Characterization of Escherichia coli and Salmonella from Victoria, Australia, Dairy Farm Environments.

Safe dairy food production starts at the farm level, with the presence of pathogens on farms potentially impacting the downstream food supply. Studies often commence with looking for pathogens in fecal material of farm animals, predominantly cows; however, pathogens may arise from other on-farm sources. In Australia, few studies have looked at the broader farm environment, particularly in relation to Escherichia coli and Salmonella. The present study characterized the genetic similarity of these pathogens from bovine, ovine, and caprine dairy farm environments and related this to the stx1, stx2, eae, or ehx virulence markers in E. coli and antibiotic resistance in Salmonella. E. coli isolates with indistinguishable genetic profiles and at least one of the virulence factors were found in multiple samples on the farms, although profiles were unique to each farm. E. coli O26 with stx1 from one bovine farm had a different fingerprint type than all of the other E. coli O26 isolates, which lacked the Shiga toxin genes. They were from a separate bovine farm and were themselves closely related. No antibiotic resistance was detected among Salmonella isolates to the 17 antibiotics tested. Three Salmonella serotypes were identified: Orion, Infantis, and Zanzibar. The published PCR serotyping method used misidentified Salmonella Zanzibar as Salmonella Javiana, which was revealed after conventional antisera-based serotyping; this illustrates the need for caution when using PCR techniques for Salmonella serotype identification. Of the three serotypes, Salmonella Orion was most prevalent and was potentially resident on the farm. This article describes the previously unreported genetic diversity of potentially pathogenic E. coli and Salmonella serotypes from the farm environments of three dairy animal species in Victoria, Australia.

Characterization of Staphylococcus aureus isolates from raw milk sources in Victoria, Australia.

BACKGROUND: Highly pathogenic strains of Staphylococcus aureus can cause disease in both humans and animals. In animal species, including ruminants, S. aureus may cause severe or sub-clinical mastitis. Dairy animals with mastitis frequently shed S. aureus into the milk supply which can lead to food poisoning in humans. The aim of this study was to use genotypic and immunological methods to characterize S. aureus isolates from milk-related samples collected from 7 dairy farms across Victoria. RESULTS: A total of 30 S. aureus isolates were collected from milk and milk filter samples from 3 bovine, 3 caprine and 1 ovine dairy farms across Victoria, Australia. Pulsed Field Gel Electrophoresis (PFGE) identified 11 distinct pulsotypes among isolates; all caprine and ovine isolates shared greater than 80 % similarity regardless of source. Conversely, bovine isolates showed higher diversity. Multi-Locus Sequence Typing (MLST) identified 5 different sequence types (STs) among bovine isolates, associated with human or ruminant lineages. All caprine and ovine isolates were ST133, or a single allele variant of ST133. Two new novel STs were identified among isolates in this study (ST3183 and ST3184). With the exception of these 2 new STs, eBURST analysis predicted all other STs to be founding members of their associated clonal complexes (CCs). Analysis of genetic markers revealed a diverse range of classical staphylococcal enterotoxins (SE) among isolates, with 11 different SEs identified among bovine isolates, compared with just 2 among caprine and ovine isolates. None of the isolates contained mecA, or were resistant to oxacillin. The only antibiotic resistance identified was that of a single isolate resistant to penicillin; this isolate also contained the penicillin resistance gene blaZ. Production of SE was observed at 16 °C and/or 37 °C in milk, however no SE production was detected at 12 °C. CONCLUSION: Although this study characterized a limited number of isolates, bovine-associated isolates showed higher genetic diversity than their caprine or ovine counterparts. This was also reflected in a more diverse SE repertoire among bovine isolates. Very little antibiotic resistance was identified among isolates in this study. These results suggest maintaining the milk cold chain will minimise any risk from SE production and highlights the need to prevent temperature abuse.

Salmonella Typhimurium and Salmonella Sofia: Growth in and Persistence on Eggs under Production and Retail Conditions.

Salmonellosis in Australia has been linked to eggs and egg products with specific serotypes associated with outbreaks. We compared attachment to and survival on egg shells and growth in eggs of two Salmonella serotypes, an egg outbreak associated Salmonella Typhimurium and a non-egg-associated Salmonella enterica ssp. II 1,4,12,27:b:[e,n,x] (S. Sofia). Experiments were conducted at combinations of 4, 15, 22, 37 and 42 °C. No significant differences occurred between the serotypes in maximum growth rates, which were significantly greater (P < 0.001) in egg yolk (0.427 log10 CFU/mL/h) compared to whole egg (0.312 log10 CFU/mL/h) and egg white (0.029 log10 CFU/mL/h). Attachment to egg shells varied by time (1 or 20 min) and temperature (4, 22 and 42 °C), with S. Typhimurium isolates attaching at higher levels (P < 0.05) than S. Sofia after 1 min at 4 °C and S. Typhimurium ATCC 14028 attaching at higher (P < 0.05) levels at 22 °C. Survival on egg shells was not significantly different across isolates. Salmonella serotypes behaved similarly regarding growth in egg contents, attachment to egg shells and survival on eggs, indicating that other factors more likely contributed to reasons for S. Typhimurium being implicated in multiple egg-associated outbreaks.

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.

Characterization of the spore-forming Bacillus cereus sensu lato group and Clostridium perfringens bacteria isolated from the Australian dairy farm environment.

BACKGROUND: The Bacillus cereus sensu lato group and Clostridium perfringens are spore-forming bacteria often associated with food spoilage and which can cause emetic and diarrheal syndromes in humans and ruminants. This study characterised the phenotypes and genotypes of 50 Bacillus cereus s. l. isolates and 26 Clostridium perfringens isolates from dairy farms environments in Victoria, Australia. RESULTS: Five of the seven B. cereus s. l. species were isolated, and analysis of the population diversity using Pulsed-Field Gel Electrophoresis (PFGE) suggested that the populations are largely distinct to each farm. Enterotoxin production by representative isolates of each B. cereus s. l. species identified was typically found to be reduced in milk, compared with broth. Among the C. perfringens isolates, only two different toxin types were identified, type A and D. Bovine and ovine farms harbored only type A whereas both type A and D were found on two of the three caprine farms. CONCLUSIONS: This study showed that the B. cereus s. l. populations on the sampled farms exhibit a broad diversity in both species and genotypes. The risk of toxin-induced diarrheal illness through consumption of contaminated milk may be limited, in comparison with other food matrices. Type A strains of C. perfringens were the most abundant on dairy farms in Victoria, however type D may be of concern on caprine farms as it can cause enterotoxemia in goats.

Prevalence and characterization of foodborne pathogens from Australian dairy farm environments.

The ability of foodborne pathogens to gain entry into food supply systems remains an ongoing concern. In dairy products, raw milk acts as a major vehicle for this transfer; however, the sources of pathogenic bacteria that contaminate raw milk are often not clear, and environmental sources of contamination or the animals themselves may contribute to the transfer. This survey examined the occurrence of 9 foodborne pathogens in raw milk and environments of 7 dairy farms (3 bovine, 3 caprine, and 1 ovine farm) in summer and autumn, in Victoria, Australia. A total of 120 samples were taken from sampling points common to dairy farms, including pasture, soil, feed, water sources, animal feces, raw milk, and milk filters. The prevalence of the Bacillus cereus group, Campylobacter, Clostridium perfringens, Cronobacter, Shiga-toxigenic Escherichia coli, Listeria, Salmonella, coagulase-positive staphylococci (CPS), and Yersinia enterocolitica across the farms was investigated. The 2 most prevalent bacteria, which were detected on all farms, were the B. cereus group, isolated from 41% of samples, followed by Cl. perfringens, which was isolated from 38% of samples. The highest occurrence of any pathogen was the B. cereus group in soil, present in 93% of samples tested. Fecal samples showed the highest diversity of pathogens, containing 7 of the 9 pathogens tested. Salmonella was isolated from 1 bovine farm, although it was found in multiple samples on both visits. Out of the 14 occurrences where any pathogen was detected in milk filters, only 5 (36%) of the corresponding raw milk samples collected at the same time were positive for the same pathogen. All of the CPS were Staphylococcus aureus, and were found in raw milk or milk filter samples from 6 of the 7 farms, but not in other sample types. Pathogenic Listeria species were detected on 3 of the 7 farms, and included 4 L. ivanovii-positive samples, and 1 L. monocytogenes-positive water sample. Shiga-toxigenic Escherichia coli were identified in fecal samples from 3 of the 7 farms and in a single raw milk sample. Cronobacter species were identified on 4 of the 7 farms, predominantly in feed samples. No Y. enterocolitica was detected. Results of this study demonstrate high standards of pathogen safety across the 7 farms, with a low incidence of pathogens detected in raw milk samples. Monitoring feed contamination levels may help control the spread of bacterial species such as Cl. perfringens and B. cereus through the farm environment, which is a natural reservoir for these organisms.

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.