The protective effect of food matrices on Listeria lytic bacteriophage P100 application towards high pressure processing.

The application of lytic phages as biocontrol agents is emerging as a promising strategy towards elimination or reduction of foodborne pathogens in a variety of food products. This technology is particularly advantageous for minimally processed and ready-to-eat (RTE) foods. In this study, the potential use of Listex™ P100 combined with high hydrostatic pressure (HPP), to enhance the control of Listeria monocytogenes in food was evaluated. For that, the effect of three pressures (200, 300 or 400 MPa; 5 min, 10 °C) on phage P100 stability was tested when inoculated in six different matrices: phosphate buffered saline (PBS, pH 7.4); apple juice (pH 3.41); orange/carrot nectar (pH 3.54); UHT whole milk (pH 6.73); and, two traditional Portuguese fermented products, "Serra da Estrela" cheese (pH 5.66) and "Alheira", a meat sausage (pH 6.07). The results showed that treatment at 400 MPa reduced phage titres to below the detection level in all matrices, whereas at milder pressures the survival of the phage was matrix dependent. "Alheira", "Serra da Estrela" cheese and UHT whole milk were shown to be baroprotective matrices that support phage P100 application in HHP up to 300 MPa; however, an accentuated phage inactivation was observed in apple and orange/carrot nectar, which may be related to the acidic pH values of these matrices. The initial phage load did not affect the inactivation rate during HHP processing (300 MPa, 5 min, 10 °C) in PBS, cheese, sausage or milk matrices, and the phage titres were stable in these matrices during storage at 4 °C for 28 days for milk and 60 days for "Alheira" and "Serra da Estrela" cheese. In addition, a baroprotective effect on phage stability was observed when PBS was supplemented with reducing sugars, dextrin, casein, and tween 80. In conclusion, at mild HHP treatment, phage P100 remained active in specific matrices and seems to present potential to be added in non-thermal inactivation of L. monocytogenes.

High hydrostatic pressure effects on Listeria monocytogenes and L. innocua: Evidence for variability in inactivation behaviour and in resistance to pediocin bacHA-6111-2.

The effect of high hydrostatic pressure (HHP) on the survival of 14 strains of Listeria monocytogenes from food or clinical origins, selected to represent different pheno and genotypes, was evaluated. Stationary phase cells were submitted to 300, 400 and 500 MPa at 10 °C, for 5 min. A high variability in the resistance of L. monocytogenes to pressure was observed, and particularly two strains isolated from food were significantly more baroresistant than the rest. Strains of L. monocytogenes resistant to one or more antibiotics exhibited significantly higher levels of survival after the high pressure treatment at 400 MPa. No correlation was found between strains' origin or thermal tolerance and resistance to HHP. The suitability of two strains of L. innocua as surrogates of L. monocytogenes, was also investigated. These exhibited significantly higher sensitivities to HHP than observed for some L. monocytogenes. The antimicrobial effect of the antilisterial bacteriocin (bacHA-6111-2) increased after L. monocytogenes cells had been exposed to pressure. The data obtained underlines the importance of strain selection for studies aiming to evaluate HHP efficacy to ensure safety of HHP-treated foods.

Survival of Listeria monocytogenes with different antibiotic resistance patterns to food-associated stresses.

The ongoing rise of antibiotic resistant microbial pathogens has become one of the major public health threats worldwide. Despite all the effort and actions taken so far, a proliferation of antibiotic resistant (AR) and multi-antibiotic resistant (MAR) strains is still observed, including in foodborne pathogens. This trend has been also noted recently for isolates of Listeria monocytogenes, a species that, although remaining largely sensitive to clinically relevant antimicrobials, has been reported to develop increased tolerance to antibiotics, particularly in isolates recovered from the food-chain. In this study we compared the ability of MAR (n=8), AR (n=18) and antibiotic susceptible (AS, n=11) L. monocytogenes strains from food and clinical origin to survive to different environmental stress conditions, including temperature (58°C), acidic stress (1% v/v lactic acid, pH3.5), and osmotic stress (37% w/v NaCl). The presence of antibiotic active efflux among MAR and AR strains, and its role on L. monocytogenes tolerance to different antimicrobial compounds was also investigated, namely; hydrogen peroxide; organic acids (acetic, citric and lactic); nisin; benzalkonium chloride (BC); and, sodium nitrite. While no significant differences were observed in the survival of the 37 strains exposed to high temperature (58°C), overall the mean logarithmic reduction of clinical strains was statistically lower after acid and salt exposure than that observed for strains of food origin; but both food and clinical strains resistant to two or three antibiotics were significantly less susceptible to acid (lactic acid 1% v/v) and osmotic stresses (37% w/v NaCl) when compared to AS strains. Using the EtBr-agar Cartwheel method, it was possible to detect efflux pumps in three of the 26 MAR and AR isolates, including one control strain; the active efflux in theses isolates was proven to be associated with fluoroquinolone resistance, and possible extrusion of BC and hydrogen peroxide. The mechanisms responsible for the possible correlation between resistance to antibiotics and to acid or salt stress in L. monocytogenes have yet to be understood.