Streptococcus agalactiae is an important pathogen causing bovine mastitis. The aim of this study was to develop a simple and specific method for direct detection of S. agalactiae from milk products. Propidium monoazide (PMA) and sodium dodecyl sulfate (SDS) were utilized to eliminate the interference of dead and injured cells in qPCR. Lysozyme (LYZ) was adopted to increase the extraction efficiency of target bacteria DNA in milk matrix. The specific primers were designed based on cfb gene of S. agalactiae for qPCR. The inclusivity and exclusivity of the assay were evaluated using 30 strains. The method was further determined by the detection of S. agalactiae in spiked milk. Results showed significant differences between the SDS–PMA–qPCR, PMA–qPCR and qPCR when a final concentration of 10 mg/ml ( R 2 = 0.9996, E = 95%) of LYZ was added in DNA extraction. Viable S. agalactiae was effectively detected when SDS and PMA concentrations were 20 μg/ml and 10 μM, respectively, and it was specific and more sensitive than qPCR and PMA–qPCR. Moreover, the SDS–PMA–qPCR assay coupled with LYZ was used to detect viable S. agalactiae in spiked milk, with a limit of detection of 3 × 10 3 cfu/ml. Therefore, the SDS–PMA–qPCR assay had excellent sensitivity and specificity for detection of viable S. agalactiae in milk.

Staphylococcus aureus (S. aureus) is one of the main pathogens in different raw milk and dairy products, which may lead to economic losses. Staphylococcus aureus is a significant and costly public health concern because it may enter the human food chain and contaminate milk causing foodborne illness. This study aimed to investigate the prevalence, antimicrobial susceptibility and virulence genes of S. aureus in raw milks. In total, 125 raw milk samples collected from goat (n = 50), buffalo (n = 25), camel (n = 25), and yak (n = 25) were collected from 5 provinces in China in 2016. Out of 125 samples, 36 (28.8%) S. aureus were isolated (16 from goat, 9 from buffalo, 6 from camel, and 5 from yak). Out of 36 S. aureus, 26 strains (26/36, 72.2%) showed antibiotics resistance, and 6 strains isolated from goats were identified as methicillin-resistant S. aureus (MRSA). The antimicrobial resistance against Penicillin G, tetracycline and gentamicin was 50% (18/36), 41.7% (15/36), and 36.1% (13/36), respectively. 19 S. aureus (52.8%) were considered as multidrug resistant. The highest prevalence of resistant S. aureus was observed in goat milk (13/36, 36.1%). Among the 36 strains, 16 isolates harbored three or more resistant genes. The resistance genes were detected in 25 S. aureus, including 13 strains in goat, 5 strains in buffalo, 4 strains in camel, and 3 strains in yak. Among the 26 resistant strains, 61.5% of isolates harbored three or more resistant genes. The resistance genes were detected in 25 S. aureus, including 13 strains in goat milk, 5 strains in buffalo milk, 4 strains in camel milk, and 3 strains in yak milk. The most predominant resistance genes were blaZ (18/26, 69.2%), aac6′-aph2″ (13/26, 50.0%), and tet(M) (10/26, 38.5%). The mecA, ant(6)-Ia and fexA gene were only detected in S. aureus from goat milk. The most predominant toxin gene were sec (8/26, 30.8%). The majority of S. aureus were multidrug resistant and carried multiple virulence genes, which may pose potential risk to public health. Our findings indicated that the prevalence and antimicrobial resistance of S. aureus was a serious concern in different raw milks in China, especially goat milks.

Introduction Streptococcus agalactiae is an important zoonotic pathogen that affects milk production and quality and poses a threat to public health. Treatment of infections with this bacterium exploits antimicrobials, to which the resistance of S. agalactiae is a growing problem. Addressing the possibility of a correlation between this pathogen’s genetic factors for antimicrobial resistance and virulence, this study attempted to identify the relevant genes. Material and Methods Antimicrobial resistance of S. agalactiae isolated from 497 Chinese bovine mastitic milk samples was detected by the broth microdilution method. Eight drug resistance genes and eleven virulence genes were detected using PCR. Results Streptococcus agalactiae was 100% susceptible to rifampicin and vancomycin, 93.33% susceptible to sulfisoxazole and sulfamethoxazole, but 100% resistant to ≥3 of the 16 antimicrobial agents, thereby being multidrug resistant, with resistance to oxacillin, tetracycline, erythromycin, clindamycin, and gentamicin being common. The ermB, ermA and lnuA genes were carried by 73.33%, 66.67% and 60.00% of the strains, respectively. The carriage rates of the glnA, clyE, hylB, bibA, iagA, and fbsA virulence genes were greater than 40%, lmb and bac were not observed in any strain, and glnA+hylB+bibA+iagA+fbsA+clyE combined virulence gene patterns were the most commonly detected. Conclusion Antimicrobial resistance of S. agalactiae is still a great concern for cattle health in China, and multidrug resistance coupled with the high positive rates of this bacterium’s strains for virulence genes indicates the importance of S. agalactiae surveillance and susceptibility tests.