Occurrence, Antimicrobial Resistance Patterns, and Genetic Characterization of Staphylococcus aureus Isolated from Raw Milk in the Dairy Farms over Two Seasons in China.

This study investigated the occurrence and resistance rates of Staphylococcus aureus strains isolated from raw milk in the dairy farms over two seasons (spring and autumn) and across four regions that included 11 provinces in China. In total, 750 raw milk samples from the 405 dairy farms were collected. Fifteen antimicrobial agents were tested for antimicrobial resistance via disk diffusion tests, and PCR tests were performed to identify drug resistance genes of S. aureus isolates. Out of 750 samples, 276 (36.8%) were positive for S. aureus, with 150 (41.1%) being positive in spring and 126 (32.7%) being positive in autumn. The occurrence rate of S. aureus in northeastern China (45%) was higher than that in western China (33%) and southern China (31.9%), respectively, and the rate significantly (p < 0.05) differed from those of western China and southern China. Of 276 isolates, 261 (94.6%) strains were resistant to more than 1 antimicrobial drug, and 193 (69.9%) strains were multidrug resistant. The blaZ (46.3%), dfrG (35.5%), and tetM (27.2%) genes were detected at a high frequency in the S. aureus strains. Our data revealed a variation (p < 0.05) in the resistance patterns in the different regions and across the two seasons. The occurrence and drug resistance rates of S. aureus isolated from raw milk obtained from dairy farms may still cause severe problems in China.

Short communication: Quantitative PCR coupled with sodium dodecyl sulfate and propidium monoazide for detection of culturable Escherichia coli in milk.

Escherichia coli has been frequently reported as a major foodborne bacterium contaminating raw milk or pasteurized milk. Therefore, the aim of this study was to explore a quantitative real-time PCR (qPCR) technique combined with sodium dodecyl sulfate (SDS) and propidium monoazide (PMA) to detect culturable E. coli in milk. An internal amplification control was also added into this reaction system as an indicator of false-negative results. The inclusivity and exclusivity of the primers were tested using DNA from 7 E. coli and 14 other bacterial strains. The concentrations of SDS and PMA were determined according to plate counts and quantitative cycle values of qPCR, respectively. A standard curve was established using series diluted E. coli DNA. The reliability and specificity of this method were further determined by the detection of E. coli in spiked milk. The results showed that the optimal concentrations of SDS and PMA were 100 µg/mL and 40 µM, respectively. A standard curve with a good linear relationship (coefficient of determination = 0.997; amplification efficiency = 100.5%) was obtained. Compared with conventional PCR and PMA-qPCR, the SDS-PMA-qPCR assay was more specific and sensitive in culturable E. coli detection. Therefore, we evaluated and improved the SDS-PMA-qPCR method for detecting culturable E. coli in milk.

Quantitative PCR coupled with sodium dodecyl sulfate and propidium monoazide for detection of viable Staphylococcus aureus in milk.

Conventional quantitative PCR (qPCR) are unable to differentiate DNA of viable Staphylococcus aureus cells from dead ones. The aim of this study was to use sodium dodecyl sulfate (SDS) and propidium monoazide (PMA) coupled with lysostaphin to detect viable Staph. aureus. The cell suspensions were treated with SDS and PMA before DNA extraction. The SDS is an anionic surfactant, which can increase the permeability of dead cells to PMA without compromising the viability of live cells. The lysostaphin was applied to improve the effectiveness of DNA extraction. The reliability and specificity of this method were further determined by the detection of Staph. aureus in spiked milk. The results showed that there were significant differences between the SDS-PMA-qPCR and qPCR when a final concentration of 200 µg/mL of lysostaphin was added in DNA extraction. The viable Staph. aureus could be effectively detected when SDS and PMA concentrations were 100 µg/mL and 40 µM, respectively. Compared with conventional qPCR, the SDS-PMA-qPCR assay coupled with lysostaphin was more specific and sensitive. Therefore, this method could accurately detect the number of viable Staph. aureus cells.

Identification and proteolytic activity quantification of Pseudomonas spp. isolated from different raw milks at storage temperatures.

Commercial milk products worldwide come not only from cows, but also from goats, buffaloes, camels, and yaks. Milk from non-bovine animals is important culturally and economically. Pseudomonas spp. are frequently linked to milk spoilage under storage temperatures. The objectives of this study were to identify Pseudomonas spp. isolated from goat, buffalo, camel, and yak milks, and to measure proteolytic activity of Pseudomonas spp. under different storage temperatures. Raw milk samples of goat (n = 50), buffalo (n = 25), camel (n = 25), and yak (n = 25) were collected from 5 provinces in China. Pseudomonas spp. were analyzed by Pseudomonas-specific 16S, universal 16S rRNA, and rpoB gene sequence analyses. Proteolytic activity on milk agar, quantification via the trinitrobenzenesulfonic acid assay at 2°C, 4°C, 7°C, 10°C and 25°C, as well as alkaline peptidase gene (aprX) identification were performed to ascertain the proteolytic activity of these isolates. Pseudomonas spp. were found in 46 samples out of total 125 samples. A total of 67 Pseudomonas spp. were identified. Of Pseudomonas isolates, we obtained extracellular peptidase activity in 7 (10.4%) at 2°C, 17 (25.4%) at 4°C, 24 (35.8%) at 7°C, 39 (58.2%) at 10°C, and 41 (61.2%) at 25°C. The results revealed that a wide diversity of Pseudomonas spp. were present in different non-bovine raw milks, with the ability to produce peptidases at storage temperatures. However, proteolytic activity varied widely among the peptidase-positive isolates. A majority of isolates from yak milk had high proteolytic activity.