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Resistance of Listeria monocytogenes, Escherichia coli O157:H7 and Campylobacter jejuni after exposure to repetitive cycles of mild bactericidal treatments

While maintaining nutritional and sensorial attributes of fresh foods mild processing technologies generally deliver microbiologically perishable food products. Currently little information exists on possible increase in the resistance of pathogens after repetitive exposure to mild (sub-lethal) treatments. Multiple strain-cocktails of Listeria monocytogenes, Escherichia coli O157:H7 and Campylobacter jejuni were exposed to 20 consecutive cycles of sub-lethal inactivation by three different techniques. Used techniques comprised inactivation with lactic acid (LA), chlorine dioxide (ClO(2)) and intense light pulses (ILP). Results showed that the selection of resistant cells was both species and technique dependent. While repetitive cycles of ClO(2) treatment did not result in increased resistance, repetitive inactivation with LA yielded L. monocytogenes culture of higher resistance in comparison to the parental culture. The increased resistance, expressed as decreased level of reduction in bacterial counts in subsequent inactivation cycles, was also observed with ILP for both L. monocytogenes and E. coli O157:H7 strains. Visual trend observations were confirmed through statistical linear regression analysis. No such effects were noted for C. jejuni which became undetectable after first 2-5 cycles. Current findings indicate the ability of foodborne pathogens to adapt to mild bactericidal treatments creating new challenges in risk assessment and more specifically in hazard analysis.

Survival of Campylobacter jejuni on raw chicken legs packed in high-oxygen or high-carbon dioxide atmosphere after the decontamination with lactic acid/sodium lactate buffer.

Quantitative risk assessment studies performed elsewhere showed the importance of reducing counts of Campylobacter jejuni on chicken carcasses for decrease of incidence of human campylobacteriosis. The current study indicated that 1.8 log CFU/g reduction of inoculated C. jejuni (6 log CFU/g) can be achieved by decontamination with lactic acid buffered with sodium lacatate (LA/NaLA, 10% w/v, pH 3.0). Subsequent packaging under modified atmosphere of 80% O(2)/20%N(2) resulted in additional reduction of approximately 1.2 log CFU/g. These results were confirmed in naturally contaminated samples (2-3 log CFU/g) resulting in immediate reduction of present C. jejuni under the limit of enumeration (1 log CFU/g). However, enrichment showed presence of C. jejuni in 10g of sample. Under 80% O(2) LA/NaLA treated C. jejuni remained detectable per 10g until day 7, after which no positive samples were found until the end of the two-weeks storage. Under 80% CO(2) LA/NaLA treated C. jejuni remained fluctuating at 10 CFU/g until the end of two-weeks storage. Control cells were reduced by approx. 1.5 log CFU/g during storage under 80% O(2)/20% N(2), whereas no reduction was observed under 80% CO(2)/20% N(2). The present study showed the potential of buffered lactic acid and high-O(2) MAP to reduce C. jejuni both on inoculated and naturally contaminated samples. The immediate effect of decontamination was further extended by additive, not synergistic, effect of 80% O(2), suggesting the practical value of the tested concept in combating C. jejuni on chicken carcasses.

Intracellular pH as an indicator of viability and resuscitation of Campylobacter jejuni after decontamination with lactic acid

The aim of the study was to determine intracellular pH (pH(i)) as an indicator of the physiological state of two Campylobacter jejuni strains (603 and 608) at the single cell level after bactericidal treatment with lactic acid (3% v/v lactic acid, pH 4.0, 0.85% w/v NaCl) and during recovery and survival using Fluorescence Ratio Imaging Microscopy (FRIM). After exposure to lactic acid solution a decline in pH(i) to 5.5 (FRIM detection limit) was observed in the majority of cells (75-100%) within 2 min. The enumeration data revealed that after 2 min of lactic acid exposure, approx. 90% of the initial population became unculturable. In the following 10 min of exposure, a further decrease in the cell count was observed resulting in 3.53 and 3.21 log CFU/ml reduction of culturable cells at the end of the treatment. On the contrary, the FRIM results revealed that the subpopulations with pH(i)>5.5 increased between 2 and 12 min of exposure to lactic acid. Removing the acid stress and incubating the cells suspension under the more favourable conditions resulted in an immediate increase in cell population with pH(i)>pH(ex) for both C. jejuni strains. Further 24 h incubation at 37 degrees C resulted in increased pH(i) and colony count (recovery study). On the contrary, 24 h incubation at suboptimal temperature of 4 degrees C, showed pH(i) decrease to pH(ex)=6.0 (no pH gradient) in the whole population of C. jejuni cells. Rather than dying, cells exposed for longer time (72 and 120 h) to 4 degrees C increased the subpopulation of the cells with positive pH gradient, mostly comprised of the cells with DeltapH>0.5, indicating the ability of C. jejuni cells to regulate their metabolic activity under suboptimal conditions.

Performance of a growth-no growth model for Listeria monocytogenes developed for mayonnaise-based salads: influence of strain variability, food matrix, inoculation level, and presence of sorbic and benzoic acid.

A previously developed growth-no growth model for Listeria monocytogenes, based on nutrient broth data and describing the influence of water activity (a(w)), pH, and acetic acid concentrations, was validated (i) for a variety of L. monocytogenes strains and (ii) in a laboratory-made, mayonnaise-based surimi salad (as an example of a mayonnaise-based salad). In these challenge tests, the influence of the inoculation level was tested as well. Also, the influence of chemical preservatives on the growth probability of L. monocytogenes in mayonnaise-based salads was determined. To evaluate the growth-no growth model performance on the validation data, four quantitative criteria are determined: concordance index, % correct predictions, % fail-dangerous, and % fail-safe. First, the growth probability of 11 L. monocytogenes strains, not used for model development, was assessed in nutrient broth under conditions within the interpolation region. Experimental results were compared with model predictions. Second, the growth-no growth model was assessed in a laboratory-made, sterile, mayonnaise-based surimi salad to identify a possible model completeness error related to the food matrix, making use of the above-mentioned validation criteria. Finally, the effect on L. monocytogenes of common chemical preservatives (sorbic and benzoic acid) at different concentrations under conditions typical of mayonnaise-based salads was determined. The study showed that the growth-no growth zone was properly predicted and consistent for all L. monocytogenes strains. A larger prediction error was observed under conditions within the transition zone between growth-no growth. However, in all cases, the classification between no growth (P = 0) and any growth (P > 0) occurred properly, which is most important for the food industry, where outgrowth needs to be prevented in all instances. The results in the sterile mayonnaise-based salad showed again that the growth-no growth zone was well predicted but that also, in real food systems, a transition zone between growth and no growth exists. This became even more obvious for lower inoculation levels. The maximum-allowed concentration of benzoic and sorbic acid in mayonnaise-based salads, according to the European Union legislation, eliminated the growth of L. monocytogenes. Concentrations of 600 and 300 ppm were already sufficient to inhibit growth at 7 and 4 degrees C, respectively, under conditions associated with mayonnaise-based salads (pH 5.6; a(w), 0.985).

Survival of lactic acid and chlorine dioxide treated Campylobacter jejuni under suboptimal conditions of pH, temperature and modified atmosphere

As mild decontamination treatments are gaining more and more interest due to increased consumer demands for fresh foods, it is of great importance to establish the influence of decontamination treatments on the subsequent bacterial behaviour under suboptimal storage conditions. For this purpose Campylobacter jejuni cells treated with lactic acid (LA, 3% lactic acid, pH 4.0, 2 min) or chlorine dioxide (ClO(2), 20 ppm, 2 min) were inoculated in Bolton broth (pH 6.0) and incubated under 80% O(2)/20% N(2), 80% CO(2)/20% N(2), air or micro-aerophilic (10% CO(2)/85% N(2)/5% O(2)) atmosphere, at 4 degrees C during 7 days. Treatment with water served as a control. The most suppressive atmosphere for the survival of C. jejuni was O(2)-rich atmosphere, followed by air, micro-aerophilic and CO(2)-rich atmosphere. The survival of C. jejuni was dependent on the type of initial decontamination treatment, with water treated cells showing the greatest survival followed by LA and ClO(2) treated cells. Intracellular pH (pH(i)) of individual C. jejuni cells was determined using Fluorescence Ratio Imaging Microscopy (FRIM). At all tested conditions, different subpopulation of the cells could be distinguished based on their pH(i) values. The pH(i) response was independent on the surrounding atmosphere since similar distribution of the subpopulations was observed for all tested atmospheres. However, the pH(i) response was dependent on the initial decontamination treatment. The investigation of intracellular parameters gave an insight into pathogen behaviour under stressful conditions at intracellular level. The results obtained in this study highlighted the importance of combining decontamination technologies with subsequent preservation techniques to the control survival and growth of foodborne pathogens.

Growth of Escherichia coli O157:H7 and Listeria monocytogenes with prior resistance to intense pulsed light and lactic acid

Previous study showed that repetitive mild decontamination treatments with intense light pulses (ILP) and lactic acid (LA) can induce increased resistance in surviving pathogenic cells. Research has evaluated the potential of increased resistance to enhance the persistence of resistant variants of Listeria monocytogenes and Escherichia coli O157:H7 under suboptimal growth conditions. Growth of resistant variants and parental strains was determined by optical density (OD) measurements in nutrient broths with different pH values and NaCl concentration, at low temperature. The real lag phase was calculated, and results indicated that intense light pulses (ILP) resistant variants needed longer time to initiate growth compared to their parental strains, for both L. monocytogenes and E. coli O157:H7 when incubated at 7 °C and 10 °C, respectively. These selected variants were of the similar resistance towards heat and low pH (no cross-tolerance). Nevertheless, lactic acid (LA) resistant variant of L. monocytogenes was cross-protected when exposed to low pH, but not when treated with heat.

Intracellular pH in Campylobacter jejuni When Treated with Aqueous Chlorine Dioxide

The aim of this study was to investigate the response of Campylobacter jejuni at single-cell level when exposed to different concentrations of chlorine dioxide (ClO(2)). The parameter of choice, intracellular pH (pH(i)), was determined by using fluorescence ratio imaging microscopy with a pH-sensitive, ratiometric 5(6)-carboxyfluorescein diacetate succinimidyl ester probe. In addition, the culturability expressed in colony counts was determined. Our results revealed that several subpopulations with different physiological states, as judged by their pH(i), were created by ClO(2) treatment. The greater the concentration of ClO(2), the smaller the subpopulation of healthy cells with pH(i) > 6.8 and the smaller the colony count as determined on nonselective agar plates. ClO(2) at concentrations (60 ppm) induced injuries that resulted in complete loss of culturability and adversely affected the ability to resuscitate under subsequent more favorable conditions. The presence of injured cells in food could present a risk for public health. Additional hurdles have to be included in food preservation to suppress the survival and recovery of injured cells.

Analysis of Intracellular pH in Escherichia coli O157:H7 to Determine the Effect of Chlorine Dioxide Decontamination

The aim of this study was to investigate the response of Escherichia coli O157:H7 when exposed to different concentrations of sanitation agent chlorine dioxide (ClO2) by determining intracellular pH (pHi). For this purpose, fluorescence ratio imaging microscopy was used together with pH-sensitive, ratiometric green fluorescent protein that was introduced in E. coli O157:H7 cells. Along with pHi, colony counts were determined during the treatment with ClO2. Results revealed several post-treatment subpopulations with different physiological states, as judged by their pHi. The fraction of cells with no pH gradient increased, and the colony count decreased as the concentration of ClO2 increased.

Organic and conventional milk – insight on potential differences

Purpose The purpose of this paper is to investigate if there is a difference in hygiene parameters of raw milk produced in organic and conventional farm of similar size. In parallel, the aim was to determine if there are differences in pasteurized organic and conventional milk samples delivered on the market. Design/methodology/approach Raw milk samples were analyzed for aerobic colony count (ACC), somatic cell count (SCC), acidity, temperature, fat and protein content. On the other side, final products of organic and conventional pasteurized milk with 2.8 percent declared milk fat were analyzed for Raman spectroscopy, color change and sensorial difference. Findings Results of raw milk analysis showed statistically significant differences in fat content, SCC, acidity, temperature and ACC (p<0.05). It is of note that ACC for organic milk were lower for approx. 1 log CFU/ml compared to conventional milk samples. Pasteurized organic milk samples had a significantly higher L* value than those samples originating from conventional farms, indicating that organic is “more white” compared to conventional milk. According to the results of triangle test, with 95 percent confidence no more than 10 percent of the population is able to detect a difference. Research limitations/implications A limitation of this research is the fact that good veterinary practices at farms, namely, animal health and adequate usage of medicine for treating the animals, animal welfare and animal feeding were not analyzed. Originality/value This study analyzed potential differences in organic and conventional milk at two important production stages of the milk chain – at receipt at dairy plant (raw milk) and perceived by consumers (final product).