversity of S. aureus in raw milk taken from 2 dairy farms in Beijing, China. Ninety (46.2%, 90/195) samples were positive for S. aureus. Resistant to penicillin (PEN) (31.3%), ciprofloxacin (18.8%) and enrofloxacin (15.6%) were the most often observed. Isolates cultured from farm B showed significantly higher resistance to penicillin (73.9%), ciprofloxacin (34.8%), enrofloxacin (34.8%), tilmicosin (17.4%), and erythromycin (17.4%) than those from farm A (p < 0.05). Totally, 94.8% S. aureus harbored at least one virulence gene and the pvl (93.8%), sec (65.6%), and sea (60.4%) genes were the most frequently detected. The pvl and sec genes were more often detected in isolates from farm A (97.3% and 84.9% respectively) than those from farm B (p < 0.05). Of all 77 staphylococcus enterotoxin (SE)-positive isolates, more than 90% could produce enterotoxins and 70.1% could produce two types. Biofilm related genes (icaA/D, clf/B, can, and fnbA) were detected in all96 isolates. All 96 isolates could produce biofilm with 8.3, 70.8, and 18.8% of the isolates demonstrating weak, moderate and strong biofilm formation, respectively. A total of 5 STs, 7 spa types (1 novel spa type t17182), 3agr types (no agrII), and 14 SmaI-pulso-types were found in this study. PFGE cluster II-CC1-ST1-t127-agr III was the most prevalent clone (56.3%). Isolates of agr III (PFGE Cluster I/II-CC1-ST1-t127/2279) had higher detection of virulence genes than those of agr I and agr IV. TheMSSA-ST398-t1456-agr I clone expressed the greatest MDRbut with no virulence genes and weakly biofilm formation. Our finding indicated a relatively high prevalence of S. aureus with less antimicrobial resistance but often positive for enterotoxigenicity and biofilm formation. This study could help identify predominant clones and provide surveillance measures to eliminate and decrease the contamination of S. aureus in raw milk of dairy cows with mastitis.

Keywords: Staphylococcus aureus; antimicrobial susceptible test; biofilm; enterotoxin production; mastitis; molecular typing; raw milk; virulence factors.

Figure 1

Dendrogram of PFGE patterns and antimicrobial susceptibility testing (AST), virulence genes, enterotoxin production, biofilm and adhesion related genes, mecA gene, and molecular characterization of 96 S. aureus isolates cultured from raw milk in Beijing China. Ninety-six isolates were grouped into 6 clusters (cluster I-VI) by PFGE patterns and all had more than 92% similarity. The results of AST were showed in different colors according to the MIC values of isolates to different antimicrobial agents. Green squares indicate susceptibility; yellow squares indicate intermediate; and red squares indicate resistance. The detection of virulence genes, enterotoxin production, biofilm and adhesion related genes, and mecA gene were summarized by a heat map. Black squares denote that the studied genes were detected in those isolates, or those isolates could produce those types of enterotoxins. White squares denote that those isolates lack these studied genes or could not produce those types of enterotoxins. BPA represents biofilm production ability. ST/CC represents sequence type of MLST and the clone complex (CC) of this ST. agr represents agr types. Antimicrobial compounds used are abbreviated as follows: TIO, Ceftiofur; CHL, chloramphenicol; CIP, ciprofloxacin; DAP, daptomycin; ENO, enrofloxacin; ERY, erythromycin; FOS, fosfomycin; GEN, gentamycin; PEN, penicillin; TET, tetracycline; TIM, tilmicosin; VAN, vancomycin. The same symbols beside farm number of $•$, ▴, ✦, ▾, ⋆, and ♦ represent isolates cultured from M11, M17, M23, M34, M87, and M91, respectively.

Figure 2

Population snapshot analyses by eBURST on 5779 S. aureus strains that belonged to 2766 STs in S. aureus MLST database. Pink circles labeled with red arrows and the ST names in black font are used for sequence types identified in this study.

Figure 3

Minimum Spanning Tree of 96 S. aureus isolates based on the MLST data. Colors indicate ST of each node. The spa types of 96 S. aureus isolates were also showed within each node. Novel spa type identified in this study was t17182.