Antonia S. Gounadaki

Study of the effect of lethal and sublethal pH and aw stresses on the inactivation or growth of Listeria monocytogenes and Salmonella Typhimurium

During food processing, microorganisms are commonly exposed to multiple sublethal or lethal stresses (commonly a(w), pH) sequentially or simultaneously. The objectives of the present study were: (i) to comparatively evaluate the survival of Listeria monocytogenes and Salmonella Typhimurium in lethal acid (pH 4.0 and 4.5 with lactic acid) or osmotic conditions (15 and 20% NaCl), applied singly, sequentially (pH then NaCl or NaCl then pH), or simultaneously at 5 and 10 degrees C; and, (ii) to quantify the effect of osmotic shifts at pH 7.0, 6.0 or 5.0 (adjusted with lactic acid) on the lag phase and growth rate of L. monocytogenes at 10 degrees C. In sequential lethal stress applications, the second stress was applied 2 or 3 days after the first for Salmonella and L. monocytogenes, respectively. Acid tolerance of L. monocytogenes was higher than osmotic tolerance and the opposite was observed for Salmonella. Higher inactivation was observed after exposure to pH 4.0 compared to pH 4.5 as well as after exposure to 20% NaCl compared to 15% NaCl. Exposure to stresses sequentially resulted in faster (P<0.05) reductions than the exposure to single or double stresses applied simultaneously. The pH then NaCl sequence was more detrimental for pathogens than the reverse sequence. Incubation temperature (5 and 10 degrees C) did not show any profound (P<0.05) effect on microbial inactivation. When L. monocytogenes was incubated at a(w) 0.930 or 0.995 at 30 degrees C, then the lag phase increased both in subsequent osmotic downshift and upshift, respectively, at 10 degrees C. Shorter lag phase and higher ability to initiate growth at lower a(w) was observed after pre-adaptation at pH 6.0 or 5.0 compared to neutral pH. The results may contribute to the review of critical limits in low pH (with lactic acid) and water activity products, considering the risk of L. monocytogenes and Salmonella survival. In addition, the present indications may address the points in processing where stricter sanitation procedures should be applied in order to minimize the risk of survivors.

Efficiency of different sanitation methods on Listeria monocytogenes biofilms formed under various environmental conditions

The resistance of Listeria monocytogenes biofilms formed under food processing conditions, against various sanitizing agents and disinfection procedures was evaluated in the present study. The first sanitation procedure included biofilm formation on stainless steel coupons (SS) placed in tryptic soy broth supplemented with 0.6% yeast extract (TSBYE) of various concentrations of NaCl (0.5, 7.5 and 9.5%) at different temperatures (5 and 20 °C). The biofilms formed were exposed to warm (60 °C) water for 20 min, or to peroxyacetic acid (2% PAA) for 1, 2, 3 and 6 min. Treatment with warm water caused no significant (P ≥ 0.05) reductions in the attached populations. Conversely, surviving bacteria on SS coupons decreased as the exposure time to 2% PAA increased and could not be detected by culture after 6 min of exposure. Biofilms formed at 20°C were more resistant to PAA than biofilms formed at 5 °C. Salt concentration in the growth medium had no marked impact on the resistance to PAA. The second sanitation procedure included biofilm formation of nonadapted (NA) and acid-adapted (AA) cells in TSBYE of pH 5.0 and 7.0 (i.e., NA-5.0, NA-7.0 and AA-5.0, AA-7.0) at 4 °C. Coupons bearing attached cells of L. monocytogenes were periodically exposed to chlorine (0.465% Cl(-)), quaternary ammonium compound (1% QAC) and 2% PAA. The resistance of attached cells to QAC, PAA and Cl(-) followed the order: AA-5.0>NA-7.0 ≥ AA-7.0>NA-5.0. The most effective sanitizer was QAC followed by PAA and Cl(-). The results can lead to the development of efficient sanitation strategies in order to eliminate L. monocytogenes from the processing environment. Furthermore, such results may explain the presence of L. monocytogenes after sanitation as a result of cell attachment history.

Listeria monocytogenes Attachment to and Detachment from Stainless Steel Surfaces in a Simulated Dairy Processing Environment

ABSTRACT The presence of pathogens in dairy products is often associated with contamination via bacteria attached to food-processing equipment, especially from areas where cleaning/sanitation is difficult. In this study, the attachment of Listeria monocytogenes on stainless steel (SS), followed by detachment and growth in foods, was evaluated under conditions simulating a dairy processing environment. Initially, SS coupons were immersed in milk, vanilla custard, and yogurt inoculated with the pathogen (107 CFU/ml or CFU/g) and incubated at two temperatures (5 and 20°C) for 7 days. By the end of incubation, cells were mechanically detached from coupons and used to inoculate freshly pasteurized milk which was subsequently stored at 5°C for 20 days. The suspended cells in all three products in which SS coupons were immersed were also used to inoculate freshly pasteurized milk (5°C for 20 days). When SS coupons were immersed in milk, shorter lag phases were obtained for detached than for planktonically grown cells, regardless of the preincubation temperature (5 or 20°C). The opposite was observed when custard incubated at 20°C was used to prepare the two types of inocula. However, in this case, a significant increase in growth rate was also evident when the inoculum was derived from detached cells. In another parallel study, while L. monocytogenes was not detectable on SS coupons after 7 days of incubation (at 5°C) in inoculated yogurt, marked detachment and growth were observed when these coupons were subsequently transferred and incubated at 5°C in fresh milk or/and custard. Overall, the results obtained extend our knowledge on the risk related to contamination of dairy products with detached L. monocytogenes cells.

Organic acids for control of Salmonella in different feed materials

BackgroundSalmonella control in animal feed is important in order to protect animal and public health. Organic acids is one of the control measures used for treatment of Salmonella contaminated feed or feed ingredients. In the present study, the efficacy of formic acid (FA) and different blends of FA, propionic acid (PA) and sodium formate (SF) was investigated. Four Salmonella strains isolated from feed were assayed for their acid tolerance. Also, the effect of lower temperatures (5°C and 15°C) compared to room temperature was investigated in rape seed and soybean meal.ResultsThe efficacy of acid treatments varied significantly between different feed materials. The strongest reduction was seen in pelleted and compound mash feed (2.5 log10 reduction) followed by rapeseed meal (1 log10 reduction) after 5 days exposure. However, in soybean meal the acid effects were limited (less than 0.5 log10 reduction) even after several weeks’ exposure. In all experiments the survival curves showed a concave shape, with a fast initial death phase followed by reduction at a slower rate during the remaining time of the experiment.No difference in Salmonella reduction was observed between FA and a blend of FA and PA, whereas a commercial blend of FA and SF (Amasil) was slightly more efficacious (0.5-1 log10 reduction) than a blend of FA and PA (Luprocid) in compound mash feed. The Salmonella Infantis strain was found to be the most acid tolerant strain followed by, S. Putten, S. Senftenberg and S. Typhimurium. The tolerance of the S. Infantis strain compared with the S. Typhimurium strain was statistically significant (p<0.05). The lethal effect of FA on the S. Typhimurium strain and the S. Infantis strain was lower at 5°C and 15°C compared to room temperatures.ConclusionsAcid treatment of Salmonella in feed is a matter of reducing the number of viable bacterial cells rather than eliminating the organism. Recommendations on the use of acids for controlling Salmonella in feed should take into account the relative efficacy of acid treatment in different feed materials, the variation in acid tolerance between different Salmonella strains, and the treatment temperature.

Effect of wine-based marinades on the behavior of Salmonella Typhimurium and background flora in beef fillets

The aim of this study was to evaluate the wine-based marinades to control the survival of acid-adapted and non-adapted Salmonella Typhimurium and background flora of fresh beef stored aerobically or under modified atmosphere. Beef slices were inoculated with a 3-strain cocktail of acid-adapted or non-adapted Salmonella Typhimurium strains DT 193, 4/74 and DSM 554 and marinated by immersion in wine (W) or wine supplemented with 0.3% thyme essential oil (WEO), for 12h at 4°C. Marinated slices were then stored under air or modified atmosphere conditions at 5°C. S. Typhimurium and background flora were followed for a 19-day period of storage. S. Typhimurium individual strains were monitored by pulsed field gel electrophoresis. Marination with wine significantly (P<0.05) reduced the background flora compared to the control (non-marinated). Furthermore, immersion of fillets in W or WEO marinades for 12h significantly (P<0.05) reduced the levels of S. Typhimurium compared to the non-marinated (control) samples by 1.1 and 1.4logCFU/g or 2.0 and 1.9logCFU/g for acid-adapted and non-adapted cells, respectively. Acid-adapted cells were more susceptible (P<0.05) to the addition of thyme essential oil in the wine marinade. The epidemic multi-drug resistant DT 193, the 4/74 and DSM 554 strains survived marination (for both W and WEO) and were detected at about similar proportions as revealed by PFGE results. Present results indicate that wine-based marinades are efficient, from a safety and shelf life stand point, in reducing pathogens levels as well as the background beef flora.

Adaptive acid tolerance response of Listeria monocytogenes strains under planktonic and immobilized growth conditions

The acid resistance of Listeria monocytogenes was evaluated: (i) after short (shock) or long-term (adaptation during growth) exposure to reduced (5.5) or neutral (7.2) pH in a liquid (broth) medium or on a solid surface (agar), and (ii) after growth on the surface of ham and turkey slices or in homogenates of these products. Three L. monocytogenes strains (serotypes 1/2a, 1/2b and 4b) were individually inoculated at: (i) 10(4)-10(5)CFU/ml in tryptic soy broth with 0.6% yeast extract (TSBYE) or on tryptic soy agar with 0.6% yeast extract (TSAYE) at pH 7.2 with 1% (+G) or without (-G) glucose of or TSBYE and TSAYE with 0.25% glucose at pH 5.5 (lactic acid) and incubated at 20°C, and (ii) 10(2)-10(3)CFU/cm(2) on ham and turkey slices (pH 6.39-6.42; formulated with potassium lactate and sodium diacetate) or in their homogenates (1:4 and 1:9; representing viscous [slurry] and liquid residues [purge], respectively), and stored at 10°C. The acid resistance of each strain was assessed in TSBYE of pH 3.5 (lactic acid) for strains growing in broth or on agar surfaces, and in TSBYE of pH 1.5 (HCl) for strains growing on ham and turkey slices or in their homogenates. Habituation at pH 5.5 for 3 or 24h at 20°C increased acid (pH 3.5) resistance of all strains compared to the control (pH 7.2). Cells grown on the surface of TSAYE-G (pH 7.2 or 5.5) showed higher resistance than cells grown in broth (TSBYE-G), whereas the opposite was observed for cells grown on TSAYE + G or in TSBYE + G. Growth of L. monocytogenes on meat product slices was markedly slower than in homogenates. Pathogen reductions following exposure to pH 1.5, after 10 and 27days of storage were strain-dependent and in the ranges of 0.5-2.5, 1.3-4.5 and 4.0-7.6 log units for cells grown on product slices in 1:4 and 1:9 homogenates, respectively. The results suggest that L. monocytogenes cells growing on food surfaces or in viscous matrices may show higher resistance to lethal acid conditions than cells growing in liquid substrates.

Modeling the effect of abrupt acid and osmotic shifts within the growth region and across growth boundaries on adaptation and growth of Listeria monocytogenes.

ABSTRACT This study aims to model the effects of acid and osmotic shifts on the intermediate lag time of Listeria monocytogenes at 10°C in a growth medium. The model was developed from data from a previous study (C. I. A. Belessi, Y. Le Marc, S. I. Merkouri, A. S. Gounadaki, S. Schvartzman, K. Jordan, E. H. Drosinos, and P. N. Skandamis, submitted for publication) on the effects of osmotic and pH shifts on the kinetics of L. monocytogenes. The predictive ability of the model was assessed on new data in milk. The effects of shifts were modeled through the dependence of the parameter h0 (“work to be done” prior to growth) induced on the magnitude of the shift and/or the stringency of the new environmental conditions. For shifts across the boundary, the lag time was found to be affected by the length of time for which the microorganisms were kept at growth-inhibiting conditions. The predicted concentrations of L. monocytogenes in milk were overestimated when the effects of this shift were not taken into account. The model proved to be suitable to describe the effects of osmotic and acid shifts observed both within the growth domain and across the growth boundaries of L. monocytogenes.

Evaluation of growth/no growth interface of Listeria monocytogenes growing on stainless steel surfaces, detached from biofilms or in suspension, in response to pH and NaCl.

The present study aimed to describe the growth/no growth interface of Listeria monocytogenes at three potential states of growth in industrial environments, namely attached, (Att), detached (Det) from a biofilm, or in a planktonic state (suspended; Plan). A 3-strain composite of L. monocytogenes cells was left to colonize stainless steel (SS) surfaces in tryptic soy broth supplemented with 0.6% yeast extract (TSBYE) at 20 °C for 72 h and then transferred to TSBYE at 30 different pH and NaCl concentrations, which were renewed every two days during incubation at 10 °C. Survival of attached population was observed at optimal conditions (pH 7.2, a(w) 0.996), whereas at 4.5-8.0% salt and/or pH<6.0, reduction of attached population on SS surfaces was observed. PFGE patterns showed that 91% of the cells colonizing the SS coupons after 30 days, at any pH and a(w) conditions, belonged to a single strain. Furthermore, the change in the probability of a single cell to initiate growth (P(in)) over time, as well as the number of cells needed (CN) for growth initiation of planktonically growing Plan and Det L. monocytogenes cells were evaluated based on MPN Tables. An ordinary logistic regression model was also used to describe the growth/no growth interface of varying inoculation levels (from <10 to 10(4)CFU/ml) of Plan and Det cells in response to pH and a(w). Although both cell types demonstrated similar growth limits at populations of 10(2)-10(4)CFU/ml, higher numbers of Det than Plan cells were needed (CN) in order to initiate growth at low a(w) and pH. Individual Plan cells reached higher maximum levels of probability of growth initiation (P(max)) and had shorter times to reach P(max)/2 (t(au)), compared to their Det counterparts. Data on growth potential of cells in suspension, attached or detached status, may assist in ranking the risk from different sources of contamination. In addition, they may establish the link between the behavior of L. monocytogenes in foods and its origin from the processing plant. The latter link is important component of biotraceability.

Listeria monocytogenes and Listeriosis

In 2003, the Hellenic Food Authority initiated a monitoring program for biological and chemical hazards in Ready-to-Eat (RTE) food products in Greece. In total, 605 products from diverse catering enterprises and retailers in Athens, Patra, Thessaloniki, andCrete were examined for the presence of pathogenic and hygiene indicator bacteria (e.g. coliforms). Detailed inspections of premises were also performed to evaluate the hygiene level of enterprises offering RTE foods, as well as their compliance to Good Hygiene Practices. Only 0.7% of analyzed samples were found positive for listeria. Different RTE meat products have been recalled for possible listeria contamination since that initial study, showing a sharp increase in the number of contaminated products (i.e., 8.5 tons) in 2005. Five deaths were also been recorded, resulting from the consumption of bacon that was presumed contaminated with listeria.

Adaptive growth responses of Listeria monocytogenes to acid and osmotic shifts above and across the growth boundaries.

The effect of acid and osmotic shifts on the growth of Listeria monocytogenes was evaluated at 10°C. Two types of shifts were tested: (i) within the range of pH and water activity (a(w)) levels that allow growth of L. monocytogenes and (ii) after habituation at no-growth conditions back to growth-permitting conditions. A L. monocytogenes cheese isolate, with high survival capacity during cheesemaking, was inoculated (10(2) CFU/ml) in tryptic soy broth supplemented with 0.6% yeast extract at six pH levels (5.1 to 7.2; adjusted with lactic acid) and 0.5% NaCl (a(w) 0.995), or four a(w) levels (0.995 to 0.93, adjusted with 0.5 to 10.5% NaCl) at pH 7.2 and grown to early stationary phase. L. monocytogenes was then shifted (at 10(2) CFU/ml) to each of the aforementioned growth-permitting pH and a(w) levels and incubated at 10°C. Shifts from no-growth to growth-permitting conditions were carried out by transferring L. monocytogenes habituated at pH 4.9 or a(w) 0.90 (12.5% NaCl) for 1, 5, and 10 days to all pH and a(w) levels permitting growth. Reducing a(w) or pH at different levels in the range of 0.995 to 0.93 and 7.2 to 5.1, respectively, decreased the maximum specific growth rate of L. monocytogenes. The lag time of the organism increased with all osmotic downshifts, as well as by the reduction of pH to 5.1. Conversely, any type of shift within pH 5.5 to 7.2 did not markedly affect the lag times of L. monocytogenes. The longer the cells were incubated at no-growth a(w) (0.90), the faster they initiated growth subsequently, suggesting adaptation to osmotic stress. Conversely, extended habituation at pH 4.9 had the opposite effect on subsequent growth of L. monocytogenes, possibly due to cell injury. These results suggest that there is an adaptation or injury rate induced at conditions inhibiting the growth of the pathogen. Thus, quantifying adaptation phenomena under growth-limiting environments, such as in fermented dairy and meat products or products preserved in brine, is essential for reliable growth simulations of L monocytogenes during transportation and storage of foods.