Control of Escherichia coli in Fresh-Cut Mixed Vegetables Using a Combination of Bacteriophage and Carvacrol.

The continual emergence of antibiotic-resistant bacteria and the slow development of new antibiotics has driven the resurgent interest in the potential application of bacteriophages as antimicrobial agents in different medical and industrial sectors. In the present study, the potential of combining phage biocontrol and a natural plant compound (carvacrol) in controlling Escherichia coli on fresh-cut mixed vegetable was evaluated. Four coliphages, designated Escherichia phage SUT_E420, Escherichia phage SUT_E520, Escherichia phage SUT_E1520 and Escherichia phage SUT_E1620, were isolated from raw sewage. Biological characterization revealed that all four phages had a latent period of 20-30 min and a burst size ranging from 116 plaque-forming units (PFU)/colony forming units (CFU) to 441 PFU/CFU. The phages effectively inhibited the growth of respective host bacteria in vitro, especially when used at a high multiplicity of infection (MOI). Based on transmission electron microscopy analysis, all phages were classified as tailed phages in the class of Caudoviricetes. Additionally, next generation sequencing indicated that none of the selected coliphages contained genes encoding virulence or antimicrobial resistance factors, highlighting the suitability of isolated phages as biocontrol agents. When a phage cocktail (~109 PFU/mL) was applied alone onto fresh-cut mixed vegetables artificially contaminated with E. coli, no bacteria were recovered from treated samples on Day 0, followed by a gradual increase in the E. coli population after 24 h of incubation at 8 °C. On the other hand, no significant differences (p < 0.05) were observed between treated and non-treated samples in terms of E. coli viable counts when carvacrol at the minimum inhibitory concentration (MIC) of 6.25 µL/mL was applied alone. When a phage cocktail at an MOI of ~1000 and MIC carvacrol were applied in combination, no E. coli were recovered from treated samples on Day 0 and 1, followed by a slight increase in the E. coli population to approximately 1.2-1.3 log CFU/mL after 48 h of incubation at 8 °C. However, total elimination of E. coli was observed in samples treated with a phage cocktail at a higher MOI of ~2000 and carvacrol at MIC, with a reduction of approximately 4 log CFU/mL observed at the end of Day 3. The results obtained in this study highlight the potential of combined treatment involving phage biocontrol and carvacrol as a new alternative method to reduce E. coli contamination in minimally processed ready-to-eat foods.

Investigating the Effects of Time and Temperature on the Growth of Escherichia coli O157:H7 and Listeria monocytogenes in Raw Cow's Milk Based on Simulated Consumer Food Handling Practices.

Consumption of raw cow's milk (RCM) is increasing in popularity in developed countries despite the associated foodborne disease risks. While previous research has focused on consumer motivations for drinking RCM, there is limited research on how consumer handling practices may impact the microbiological safety of RCM. In this study, consumer handling practices associated with transport, storage, and freezing and thawing were simulated to investigate the impact of time and temperature variables on the concentrations of either Escherichia coli O157:H7 or Listeria monocytogenes in RCM. We found that the type of storage during simulated transport had a large (η2 = 0.70) and significant (p < 0.001) effect on both pathogens. The refrigeration temperature also had a large (η2 = 0.43) and significant (p < 0.001) effect on both pathogens during refrigerated storage. The interaction between pathogen species and initial pathogen inoculum level had a large (η2 = 0.20) and significant (p = 0.012) effect on the concentration of the pathogens during ambient temperature storage. We found that freezing and thawing practices did not have a significant effect on the pathogens (p > 0.05). However, we were able to recover L. monocytogenes, but not E. coli O157:H7, from RCM after freezing for 365 days. The results from this study highlight that consumer transport and storage practices can have significant effects on the growth of E. coli O157:H7 and L. monocytogenes in RCM. Consumer food handling practices should be considered when developing public health strategies aimed at reducing the risks of RCM consumption.

Phage inhibition of Escherichia coli in ultrahigh-temperature-treated and raw milk.

Escherichia coli can contaminate raw milk during the milking process or via environmental contamination in milk-processing facilities. Three bacteriophages, designated EC6, EC9, and EC11, were investigated for their ability to inhibit the growth of three strains of E. coli in ultrahigh-temperature (UHT) treated and raw bovine milk. A cocktail of the three phages completely inhibited E. coli ATCC 25922 and E. coli O127:H6 in UHT milk at 25 °C and under refrigeration temperatures (5-9 °C). The phage cocktail produced similar results in raw milk; however, E. coli ATCC 25922 and O127:H6 in raw milk controls also declined to below the level of detection at both temperatures. This observation indicated that competition by the raw milk microbiota might have contributed to the decline in viable E. coli cells. A cocktail containing EC6 and EC9 completely inhibited E. coli O5:H-, an enterohemorrhagic strain, in UHT milk at both temperatures. In raw milk, the phage cocktail initially inhibited growth of E. coli O5:H- but regrowth occurred following incubation for 9 h at 25 °C and 144 h at 5-9 °C. In contrast to the other E. coli strains, O5:H- was not inhibited in the raw milk controls. This study demonstrates that bacteriophages are effective biocontrol agents against E. coli host strains in UHT and raw bovine milk at various storage temperatures.

A pilot study of the microbiological quality of culturally diverse, ready-to-eat foods from selected retail establishments in Melbourne, Australia.

In recent years, there has been an increasing number of foodborne outbreaks linked to the consumption of culturally diverse foods. This appears to be because of the increasing quantity of culturally diverse foods available and a preference to store these foods, some of which are considered potentially hazardous, at ambient temperature. This practice may contravene temperature requirements defined by the Food Standards Code. A lack of understanding of the hazardous nature of some culturally prepared foods also poses difficulties in applying the Australian food safety legislation by regulators. This pilot study examined the normal microbiota of four culturally diverse foods: nem chua, che dau trang, kueh talam, and bánh tét nhân man, which are traditionally stored and consumed at ambient temperature. Challenge testing was conducted to investigate the ability of these foods to support the growth of foodborne bacterial pathogens. Two of the products (kueh talam and che dau) were found to be microbiologically unsatisfactory because of the high standard plate counts. Challenge testing indicated that kueh talam, che dau, and bánh tét nhân man were able to support the growth of Bacillus cereus, Escherichia coli, Staphylococcus aureus, and Salmonella (1-2 log increases over 6 hours at 25 degrees C), suggesting that these foods may require temperature control during storage. However, nem chua was unable to support the growth of test bacteria, probably because of its acidic nature (pH 4.5), suggesting that ambient storage of this food may be safe. This study provided some preliminary evidence to support the need for further sampling and challenge testing of these products.