Prevalence, seasonality, and growth of enterococci in raw and pasteurized milk in Victoria, Australia.

This study investigated the prevalence, seasonality, and species variety of enterococci present in raw milk factory silos and pasteurized milk in 3 dairying regions in Victoria, Australia, over a 1-yr period. Additionally, the growth ability of thermoduric enterococci isolated in this study (Enterococcus faecalis, E. faecium, E. hirae, and E. durans) was determined in milk at temperatures likely to occur during storage, transport, and distribution, and before domestic consumption (4 and 7°C). Enterococci were detected in 96% of 211 raw milk samples, with an average count of 2.48 log10 cfu/mL. Counts were significantly lower in winter than summer (average 1.84 log10 cfu/mL) and were different between factories but not regions. Enterococcus faecalis was the most prevalent species isolated from raw milk in every factory, comprising between 61.5 and 83.5% of enterococcal species across each season. Enterococci were detected in lower numbers in pasteurized milk than in raw milk and were below the limit of detection on spread plates (<10 cfu/mL) after factory pasteurization. Residual viable cells were only detected following enrichment using 100-mL samples of milk, with 20.8% of the samples testing positive; this equated to a decrease in the average raw milk enterococci count of >4 log10 cfu/mL following pasteurization. Although E. faecalis predominated in raw milk and E. durans was found in only 2.9% of raw milk samples, E. durans was the most prevalent species detected in pasteurized milk. The detection of enterococci in the pasteurized milk did not correlate with higher enterococci counts in the raw milk. This suggested that the main enterococci populations in raw milk were heat-sensitive and that thermoduric enterococci survived pasteurization in a small numbers of instances. All of the thermoduric enterococci that were assessed for growth at likely refrigeration temperatures were able to grow at both 4 and 7°C in sterile milk, with generation times of 35 to 41h and 16 to 22h, respectively. Thermoduric enterococci were detected in pasteurized milk stored at 4°C for 2 wk (typically 1 to 9 cells/100mL, up to 2.82 log10 cfu/mL), demonstrating the potential of enterococci to survive pasteurization and contribute to milk spoilage at refrigeration temperatures. This is particularly relevant for milk that is aseptically packaged to exclude gram-negative psychrotrophic bacteria and kept above the recommended storage temperature of ≤5°C.

Genotyping of present-day and historical Geobacillus species isolates from milk powders by high-resolution melt analysis of multiple variable-number tandem-repeat loci.

Spores of thermophilic Geobacillus species are a common contaminant of milk powder worldwide due to their ability to form biofilms within processing plants. Genotyping methods can provide information regarding the source and monitoring of contamination. A new genotyping method was developed based on multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) in conjunction with high-resolution melt analysis (MLV-HRMA) and compared to the currently used method, randomized amplified polymorphic DNA PCR (RAPD-PCR). Four VNTR loci were identified and used to genotype 46 Geobacillus isolates obtained from retailed powder and samples from 2 different milk powder processing plants. These 46 isolates were differentiated into 16 different groups using MLV-HRMA (D = 0.89). In contrast, only 13 RAPD-PCR genotypes were identified among the 46 isolates (D = 0.79). This new method was then used to analyze 35 isolates obtained from powders with high spore counts (>10(4) spores · g(-1)) from a single processing plant together with 27 historical isolates obtained from powder samples processed in the same region of Australia 17 years ago. Results showed that three genotypes can coexist in a single processing run, while the same genotypes observed 17 years ago are present today. While certain genotypes could be responsible for powders with high spore counts, there was no correlation to specific genotypes being present in powder plants and retailed samples. In conclusion, the MLV-HRMA method is useful for genotyping Geobacillus spp. to provide insight into the prevalence and persistence of certain genotypes within milk powder processing plants.

Heat resistance of thermoduric enterococci isolated from milk.

Enterococci are reported to survive pasteurisation but the extent of their survival is unclear. Sixty-one thermoduric enterococci isolates were selected from laboratory pasteurised milk obtained from silos in six dairy factories. The isolates were screened to determine log(10) reductions incurred after pasteurisation (63°C/30 min) and ranked from highest to lowest log(10) reduction. Two isolates each of Enterococcus faecalis, Enterococcus faecium, Enterococcus durans and Enterococcus hirae, exhibiting the median and the greatest heat resistance, as well as E. faecalis ATCC 19433, were selected for further heat resistance determinations using an immersed coil apparatus. D values were calculated from survival curves plotted from viable counts obtained after heating isolates in Brain Heart Infusion Broth at 63, 69, 72, 75 and 78°C followed by rapid cooling. At 72°C, the temperature employed for High Temperature Short Time (HTST) pasteurisation (72°C/15s), the D values extended from 0.3 min to 5.1 min, depending on the isolate and species. These data were used to calculate z values, which ranged from 5.0 to 9.8°C. The most heat sensitive isolates were E. faecalis (z values 5.0, 5.7 and 7.5°C), while the most heat resistant isolates were E. durans (z values 8.7 and 8.8°C), E. faecium (z value 9.0°C) and E. hirae (z values 8.5 and 9.8°C). The data show that heat resistance in enterococci is highly variable.