Peroxyacetic acid and acidified sodium chlorite reduce microbial contamination on whole chicken carcasses obtained from two processing points.

Chicken meat is frequently contaminated with zoonotic bacterial pathogens such as Campylobacter spp and Salmonella spp. These two bacterial genera are commonly linked with cases of human gastrointestinal disease, thus mitigating their presence in the poultry meat supply chain is paramount. Here, the efficacy of two sanitizers, peroxyacetic acid (PAA) and acidified sodium chlorite (ASC), was tested using whole chicken carcasses obtained either prior to the inside/outside wash or the post-immersion spin chill steps of processing. Two concentrations of PAA (100 and 200 ppm) and ASC (450 and 900 ppm) were tested, and both significantly reduced total viable bacteria and Campylobacter counts per carcass. Both sanitizers also reduced the prevalence of Salmonella on whole carcasses from both processing steps. Log reduction of both the total viable and Campylobacter counts were, however, temperature and processing stage dependent. The efficacy of both PAA and ASC were also compared with sodium hypochlorite. No significant difference between the three sanitizers was observed for the reduction of TVC, Campylobacter or Salmonella using carcasses obtained at either processing step. These results demonstrate that PAA or ASC could be implemented as a replacement or used in addition to sodium hypochlorite to effectively reduce bacteria on whole carcasses.

Salmonella on Australian cage egg farms: Observations from hatching to end of lay.

Single-aged caged layer hen flocks were monitored for Salmonella over the course of their lifetime. Chicks from both flocks were Salmonella negative at hatch and remained negative during rearing. Pullets were transported to production farms at 15 weeks of age. Pre-population dust swabs collected from both production sheds had a high percentage of Salmonella positive samples (80 and 90%). Flocks were sampled at regular intervals until 70-72 weeks of age. The proportion of Salmonella positive samples and mean load detected on eggs was low on both farms. Analysis of dust samples revealed that Salmonella persisted in dust over 8 weeks. Dust total moisture content and water activity appears to influence bacterial persistence. On egg grading equipment, only suction cups prior to egg washing were Salmonella positive (mean proportion Salmonella positive samples 0.13 ± 0.07; mean load of 18.6 ± 12.31 MPN/ml). An egg washing experiment demonstrated that while washing reduced the total Salmonella load from eggshell surfaces, no effect was observed for shell pores. These results demonstrate that despite environmental contamination on farm, Salmonella contamination of eggs is low and is further minimized by washing.

The effects of varied food acid ratios and egg components on Salmonella Typhimurium culturability from raw egg-based sauces.

Raw egg-based sauces, such as mayonnaise and aioli, are frequently identified as sources of Salmonella during outbreaks of human cases of foodborne gastrointestinal disease. In this study, we surveyed aioli and mayonnaise recipes from different popular food websites to identify potential risk factors that may lead to the survival of Salmonella Typhimurium. In laboratory experiments, different ratios of food acids were used to determine if lemon juice, vinegar, or a combination of both restricted Salmonella Typhimurium culturability. We found that as long as the pH was below 4.2, bacterial culturability was limited. The use of whole egg alone or in combination with egg yolk was also investigated. Sauce preparations containing whole egg exhibited higher pH and supported Salmonella Typhimurium culturability longer than those containing yolk only. Ten restaurant prepared sauces were also obtained to further characterize the effect of preparation variability. Sauce preparations with a pH ≤ 3.8 did not support bacterial culturability after 4 h incubation at any temperature. The higher the pH the longer Salmonella Typhimurium remained culturable. Based on this study, it is recommended that raw egg-based foods are acidified, then stored at room temperature for at least 4 h prior to consumption.

From hatch to egg grading: monitoring of Salmonella shedding in free-range egg production systems.

Human cases of salmonellosis are frequently liked with the consumption of contaminated table eggs. Recently, there has been an increase in consumer demand for cage-free eggs precipitating the need for a greater understanding of Salmonella dynamics in free-range production systems. A longitudinal study was conducted to determine the points in production where birds are most likely to be exposed to Salmonella and where the risk of egg contamination is highest. In this study, two free-range flocks were sampled from hatch to the end of production. At hatch, all chicks were Salmonella negative and remained negative during rearing. During production, the proportion of positive samples was low on both farms. Salmonella positive samples were detected intermittently for Flock A. Dust, nest box, and egg belt swabs had the highest proportion of positive samples and highest overall loads of Salmonella. The egg grading floor was swabbed at different points following the processing of eggs from Flock A. Only the suction cups that handle eggs prior to egg washing tested positive for Salmonella. Swabs collected from machinery handling eggs after washing were Salmonella negative. During production, positive samples from Flock B were observed at only single time point. Dust has been implicated as a source of Salmonella that can lead to flock to flock contamination. Bulk dust samples were collected and tested for Salmonella. The proportion of positive dust samples was low and is likely due to physical parameters which are not likely to support the survival of Salmonella in the environment.

In vivo passage of Salmonella Typhimurium results in minor mutations in the bacterial genome and increases in vitro invasiveness.

Eggs and raw or undercooked egg-containing food items are frequently identified as the bacterial source during epidemiolocal investigation of Salmonella outbreaks. Multi-locus variable number of tandem repeats analysis (MLVA) is a widely used Salmonella typing method enabling the study of diversity within populations of the same serotype. In vivo passage, however, has been linked with changes in MLVA type and more broadly the Salmonella genome. We sought to investigate whether in vivo passage through layer hens had an effect on MLVA type as well as the bacterial genome and whether any mutations affected bacterial virulence. Layer hens were infected with either Salmonella Typhimurium DT9 (03-24-11-11-523) as part of a single infection or were co-infected with an equal amount of Salmonella Mbandaka. Salmonella shedding in both single and co-infected birds was variable over the course of the 16-week experiment. Salmonella Typhimurium and Salmonella Mbandaka were identified in feces of co-infected birds. Salmonella colonies isolated from fecal samples were subtyped using MLVA. A single change in SSTR-6 was observed in Salmonella Typhimurium strains isolated from co-infected birds. Isolates of Salmonella Typhimurium of both the parent (03-24-11-11-523) and modified (03-24-12-11-523) MLVA type were sequenced and compared with the genome of the parent strain. Sequence analysis revealed that in vivo passaging resulted in minor mutation events. Passaged isolates exhibited significantly higher invasiveness in cultured human intestinal epithelial cells than the parent strain. The microevolution observed in this study suggests that changes in MLVA may arise more commonly and may have clinical significance.

Anti-bacterial and anti-biofilm activity of commercial organic acid products against Salmonella enterica isolates recovered from an egg farm environment.

This study evaluated the antibacterial activity of commercially available organic acid water additives against Salmonella enterica isolates and examined the susceptibility of Salmonella Typhimurium biofilms to these products. Three commercial organic acid products (A, B, and C) were evaluated for minimum inhibitory and bactericidal concentrations against isolates of S. enterica serovars. Three- and five-day-old S. Typhimurium biofilms were formed at 22 ± 2°C using an MBEC™ assay system and exposed for 30 min or 90 min at 0.2% and 0.4% concentrations. No significant difference among serovars for inhibitory and bactericidal concentrations was detected. Two products (A and C) significantly reduced viable cells from biofilms of both ages in a dose- and time-dependent manner. Increased biofilm age did not enhance resistance towards organic acid treatments. None of the products completely eliminated biofilm cells at any concentration or exposure time. Product composition, exposure time, and concentration of organic acid products were important factors in reducing viable biofilm cells. This study has expanded our understanding about the susceptibility of Salmonella biofilms to commercial organic acid products. These findings have implications in the usage, development, and optimization of organic acid products.

Correlating bacterial shedding with fecal corticosterone levels and serological responses from layer hens experimentally infected with Salmonella Typhimurium.

Salmonella Enteriditis and Salmonella Typhimurium are commonly isolated during egg-related outbreaks of salmonellosis and represent a significant international public health issue. In Australia, Salmonella Typhimurium is the most common serovar identified in egg product related foodborne outbreaks. While a number of studies have investigated Salmonella shedding and host responses to infection, they have been conducted over a short time period. The present study sought to characterise bacterial shedding and host responses to infection in hens infected with only Salmonella Typhimurium or co-infected with both Salmonella Typhimurium and Salmonella Mbandaka over a 16 week period. Salmonella shedding was quantified using the most probable number and qPCR methods and was highly variable over the course of the experiment. On day 1, fecal corticosterone metabolites in birds infected with Salmonella Typhimurium (674.2 ± 109.3 pg/mg) were significantly higher than control (238.0 ± 12.62 pg/mg) or co-infected (175.4 ± 8.58 pg/mg) birds. The onset of lay occurred between weeks 6-8 post-infection (pi) and Fecal corticosterone metabolite (FCM) concentrations increased in both control and co-infected birds. Antibody responses to infection were monitored in both serum and yolk samples. Salmonella Typhimurium specific antibody was lower in co-infected animals than monoinfected animals. Bacterial loads in internal organs were characterised to determine persistence. Spleen, liver and caecal tonsils were positive for bacteria in both groups, indicating that Salmonella was not cleared from the birds and internal organ colonization could serve as a reservoir for continued bacterial shedding.

Salmonella enterica isolates from layer farm environments are able to form biofilm on eggshell surfaces.

This study examined the eggshell biofilm forming ability of Salmonella enterica isolates recovered from egg farms. Multicellular behaviour and biofilm production were examined at 22 and 37°C by Congo red morphology and the crystal violet staining assay. The results indicated that the biofilm forming behaviour of Salmonella isolates was dependent on temperature and associated with serovars. Significantly greater biofilm production was observed at 22°C compared with 37°C. The number of viable biofilm cells attached to eggshells after incubation for 48 h at 22°C was significantly influenced by serovar. Scanning electron microscopic examination revealed firm attachment of bacterial cells to the eggshell surface. The relative expression of csgD and adrA gene was significantly higher in eggshell biofilm cells of S. Mbandaka and S. Oranienburg. These findings demonstrate that Salmonella isolates are capable of forming biofilm on the eggshell surface and that this behaviour is influenced by temperature and serovar.

Murine cytomegalovirus strains co-replicate at multiple tissue sites and establish co-persistence in salivary glands in the absence of Ly49H-mediated competition.

Infection with multiple genetically distinct strains of pathogen is common and can lead to positive (complementation) or negative (competitive) within-host interactions. These interactions can alter aspects of the disease process and help shape pathogen evolution. Infection of the host with multiple strains of cytomegalovirus (CMV) occurs frequently in humans and mice. Profound, NK-cell-mediated (apparent) competition has been identified in C57BL/6 mice, and prevented the replication and shedding of certain co-infecting CMV strains. However, the frequency of such strong competition has not been established. Other within-host interactions such as complementation or alternative forms of competition remain possible. Moreover, high rates of recombination in both human CMV and murine CMV (MCMV) suggest prolonged periods of viral co-replication, rather than strong competitive suppression. An established model was employed to investigate the different possible outcomes of multi-strain infection in other mouse strains. In this study, co-replication of up to four strains of MCMV in the spleen, liver and salivary glands was observed in both MCMV-susceptible and MCMV-resistant mice. In the absence of apparent competition, no other forms of competition were unmasked. In addition, no evidence of complementation between viral strains was observed. Importantly, co-replication of MCMV strains was apparent for up to 90 days in the salivary glands. These data indicated that competition was not the default outcome of multi-strain CMV infection. Prolonged, essentially neutral, co-replication may be the norm, allowing for multi-strain transmission and prolonged opportunities for recombination.

Natural killer cell dependent within-host competition arises during multiple MCMV infection: consequences for viral transmission and evolution.

It is becoming increasingly clear that many diseases are the result of infection from multiple genetically distinct strains of a pathogen. Such multi-strain infections have the capacity to alter both disease and pathogen dynamics. Infection with multiple strains of human cytomegalovirus (HCMV) is common and has been linked to enhanced disease. Suggestions that disease enhancement in multi-strain infected patients is due to complementation have been supported by trans-complementation studies in mice during co-infection of wild type and gene knockout strains of murine CMV (MCMV). Complementation between naturally circulating strains of CMV has, however, not been assessed. In addition, many models of multi-strain infection predict that co-infecting strains will compete with each other and that this competition may contribute to selective transmission of more virulent pathogen strains. To assess the outcome of multi-strain infection, C57BL/6 mice were infected with up to four naturally circulating strains of MCMV. In this study, profound within-host competition was observed between co-infecting strains of MCMV. This competition was MCMV strain specific and resulted in the complete exclusion of certain strains of MCMV from the salivary glands of multi-strain infected mice. Competition was dependent on Ly49H(+) natural killer (NK) cells as well as the expression of the ligand for Ly49H, the MCMV encoded product, m157. Strains of MCMV which expressed an m157 gene product capable of ligating Ly49H were outcompeted by strains of MCMV expressing variant m157 genes. Importantly, within-host competition prevented the shedding of the less virulent strains of MCMV, those recognized by Ly49H, into the saliva of multi-strain infected mice. These data demonstrate that NK cells have the strain specific recognition capacity required to meditate within-host competition between strains of MCMV. Furthermore, this within-host competition has the capacity to shape the dynamics of viral shedding and potentially select for the transmission of more virulent virus strains.

Pathogenicity of Salmonella strains isolated from egg shells and the layer farm environment in australia.

In Australia, the egg industry is periodically implicated during outbreaks of Salmonella food poisoning. Salmonella enterica serovar Typhimurium and other nontyphoidal Salmonella spp., in particular, are a major concern for Australian public health. Several definitive types of Salmonella Typhimurium strains, but primarily Salmonella Typhimurium definitive type 9 (DT9), have been frequently reported during egg-related food poisoning outbreaks in Australia. The aim of the present study was to generate a pathogenicity profile of nontyphoidal Salmonella isolates obtained from Australian egg farms. To achieve this, we assessed the capacity of Salmonella isolates to cause gastrointestinal disease using both in vitro and in vivo model systems. Data from in vitro experiments demonstrated that the invasion capacity of Salmonella serovars cultured to stationary phase (liquid phase) in LB medium was between 90- and 300-fold higher than bacterial suspensions in normal saline (cultured in solid phase). During the in vivo infection trial, clinical signs of infection and mortality were observed only for mice infected with either 10(3) or 10(5) CFU of S. Typhimurium DT9. No mortality was observed for mice infected with Salmonella serovars with medium or low invasive capacity in Caco-2 cells. Pathogenicity gene profiles were also generated for all serovars included in this study. The majority of serovars tested were positive for selected virulence genes. No relationship between the presence or absence of virulence genes by PCR and either in vitro invasive capacity or in vivo pathogenicity was detected. Our data expand the knowledge of strain-to-strain variation in the pathogenicity of Australian egg industry-related Salmonella spp.

Dust sprinkling as an effective method for infecting layer chickens with wild-type Salmonella Typhimurium and changes in host gut microbiota.

Role of dust in Salmonella transmission on chicken farms is not well characterised. Salmonella Typhimurium (ST) infection of commercial layer chickens was investigated using a novel sprinkling method of chicken dust spiked with ST and the uptake compared to a conventional oral infection. While both inoculation methods resulted in colonisation of the intestines, the Salmonella load in liver samples was significantly higher at 7 dpi after exposing chicks to sprinkled dust compared to the oral infection group. Infection of chickens using the sprinkling method at a range of doses showed a threshold for colonisation of the gut and organs as low as 1000 CFU/g of dust. Caecal content microbiota analysis post-challenge showed that the profiles of chickens infected by the sprinkling and oral routes were not significantly different; however, both challenges induced differences when compared to the uninfected negative controls. Overall, the study showed that dust sprinkling was an effective way to experimentally colonise chickens with Salmonella and alter the gut microbiota than oral gavage at levels as low as 1000 CFU/g dust. This infection model mimics the field scenario of Salmonella infection in poultry sheds. The model can be used for future challenge studies for effective Salmonella control.

A live attenuated Salmonella Typhimurium vaccine dose and diluent have minimal effects on the caecal microbiota of layer chickens.

Among the Salmonella reduction strategies in poultry production, one option is to use a Salmonella vaccine. The aim of vaccinating layer flocks is to reduce the shedding of wild-type Salmonella in the poultry environment, thereby reducing the contamination of poultry products (eggs and meat). Nutritive diluent and a higher dose of vaccine may enhance its colonization potential in the gut of chickens. In this study, a commercially available live attenuated vaccine (Vaxsafe® ST) was reconstituted in different media and delivered orally to day-old chicks at three different doses (107, 108, and 109 CFU/chick). Gut colonization of the vaccine strain and the effects of vaccination on gut microbiota were assessed in commercial-layer chickens. The vaccine diluent and dosage minimally affected microbiota alpha diversity. Microbiota beta diversity was significantly different (P < 0.05) based on the vaccine diluent and dose, which indicated that the vaccinated and unvaccinated chickens had different gut microbial communities. Differences were noted in the abundance of several genera, including Blautia, Colidextribacter, Dickeya, Enterococcus, Lactobacillus, Pediococcus, and Sellimonas. The abundance of Colidextribacter was significantly lower in chickens that received vaccine reconstituted in Marek's and water diluents, while Lactobacillus abundance was significantly lower in the water group. The highest vaccine dose (109 CFU/chick) did not significantly alter (P > 0.05) the abundance of microbial genera. Chicken age affected the microbiota composition more significantly than the vaccine dose and diluent. The abundance of Lactobacillus, Blautia, Caproiciproducens, Pediococcus, and Colidextribacter was significantly higher on day 14 compared with day 7 post-vaccination. The Salmonella Typhimurium vaccine load in the caeca was not significantly affected by diluent and vaccine dose; however, it was significantly lower (P < 0.0001) on day 14 compared with day 7 post-vaccination. Overall, the S. Typhimurium vaccine minimally affected the gut microbiota structure of layer chicks, whereas changes in microbiota were more significant with chicken age.

Comparison of peroxyacetic acid and acidified sodium chlorite at reducing natural microbial contamination on chicken meat pieces.

The spin-chill process at poultry processing plants involves the immersion of chicken carcasses in cold water (<5°C) often containing sodium hypochlorite which significantly contributes to the reduction of bacterial loads. Cutting carcasses into pieces, however, has been linked with increases in Campylobacter and Salmonella counts. Here, the efficacy of PAA and ASC on reducing bacteria on skin-on, bone-in thigh cuts was investigated. Three concentrations of ASC (60, 112, and 225 ppm) and PAA (50, 75, 100 ppm) were used. Thighs were dipped into sanitizer and tested for total viable bacterial counts, Campylobacter load, and prevalence of Salmonella. The efficacy of PAA and ASC was also compared with chlorine (8 ppm). All sanitizers exhibited a greater log reduction compared with water. PAA at both 75 and 100 ppm resulted in significantly higher log reductions compared with the water only. PAA at 100 ppm and 225 ppm ASC were the most effective at reducing Campylobacter. All wash treatments reduced the proportion of Salmonella positive samples, but the greatest reduction was observed for 225 ppm ASC. Both concentrations of ASC resulted in a greater reduction in total viable counts compared with chlorine.

Investigation of a gel-based delivery method for the administration of a live, attenuated Salmonella Typhimurium vaccine.

Poultry vaccines are often administered using water as a suspension media and applied using an oral or coarse spray method. Gel-based vaccine diluents have been developed as an alternative vaccine delivery method. Gels are more viscous, and droplets adhere more effectively to feathers giving the vaccine a longer time to be ingested (through preening). Application of gel diluents with live bacterial vaccines, however, is limited. The present study tested a gel diluent prepared in various media, using a live, attenuated Salmonella Typhimurium vaccine, Vaxsafe ST. Reconstitution in gel diluent did not negatively affect vaccine viability or motility. The invasive capacity of vaccine suspended in gel diluent into cultured intestinal epithelial cells was also tested. Results demonstrated that vaccine suspended in gel diluent retained invasiveness. Day old chicks were orally administered with Vaxsafe ST suspended in gel diluent to characterize in vivo colonization capacity of the vaccine. The results revealed that the VaxSafe ST suspended in gel diluent could efficiently colonize the caeca of chicks, which is needed for the development of effective immunity.

Effects of Sublethally Injured Campylobacter jejuni in Mice.

Globally, Campylobacter spp. are the most common food-associated bacterial cause of human gastrointestinal disease. Campylobacteriosis is primarily associated with the consumption of contaminated chicken meat. Chemical decontamination of chicken carcasses during processing is one of the most effective interventions to mitigate Campylobacter contamination. Following exposure to sanitizers, however, sublethally injured populations of bacteria may persist. The risk that sublethally injured Campylobacter pose for public health is unknown. Furthermore, the virulence potential of sublethally injured Campylobacter jejuni during prolonged storage in relation to host pathogenesis and the host immune response has not been well established. Therefore, we evaluated the effects of sublethally injured C. jejuni on the host, after storage in chicken meat juice. C57BL/6 mice were infected with two C. jejuni chicken meat isolates or the ATCC 33291 strain that had been stored in the chicken meat juice, after exposure to chlorine or acidified sodium chlorite (ASC). Although chlorine exposure was unable to reduce intestinal colonization by C. jejuni, exposure to ASC significantly reduced the intestinal colonization and tissue translocation in mice. The expression of pro- and anti-inflammatory cytokine genes for interleukin-6 (IL-6), IL23a, and IL-10, Toll-like receptor 2 (TLR2) and TLR4 genes, and host stress response genes (CRP, MBL1, and NF-κB1) were significantly reduced following the exposure to ASC. Our results demonstrated that sublethally injured C. jejuni has reduced virulence potential and colonization in mice. The data contribute toward clarification of the importance of chemical decontamination during processing to minimize human campylobacteriosis. IMPORTANCE Campylobacter is the most common cause of bacterial gastrointestinal disease, and consumption of contaminated poultry is frequently identified as the source of bacteria. The survivability and virulence potential of sublethally injured Campylobacter following exposure to chemicals which are commonly used to eliminate Campylobacter during the poultry meat processing are of concern to the food industry, government health officials, and consumers. Here, we demonstrate that sublethally injured Campylobacter jejuni has reduced bacterial virulence and colonization potential in mice.

Refrigeration of eggs influences the virulence of Salmonella Typhimurium.

Salmonella Typhimurium is a human pathogen associated with eggs and egg-derived products. In Australia, it is recommended that eggs should be refrigerated to prevent condensation that can enhance bacterial penetration across the eggshell. Except for the United States, the guidelines on egg refrigeration are not prescriptive. In the current study, in-vitro and in-vivo experiments were conducted to understand the role of egg storage temperatures (refrigerated vs ambient) on bacterial load and the virulence genes expression of Salmonella Typhimurium. The in-vitro egg study showed that the load of Salmonella Typhimurium significantly increased in yolk and albumen stored at 25 °C. The gene expression study showed that ompR, misL, pefA, spvA, shdA, bapA, and csgB were significantly up-regulated in the egg yolk stored at 5 °C and 25 °C for 96 h; however, an in-vivo study revealed that mice infected with egg yolk stored at 25 °C, developed salmonellosis from day 3 post-infection (p.i.). Mice fed with inoculated egg yolk, albumen, or eggshell wash stored at refrigerated temperature did not show signs of salmonellosis during the period of the experiment. Data obtained in this study highlighted the importance of egg refrigeration in terms of improving product safety.

Transcriptomic response of Campylobacter jejuni following exposure to acidified sodium chlorite.

Chemical decontamination during processing is used in many countries to mitigate the Campylobacter load on chicken meat. Chlorine is a commonly used sanitizer in poultry processing to limit foodborne bacterial pathogens but its efficacy is limited by high bacterial loads and organic material. Acidified sodium chlorite (ASC) is a potential alternative for poultry meat sanitization but little is known about its effects on the cellular response of Campylobacter. In this study, the sensitivity of C. jejuni isolates to ASC was established. RNAseq was performed to characterize the transcriptomic response of C. jejuni following exposure to either chlorine or ASC. Following chlorine exposure, C. jejuni induced an adaptive stress response mechanism. In contrast, exposure to ASC induced higher oxidative damage and cellular death by inhibiting all vital metabolic pathways and upregulating the genes involved in DNA damage and repair. The transcriptional changes in C. jejuni in response to ASC exposure suggest its potential as an effective sanitizer for use in the chicken meat industry.

Acidification and extended storage at room temperature of mayonnaise reduce Salmonella Typhimurium virulence and viability.

Despite food safety recommendations, raw egg-based foods, such as mayonnaise, are frequently identified as the source of Salmonella during outbreaks. Acidification and storage temperature have been linked with reduced bacterial culturability. Raw egg-based sauces stored at 25 °C have historically been linked with faster decline of Salmonella culturability than preparations stored at 5 °C. This study aimed to determine whether reduced culturability in acidified mayonnaise correlated with reduced in vitro bacterial motility, invasiveness and viability as well as disease-causing capacity in BALB/c mice. Acidification of mayonnaise and incubation at 25 °C for 4 h significantly reduced culturability of Salmonella Typhimurium DT9 but was dependent on initial bacterial load. Bacteria recovered from acidified mayonnaise exhibited reduced invasiveness into polarized cultured intestinal epithelial cells and 12 h post inoculation were no longer invasive suggesting a reduced capacity to cause disease. To confirm this, BALB/c mice were inoculated with Salmonella Typhimurium contaminated mayonnaise stored at 5 °C or 25 °C for 12, 24, 48, 72, and 96 h. Mice inoculated with mayonnaise incubated at 5 °C for 12 and 24 h exhibited mild to moderate disease symptoms; all other mayonnaise treatment groups did not exhibit disease symptoms. In acidified mayonnaise, Salmonella Typhimurium DT9 exhibited a global downregulation of metabolism, stress response, and virulence genes upon addition to mayonnaise. After 4 h of incubation at both 5 °C and 25 °C, however, the vast majority of genes were upregulated which was maintained over the 96-hour experiment suggesting that bacteria were severely stressed. Salmonella Typhimurium DT9 cells were isolated from mayonnaise samples and ATP production was quantified. At both 5 °C and 25 °C, ATP production decreased in acidified mayonnaise preparations. At 25 °C, ATP production decreased more rapidly than at 5 °C. After 24 h, ATP production of bacteria in mayonnaise stored at 25 °C was not significantly different from the dead control group. Thus, the current recommendation of only serving freshly prepared raw egg-sauces or refrigerating immediately after preparation, could be placing consumers at higher risk for contracting salmonellosis.

Chlorine Induces Physiological and Morphological Changes on Chicken Meat Campylobacter Isolates.

Broiler chickens frequently become colonized by Campylobacter species. As a consequence, Campylobacter, can enter the poultry meat supply chain and represents a significant risk for human public health. A number of on-farm biosecurity and processing measures are used to mitigate the load of Campylobacter on chicken meat. In many countries, chlorine is commonly used as a biocide in processing plants to reduce bacterial loads on poultry carcasses but there is limited evidence of its effectiveness on Campylobacter. In this study, 116 Campylobacter isolates (89 C. jejuni and 27 C. coli) were isolated from poultry meat carcasses prior to the inside/outside wash step and used in in vitro assays exploring the efficacy of chlorine. A high proportion of isolates exhibited MIC and MBC values of 128 ppm but organic material present in the broth likely affected this result. Thus, additional bactericidal assays (time kill and chlorine inactivation) were used to characterize the response of C. jejuni isolates to different concentrations of chlorine. At 106 CFU, C. jejuni was found to be highly sensitive to concentrations of chlorine and was inhibited at low concentrations (0.2-2.0 ppm). At a higher bacterial load (108 CFU), variation in the response of different C. jejuni isolates was observed. One isolate was growth inhibited at 1.8 ppm while another required 16 ppm. At 108 CFU, C. jejuni could be resuscitated following exposure to chlorine highlighting a potential limitation of chlorine use. Analysis of UV leakage indicated that high chlorine concentrations resulted in increased 280 nm absorbance values suggesting bacterial membrane damage. Scanning electron and transmission electron microscopy were performed to characterize the morphological effects of chlorine exposure. Some effects of chlorine exposure included changes in shape (coccoid, or elongated), cellular degeneration, and shriveled bacterial cells. Interestingly, C. jejuni cells with normal morphology were also observed in the chlorine exposed group and represent a population of cells that could be resuscitated. This study is useful for the chicken meat industry and provides data for future optimization of chlorine use in reducing Campylobacter loads.