Heat-injured Cells Research Articles

Optimisation of Saccharomyces cerevisiae BRYC 501 ascospore formation and recovery for heat inactivation experiments

Why was the work done: Ascospores from Saccharomyces cerevisiae BRYC 501 are useful biological indicators for validating the pasteurisation of beer. Ascospores are formed by yeast in response to low nutrient conditions and are characterised by greater resistance to stressors including desiccation, freezing and, during pasteurisation, high temperatures. Accordingly, the sporulation temperature of yeast ascospores, their shelf life, heat resistance and recovery need to be investigated. How was the work done: The role of temperature in sporulation was investigated at 25, 27 and 30°C by measurement of the rate of sporulation over ten days. Shelf life of ascospores was established in deionised water at 1-5°C over 120 days by the measurement of heat resistance in McIlvaine's buffer. The recovery conditions of ascospores were determined using six agars after heat inactivation experiments in McIlvaine's buffer, alcohol free and lager beer. What are the main findings: The sporulation rate of S. cerevisiae ascospores was highest at 27°C, and lowest at 30°C. Heat resistance of ascospores formed at 30°C was low with a D60 value of <3 minutes, whereas at 25 and 27°C the D60 was 8.8 and 9.2 minutes. The shelf life of ascospores was 60 days after an initial 14 days of maturation at both 25 and 27°C. The variation of heat resistance for ascospores grown at 25°C was shown to be lower than those grown at 27°C. Use of yeast extract-peptone-glucose agar to recover heat injured cells was > 200% higher than with the control agar. Why is the work important: Sporulation should be performed at 25°C to produce the maximum number of spores for heat inactivation experiments. Spores can be maintained for 60 days, enabling several experiments to be performed. After heat inactivation experiments, ascospores should be recovered on YEPG agar for 10 days at 27°C

Fate of osmotically adapted and biofilm Listeria monocytogenes cells after exposure to salt, heat, and liquid smoke, mimicking the stresses induced during the processing of hot smoked fish

The study aimed to investigate the response of osmotically adapted and detached biofilm Listeria monocytogenes cells following sequential stresses that occur during the processing of hot smoking, such as heating and smoke application. Thermal resistance of L. monocytogenes was significantly affected by previous osmotic adaptation of the cells. D60oC-values of osmotically adapted L. monocytogenes cells were significantly higher than control cells. The osmotically adapted and subsequently heat-injured cells were more resistant to PALCAM and less resistant to TSAYE with 5.00% NaCl (TSAYE/NaCl) than control cells. Detached biofilm cells were more thermotolerant and less resistant to PALCAM and TSAYE/NaCl than control cells. The sequential effect of smoking against heat-treated (60 °C, 20 min) and osmotically adapted or detached L. monocytogenes biofilm cells was investigated using two liquid smoke extracts (L9 and G6). L9 led to significantly higher reductions (>3.00-Log CFU) compared to G6. The heat-treated, detached biofilm cells revealed resistance to L9, presumably due to metabolic downregulation and physical protection by the extracellular polymeric substances (EPS). These data highlight the potential of the food industry to make informed decisions for using safe heat treatments during hot smoking to effectively inactivate L. monocytogenes and maintain rigorous environmental sanitation practices to control biofilm cells.

Supercharged MPNs? Automated Determination of High-Throughput Most Probable Number (htMPN) Using Chip-Based 3D Digital PCR.

Surface plating on agar and most probable number (MPN) are the standard methods for determining bacterial viability but both have limitations. Here we present a novel cell count method, high-throughput MPN (htMPN), that uses a chip-based digital PCR instrument to accelerate and to improve the quantification of viable or sublethally injured cells. This method tracks growth of up to 20,000 individual bacterial cells on a single chip. Single cells were grown in the individual wells of the chip at their optimal temperature until the cell density was high enough to detect the fluorescent signal with cell-permeant or cell-impermeant DNA-intercalating fluorescent dyes. This method based on microfluidic devices implemented in digital PCR equipment was equivalent to surface plating in determining cell counts of Escherichia coli, Salmonella enterica serovar Typhimurium, Fructilactobacillus sanfranciscensis, Pseudomonas putida, and vegetative cells but not spores of Bacillus subtilis. Viable E. coli could be enumerated within 7 h. Culture of strict aerobes was restricted to strains that are capable of nitrate respiration; organisms requiring complex media that also contain double-stranded DNA were detected after treatment of growth media with DNase before inoculation. Our approach not only monitors the frequency distribution of bacterial growth and determines cell counts with high reliability but also detected heat-injured cells of S. Typhimurium that escaped detection by the surface plating. Overall, the method accelerates detection of viable bacterial cells, facilitates automation, and offers new possibilities for the analysis of individual bacterial cells. IMPORTANCE htMPN uses chip-based fluorescence acquisition and is a simple and compact tool for automatic viable cell enumeration with applications in microbiological research. This method applies to a wide range of anaerobic or facultative anaerobic species and improves accuracy by reducing the number of pipetting steps. In addition, the method offers an additional tool for single-cell microbiology. The single cell time-to-detection times have been used as an important criterion for the physiological state of bacterial cells after sublethal stress, and htMPNs support the acquisition of such data with an unprecedented number of cells. In particular, htMPN provides an anaerobic environment and enables a long incubation time to increase the recovery rate of sublethally injured cells. Given its reproducibility and reliability, our approach can potentially be applied to quantify viable cells in samples from environmental, clinical, or food samples to reduce the risk of underestimation of the number of viable bacterial cells.

Determination and validation of D-values for Listeria monocytogenes and Shiga toxin–producing Escherichia coli in cheese milk

Certain cheeses can be legally produced in the United States using raw milk, but they must be aged for at least 60 d to reduce pathogen risks. However, some varieties, even when aged for 60 d, have been shown to support growth of Listeria monocytogenes or survival of Shiga toxin-producing Escherichia coli (STEC). Thermization, as a subpasteurization heat treatment, has been proposed as a control to reduce the risk of pathogens in raw cheese milk while retaining some quality attributes in the cheese. However, the temperature and time combinations needed to enhance safety have not been well characterized. The objective of this research was to determine and validate decimal reduction values (D-values) for L. monocytogenes and STEC at thermization temperatures 65.6, 62.8, and 60.0°C; a D-value at 57.2°C was also determined for L. monocytogenes only. Nonhomogenized, pasteurized whole-milk samples (1 mL) were inoculated with 8-log cfu/mL L. monocytogenes or STEC (5- or 7-strain mixtures, respectively), vacuum-sealed in moisture-impermeable pouches, and heated via water bath submersion. Duplicate samples were removed at appropriate intervals and immediately cooled in an ice bath. Surviving bacteria were enumerated on modified Oxford or sorbitol MacConkey overlaid with tryptic soy agar to aid in the recovery of heat-injured cells. Duplicate trials were conducted, and survival data were used to calculate thermal inactivation rates. D65.6°C-, D62.8°C-, and D60.0°C-values of 17.1 and 7.2, 33.8 and 16.9, and 146.6 and 60.0 s were found for L. monocytogenes and STEC, respectively, and a D57.2°C-value of 909.1 s was determined for L. monocytogenes. Triplicate validation trials were conducted for each test temperature using 100 mL of milk inoculated with 3 to 4 log cfu/mL of each pathogen cocktail, A 3-log reduction of each pathogen was achieved faster in larger volumes than what was predicted by D-values (D-values were fail-safe). Data were additionally compared with published results from 21 scientific studies investigating L. monocytogenes and STEC in whole milk heated to thermization temperatures (55.0-71.7°C). These data can be used to give producers of artisanal raw-milk cheese flexibility in designing thermal processes to reduce L. monocytogenes and STEC populations to levels that are not infectious to consumers.

Short communication: Evaluating the recovery potential of injured cells of Listeria innocua under product temperature-abuse conditions and passage through simulated gastrointestinal fluids

Ice cream handling and serving conditions on the consumer side may result in temperature abuse before consumption. Under some extreme conditions, even the sporadic presence of injured bacterial cells might pose a health risk due to the possibility of recovery of those cells. We conducted this investigation to evaluate the potential of injured cells of Listeria innocua to recover under ice cream temperature abuse conditions and on exposure to simulated gastrointestinal (GI) fluids. Ice cream mix samples (42% total solids), spiked with 4 log10 cfu/g of Listeria innocua, were thermally treated at 69°C for 30 min. Potential heat-injured cells were recovered in buffered Listeria broth (BLEB), followed by isolation on Listeria-specific modified Oxford agar (MOX). The ice cream mix samples, containing potentially injured cells of Listeria innocua, were followed through overnight aging (7°C), freezing (-3.3°C), and overnight hardening (-40°C) steps to obtain the final ice cream samples. To simulate temperature abuse conditions, the samples were held for 12 h at 4.4°C, followed by 30 min at room temperature (22°C); this treatment was considered the first cycle of temperature abuse. To generate a worst-case scenario, the samples were exposed to 3 such consecutive temperature abuse cycles. At the end of each cycle, direct plating was done on MOX to recover viable cells, and BLEB enrichment verified the presence of potential injured cells. In addition, the ice cream samples, containing potential injured cells, were passed through simulated GI fluids. As a first step, samples were mixed (1:1) with simulated gastric fluids (pH 1.0 and 2.0 before mixing) and held at 37°C in a shaker incubator. Samples drawn at 15, 30, and 60 min were analyzed for viable and potential injured cells. To study the effect of sequential transit through simulated intestinal fluid, a mixture of ice cream and gastric fluid (1:1) from the gastric fluid experiment above was added to simulated intestinal fluid (pH 6.8) and held at 37°C. Samples were analyzed at 30 and 360 min for viable and potential injured cells. Three trials were conducted and the samples collected in duplicates. The temperature abuse or GI fluid exposure studies did not result in the recovery of potential injured cells of Listeria innocua in the ice cream samples under the conditions tested. Exposure to gastric fluids, however, did not eliminate the potential injured cells. Further studies are necessary to understand the exact risk implications of these findings.

Detection Time Distribution of Microcolonies Formed by Individual Heat-Injured Cells of Escherichia coli.

The microcolony formation at 30℃ on an enriched minimal salts agar plates by individual Escherichia coli cells heated at 50℃ was monitored with a time-lapse shadow image analysis system, MicroBio μ3DTM AutoScanner. While the time course of microcolony count detected every half an hour for the unheated cells seemingly demonstrated a normal distribution, that for the heated cell population demonstrated totally the growth delay probably resulting from cell injury and also interestingly distributed in its rather deformed pattern with a tailing. Those patterns of the cumulative counts of appearing microcolonies during the post-heating cultivation period were expressed in three different mathematical models. This approach may be proposed as a rapid cultivation method predictable for enumeration of viable and repairable injured cells in practical use.

Variability in lag duration of Listeria monocytogenes strains in half Fraser enrichment broth after stress affects the detection efficacy using the ISO 11290-1 method

A collection of 23 Listeria monocytogenes strains of clinical and food origin was tested for their ability to recover and grow out in half Fraser enrichment broth following the ISO 11290-1:2017 protocol. Recovery of sub-lethally heat-injured cells in half Fraser broth was compared to reference cells with no stress pre-treatment. The enrichments were followed over time by plate counts and the growth parameters were estimated with the 3-phase model which described the data best. The reference cells without stress pre-treatment showed a short lag duration, which ranged from 1.4 to 2.7 h. However, significant variation in the ability to recover after 60 °C heat stress was observed among the tested strains and resulted in a lag duration from 4.7 to 15.8 h. A subset of strains was also exposed to low-temperature acid stress, and the lag duration showed to be also stress dependent. Scenario analyses and Monte Carlo simulations were carried out using the growth parameters obtained in the enrichments. This demonstrated that when starting with one cell, the detection threshold for efficient transfer of at least one cell to the secondary enrichment step, i.e. 2 log10 CFU/ml, was not reached by 11 of 23 strains tested (48%) after exposure to 60 °C heat stress. Increasing the incubation time from 24 to 26 h and the transfer volume from 0.1 to 1.0 ml can increase the average probability to transfer at least one cell to the secondary enrichment step from 79.9% to 99.0%. When optimizing enrichment procedures, it is crucial to take strain variability into account as this can have a significant impact on the detection efficacy.

Development of an Effective Two-Step Enrichment Process to Enhance Bax System Detection of Healthy and Injured Salmonella Enteritidis in Liquid Whole Egg and Egg Yolk

The BAX system for pathogen detection has been highly accurate in a variety of food products. However, false-negative results have been reported for the detection of pathogens in liquid egg products because of failed pathogen resuscitation and the existence of inhibitory components. In this study, a short-time enrichment step was used to simultaneously resuscitate the target cells to the detection level and to dilute the inhibitory components to reduce detection interference. The MP medium (BAX system) enabled faster multiplication of healthy Salmonella cells than did buffered peptone water (BPW) in tested liquid whole egg and egg yolk. However, MP failed to resuscitate heat-injured cells even after 24 h of incubation. Therefore, MP was replaced with BPW as the enrichment broth for the BAX system. However, the use of BPW for a one-step enrichment was not effective for removal of PCR inhibitors in egg yolk, and unstable detection results were obtained. To improve detection accuracy, a second step of enrichment with brain heart infusion was added. This two-step enrichment process shortened the enrichment time to 14 h and greatly increased the number of samples in which the pathogen was detected during the same enrichment time, especially in the liquid egg yolk samples. The validation study revealed 100% diagnostic accuracy of the two-step enrichment process plus the BAX system. These results indicate that a two-step enrichment process added to the BAX system can improve the detection of pathogenic Salmonella in liquid egg products.

Short communication: Entrapment of Listeria cells within air pockets of ice cream mix matrix may lead to potentially heat-injured cells

In our previous study, we observed the sporadic presence of potentially heat-injured cells of Listeria innocua in ice cream mix following a selective enrichment protocol. Although injured cells have not yet been reported to cause any disease outbreaks, it is important to understand their presence in heat-treated food matrices. In this study, we propose a possible protective role of air pockets that may help explain the sporadic presence of potentially heat-injured cells following heat treatment. Challenge studies were conducted by inoculating ice cream mix samples (42% total solids, 16.3% fat, 22.2% total sugar, and 3.4% protein) with Listeria innocua (an established surrogate) at a mean spiking level of 4.0 log cfu/g. The inoculated samples were heat-treated at 69°C for 30 min and potentially heat-injured cells were detected using buffered Listeria enrichment broth, followed by plating on modified Oxford and Rapid'LMono agars. Scanning electron microscopy and atomic force microscopy were conducted on the air-dried, spiked ice cream mix samples, before and after the thermal treatment stages. Although direct plating did not reveal any intact cells in the heat-treated ice cream mix, a more sensitive enrichment protocol was able to identify cells that were potentially heat-injured. The scanning electron micrographs showed air pockets of different sizes in the ice cream mix samples. The spiked mix samples before heat treatment showed some Listeria cells unevenly distributed in the mix matrix and some entrapped within the larger air pockets. After heat treatment, scanning electron and atomic force micrographs showed cells entrapped only within the larger air pockets. The mix matrix, however, did not show any Listeria cells. Confirmation of Listeria at all stages of analysis was done using MALDI-TOF. These observations suggest that the Listeria cells could be entrapped within the larger air pockets and thus may undergo inadequate thermal effect. This could have resulted in their detection as potentially heat-injured cells, as evident under the conditions of the experiment. These results are preliminary observations and further studies are necessary to draw conclusions and understand the true implications of these findings.

Human amniotic mesenchymal stem cells and their paracrine factors promote wound healing by inhibiting heat stress-induced skin cell apoptosis and enhancing their proliferation through activating PI3K/AKT signaling pathway

BackgroundIncreasing evidence has shown that mesenchymal stem cells (MSCs) yield a favorable therapeutic benefit for thermal burn skin wounds. Human amniotic MSCs (hAMSCs) derived from amniotic membrane have multilineage differentiation, immunosuppressive, and anti-inflammatory potential which makes them suitable for treating skin wounds. However, the exact effects of hAMSCs on the healing of thermal burn skin wounds and their potential mechanisms are not explored.MethodshAMSCs were isolated from amniotic membrane and characterized by RT-PCR, flow cytometry, immunofluorescence, and tumorigenicity test. We assessed the effects of hAMSCs and hAMSC conditional medium (CM) on wound healing in a deep second-degree burn injury model of mice. We then investigated the biological effects of hAMSCs and hAMSC-CM on the apoptosis and proliferation of heat stress-injured human keratinocytes HaCAT and dermal fibroblasts (DFL) both in vivo and in vitro. Next, we explored the underlying mechanisms by assessing PI3K/AKT and GSK3β/β-catenin signaling pathways in heat injured HaCAT and DFL cells after hAMSCs and hAMSC-CM treatments using PI3K inhibitor LY294002 and β-catenin inhibitor ICG001. Antibody array assay was used to identify the cytokines secreted by hAMSCs that may activate PI3K/AKT signaling pathway.ResultsOur results showed that hAMSCs expressed various markers of embryonic stem cells and mesenchymal stem cells and have low immunogenicity and no tumorigenicity. hAMSC and hAMSC-CM transplantation significantly promoted thermal burn wound healing by accelerating re-epithelialization with increased expression of CK19 and PCNA in vivo. hAMSCs and hAMSC-CM markedly inhibited heat stress-induced apoptosis in HaCAT and DFL cells in vitro through activation of PI3K/AKT signaling and promoted their proliferation by activating GSK3β/β-catenin signaling. Furthermore, we demonstrated that hAMSC-mediated activation of GSK3β/β-catenin signaling was dependent on PI3K/AKT signaling pathway. Antibody array assay showed that a panel of cytokines including PAI-1, C-GSF, periostin, and TIMP-1 delivered from hAMSCs may contribute to the improvement of the wound healing through activating PI3K/AKT signaling pathway.ConclusionOur results demonstrated that hAMSCs and hAMSC-CM efficiently cure heat stress-induced skin injury by inhibiting apoptosis of skin cells and promoting their proliferation through activating PI3K/AKT signaling pathway, suggesting that hAMSCs and hAMSC-CM may provide an alternative therapeutic approach for the treatment of skin injury.

Transcriptional analysis on heat resistance and recovery from thermal damage in Salmonella under high salt condition

Sodium chloride maintains osmotic pressure of living cells including bacteria. Heat treatment is common for decontamination of bacteria in food. In this study, effects of NaCl on heat resistance of Salmonella Typhimurium were investigated. After cultivation in TSB containing 0.5% (TSB), 4% (4SC) and 8% (8SC) NaCl, S. Typhimurium cells were heated at 60 °C for 20 min. Total viable counts including intact cells and injured but recoverable cells determined by the plating method using TSA of S. Typhimurium cultured in 4SC and 8SC were significantly higher than those of the cells cultured in TSB. Meanwhile, changes of gene transcription were analyzed by DNA microarray. Transcription of genes involved in the colanic acid synthesis largely increased after cultivation in 4SC and 8SC. The amount of colanic acid significantly increased in the cells cultured in 4SC compared to that in M9-glucose medium. After recovery culture for 3 h, the genes involved in the phage shock response strongly up-regulated, suggesting contribution of these gene products in recovery of heat injured cells. The outcome of this study contributes to understand the mechanism of cross protection in Salmonella.

Listeria cross contamination levels in raw ice cream mix can serve as a predictor of their potential presence as heat-injured cells

Listeriosis is a life-threatening infection caused by foods contaminated with Listeria monocytogenes. Some of the major ice cream recalls in recent years reaffirm the ability of this food-borne pathogen to survive in diverse dairy processing environments and cause cross contamination. Inspection reports revealed certain lapses in implementing adequate hygienic practices for Listeria persistence in the processing environment, leading to cross contamination of ice cream. The higher levels of cross contamination of raw ice cream mix might result in random heat-injured cells when exposed to minimum heat treatment (69°C for 30 min). These heat-injured cells could later recover under abusive storage and handling conditions and pose a health risk. Evidence about the presence of injured cells in ice cream mix may thus prove useful to establish the overall Listeria risk, which was the aim of this study. Challenge studies were conducted to evaluate the dose-dependent presence of heat-injured cells of Listeria. Ice cream mix formulations of 4 different types (36, 40, 42, and 45% total solids) were inoculated at 2.0, 3.0, and 4.0 log cfu/g levels of Listeria innocua (an established surrogate). The dose levels were selected based on a likely cross contamination on the raw side from environmental Listeria, especially due to their resident nature and growth in harborage sites. The samples were exposed to minimum heat treatment (69°C for 30 min) and the survivors, including heat-injured cells, were enumerated using standard protocols. A binary logistic regression model was fitted for evaluating the severity of risk. The influence of total solids, water activity, and pH variability were also studied on Listeria survival. The enrichment protocol, using buffered Listeria enrichment broth, followed by plating on modified oxford agar and Rapid L'mono medium, revealed the random presence of heat-injured cells in buffered Listeria enrichment broth, only at the highest dose level of 4+ logs. Any potential risk from heat-injured cells was thus limited only to the highest levels of cross contamination, irrespective of the type of the mix. Significantly, none of the pasteurized ice cream mix samples supported the recovery of any heat-injured cells of Listeria during 72 h holding at 7°C, even at the highest dose level of 4+ logs, under the conditions of experimentation. The level of cross contamination (dose) emerged as a predictor of the potential presence of heat-injured cells of Listeria exposed to minimum pasteurization treatment.

536 Glucocorticoid Prevents Apoptosis of Heat-injured Fibroblast Cells in Vitro Experiment Model

536 Glucocorticoid Prevents Apoptosis of Heat-injured Fibroblast Cells in Vitro Experiment Model

Role of Phage Shock Protein in Recovery of Heat-injured Salmonella.

Sublethally heat-injured cells of Salmonella in food can recover under favorable conditions, leading to foodborne illness. To elucidate the molecular mechanism of recovery from heat injury, the global changes in gene transcription of Salmonella Typhimurium were investigated in previous study. In this study, the functions of genes involved in phage shock response (viz., phage shock protein (psp) genes), the transcription levels of which were found in previous study to be increased during recovery from heat injury, were investigated in recovering cells. The increase in pspABCDEFG transcription levels during the recovery process was confirmed by qRT-PCR. To understand the role of psp genes in heat injury recovery, a pspA deletion mutant (ΔpspA) and a pspA-overexpressing strain (S. Typhimurium pBAD30/pspA (+) ) were constructed. ΔpspA showed slightly lower viable counts and membrane potential than those of the wild-type strain during recovery. On the other hand, there was no significant difference in the viable counts between S. Typhimurium pBAD30/pspA (+) and the control strains S. Typhimurium pBAD30/pspA (-) and S. Typhimurium pBAD30 (+) during recovery. It would seem that a lack of PspA protein alone somewhat affects the recovery of S. Typhimurium from heat injury, but overexpression of PspA alone is not sufficient to overcome this effect.

Resuscitation and growth kinetics of sub-lethally injured Listeria monocytogenes strains following fluorescence activated cell sorting (FACS)

This study investigated the effect of acid (pH4.2), osmotic (10% NaCl) and heat (55°C for 30min) stress induced injury on Listeria monocytogenes strains ATCC19115, 69, 159/10 and 243 using differential plating and flow cytometry coupled with membrane integrity indicators, thiazole orange (TO) and propidium iodide (PI) staining. Growth kinetics of injured cells sorted by fluorescence activated cell sorting (FACS) were studied at 4, 25 and 37°C. The percentage of cell injury detectable by both flow cytometry and differential plating varied significantly among strains and stress treatments (p<0.0001). Based on flow cytometry and TO/PI staining, acid stress caused the highest level of injury followed by heat and osmotic stress. Following cell sorting, acid and osmotic stress injured cells were capable of resuscitation and re-growth while heat injured cells (except for strain 69) were incapable of re-growth despite having a high level of membrane intact cells. The lag phase duration (λ) of sorted stress injured cells resuscitated in brain heart infusion (BHI) broth was significantly influenced by strain variations (p<0.0001), stress treatments (p=0.007) and temperature of resuscitation (p≤0.001). Following repair, the maximum specific growth rate (μmax) of resuscitated cells was not different from untreated control cells regardless of strain differences and stress treatments. Only temperature had a significant effect (p<0.0001) on growth rate. Sorted cells were also capable of growth at 4°C, with the time to detectable growth (≥1.40Log10CFUml−1) ranging from 3 to 15days. Overall, re-growth potential of sorted cells showed that while membrane integrity was a good indicator of cell injury and viability loss for acid and osmotic stress, it was not a sufficient indicator of heat stress injury. Once injured cells repair the cellular damage, their growth rate is not different from non-injured cells regardless of form of stress and strain differences. Thus highlighting the potential food safety risks of stress injured L. monocytogenes cells.

Differentiation of Different Mixed Listeria Strains and Also Acid-Injured, Heat-Injured, and Repaired Cells of Listeria monocytogenes Using Fourier Transform Infrared Spectroscopy

Fourier transform infrared (FT-IR) spectroscopy was used to differentiate mixed strains of Listeria monocytogenes and mixed strains of L. monocytogenes and Listeria innocua. FT-IR spectroscopy was also applied to investigate the hypothesis that heat-injured and acid-injured cells would return to their original physiological integrity following repair. Thin smears of cells on infrared slides were prepared from cultures for mixed strains of L. monocytogenes, mixed strains of L. monocytogenes and L. innocua, and each individual strain. Heat-injured and acid-injured cells were prepared by exposing harvested cells of L. monocytogenes strain R2-764 to a temperature of 56 ± 0.2°C for 10 min or lactic acid at pH 3 for 60 min, respectively. Cellular repair involved incubating aliquots of acid-injured and heat-injured cells separately in Trypticase soy broth supplemented with 0.6% yeast extract for 22 to 24 h; bacterial thin smears on infrared slides were prepared for each treatment. Spectral collection was done using 250 scans at a resolution of 4 cm(-1) in the mid-infrared wavelength region. Application of multivariate discriminant analysis to the wavelength region from 1,800 to 900 cm(-1) separated the individual L. monocytogenes strains. Mixed strains of L. monocytogenes and L. monocytogenes cocultured with L. innocua were successfully differentiated from the individual strains when the discriminant analysis was applied. Different mixed strains of L. monocytogenes were also successfully separated when the discriminant analysis was applied. A data set for injury and repair analysis resulted in the separation of acid-injured, heat-injured, and intact cells; repaired cells clustered closer to intact cells when the discriminant analysis (1,800 to 600 cm(-1)) was applied. FT-IR spectroscopy can be used for the rapid source tracking of L. monocytogenes strains because it can differentiate between different mixed strains and individual strains of the pathogen.

Real-time PCR method combined with immunomagnetic separation for detecting healthy and heat-injured Salmonella Typhimurium on raw duck wings

Conventional culture detection methods are time consuming and labor-intensive. For this reason, an alternative rapid method combining real-time PCR and immunomagnetic separation (IMS) was investigated in this study to detect both healthy and heat-injured Salmonella Typhimurium on raw duck wings. Firstly, the IMS method was optimized by determining the capture efficiency of Dynabeads® on Salmonella cells on raw duck wings with different bead incubation (10, 30 and 60min) and magnetic separation (3, 10 and 30min) times. Secondly, three Taqman primer sets, Sal, invA and ttr, were evaluated to optimize the real-time PCR protocol by comparing five parameters: inclusivity, exclusivity, PCR efficiency, detection probability and limit of detection (LOD). Thirdly, the optimized real-time PCR, in combination with IMS (PCR–IMS) assay, was compared with a standard ISO and a real-time PCR (PCR) method by analyzing artificially inoculated raw duck wings with healthy and heat-injured Salmonella cells at 101 and 100CFU/25g. Finally, the optimized PCR–IMS assay was validated for Salmonella detection in naturally contaminated raw duck wing samples. Under optimal IMS conditions (30min bead incubation and 3min magnetic separation times), approximately 85 and 64% of S. Typhimurium cells were captured by Dynabeads® from pure culture and inoculated raw duck wings, respectively. Although Sal and ttr primers exhibited 100% inclusivity and exclusivity for 16 Salmonella spp. and 36 non-Salmonella strains, the Sal primer showed lower LOD (103CFU/ml) and higher PCR efficiency (94.1%) than the invA and ttr primers. Moreover, for Sal and invA primers, 100% detection probability on raw duck wings suspension was observed at 103 and 104CFU/ml with and without IMS, respectively. Thus, the Sal primer was chosen for further experiments. The optimized PCR–IMS method was significantly (P=0.0011) better at detecting healthy Salmonella cells after 7-h enrichment than traditional PCR method. However there was no significant difference between the two methods with longer enrichment time (14h). The diagnostic accuracy of PCR–IMS was shown to be 98.3% through the validation study. These results indicate that the optimized PCR–IMS method in this study could provide a sensitive, specific and rapid detection method for Salmonella on raw duck wings, enabling 10-h detection. However, a longer enrichment time could be needed for resuscitation and reliable detection of heat-injured cells.

Comparison of Enrichment Broths for the Recovery of Healthy and Heat-Injured Salmonella Typhimurium on Raw Duck Wings

This study was performed to optimiz eSalmonella Typhimurium recovery from raw duck wings with five nonselective broths (buffered peptone water, tryptic soy broth, lactose broth, universal preenrichment broth, nutrient broth) and four selective broths (selenite broth, BAX System MP media [MP], Salmonella AD media [AD], ONE broth-Salmonella [OB]). Healthy or heat-injured (50 and 85% injury) cells were inoculated at a level of 10(2), 10(1), or 10(0) CFU/25 g on raw duck wings. Growth was modeled using DMfit with four growth parameters: lag-phase duration, maximum growth rate, doubling time, and maximum population density. Most enrichments were able to recover Salmonella Typhimurium to greater than 6 log CFU/ml. AD, MP, and OB had significantly (P < 0.05) higher maximum growth rate (0.9 to 1.0/h) and lower doubling time (0.7 to 0.8 h). Buffered peptone water, AD, MP, and OB recovered healthy and 50%-injured cells at low inoculum levels to more than 6.0 log CFU/ml; OB achieved the greatest recovery (7.6 and 7.9 log CFU/ml), following 24 h of incubation. The 85%-injured cells at 10(0) and 10(1) CFU/25 g, however, were only recovered in OB, reaching 7.3 and 7.5 log CFU/ml, respectively. These results suggest that OB may be an appropriate enrichment broth for the recovery of Salmonella Typhimurium from raw duck wings in standard diagnostic tests or other rapid detection methods, to avoid false-negative results.

Thermal inactivation of desiccation-adapted Salmonella spp. in aged chicken litter.

Thermal inactivation of desiccation-adapted Salmonella spp. in aged chicken litter was investigated in comparison with that in a nonadapted control to examine potential cross-tolerance of desiccation-adapted cells to heat treatment. A mixture of four Salmonella serovars was inoculated into the finished compost with 20, 30, 40, and 50% moisture contents for a 24-h desiccation adaptation. Afterwards, the compost with desiccation-adapted cells was inoculated into the aged chicken litter with the same moisture content for heat treatments at 70, 75, 80, 85, and 150°C. Recovery media were used to allow heat-injured cells to resuscitate. A 5-log reduction in the number of the desiccation-adapted cells in aged chicken litter with a 20% moisture content required >6, >6, ∼4 to 5, and ∼3 to 4 h of exposure at 70, 75, 80, and 85°C, respectively. As a comparison, a 5-log reduction in the number of nonadapted control cells in the same chicken litter was achieved within ∼1.5 to 2, ∼1 to 1.5, ∼0.5 to 1, and <0.5 h at 70, 75, 80, and 85°C, respectively. The exposure time required to obtain a 5-log reduction in the number of desiccation-adapted cells gradually became shorter as temperature and moisture content were increased. At 150°C, desiccation-adapted Salmonella cells survived for 50 min in chicken litter with a 20% moisture content, whereas control cells were detectable by enrichment for only 10 min. Our results demonstrated that the thermal resistance of Salmonella in aged chicken litter was increased significantly when the cells were adapted to desiccation. This study also validated the effectiveness of thermal processing being used for producing chicken litter free of Salmonella contamination.

Growth Kinetics and Model Comparison of Cronobacter sakazakii in Reconstituted Powdered Infant Formula

Cronobacter sakazakii is a life-threatening bacterium, infrequently implicated in illnesses associated with the consumption of powdered infant formula (PIF). It can cause rare but invasive infections in neonatal infants who consume contaminated PIF. The objective of this research was to investigate the growth kinetics and develop mathematical models to predict the growth of heat-injured C. sakazakii in reconstituted PIF (RPIF). RPIF, inoculated with a 6-strain cocktail of non-heat-treated (uninjured) or heat-injured C. sakazakii, was incubated at different temperatures to develop growth models. Except for storage at 6 °C, C. sakazakii grew well at all test temperatures (10 to 48 °C). Uninjured C. sakazakii exhibited no observable lag phase, while a lag phase was apparent in heat-treated cells. A simple 3-parameter logistic equation was used to fit growth curves for non-heat-treated cells, while both Baranyi and Huang models were suitable for heat-treated C. sakazakii. Calculated minimum and maximum growth temperatures were 6.5 and 51.4 °C for non-heat-treated cells, and 6.9 and 50.1 °C for heat-treated cells of C. sakazakii in RPIF, respectively. There was no significant difference between growth rates of non-heat-treated and heat-injured cells in RPIF. For heat-treated cells of C. sakazakii, the lag phase was temperature-dependent and very short (between 25 °C and 48 °C). These results suggest that both non-heat-treated and heat-injured C. sakazakii cells may present a risk to infants if the pathogens are not completely destroyed by heat in RPIF and then exposed to subsequent temperature abuse. C. sakazakii is a life-threatening bacterium found in powdered infant formula (PIF). This study shows that the uninjured bacterium exhibits very short or no lag phase if not refrigerated and can grow well in reconstituted PIF (RPIF), while the heat-injured cells can multiply at an equivalent rate following metabolic recovery. Temperature abuse may allow C. sakazakii to grow and endanger infants fed with RPIF. Predictive models developed in this study can be used to estimate the growth and conduct risk assessments of this pathogen.