Contact time has limited impact on the efficacy of disinfectant towelettes when tested under conditions reflective of realistic use.

BACKGROUND: Disinfectant towelettes are increasingly being used as a means to prevent transmission of clinically important pathogens which could lead to healthcare-associated infections (HAIs). However, the efficacy of disinfectant towelette products when tested under realistic use conditions is understudied. A test model was designed to replicate realistic wiping conditions. The objective of this study was to determine the impact of varied contact time on disinfectant towelette efficacy under these conditions. METHODS: Five product types were tested against Staphylococcus aureus (ATCC 6538) and Pseudomonas aeruginosa (ATCC 15,442) at five contact times (30 s, one min, two min, three min, and 10 min) on hard, non-porous laminate templates to determine the impact of contact time on disinfectant towelette efficacy when tested under realistic use. RESULTS: Product type had a significant impact on the efficacy of disinfectant towelettes when tested under conditions reflective of realistic use. The effect of contact time was limited and no differences in efficacy were seen at a contact time of one min compared with the other contact times tested. Only one disinfectant towelette product achieved a mean 5-log reduction under the tested conditions. CONCLUSION: Efficacy of disinfectant towelettes was primarily impacted by product type when applied in a model designed to replicate realistic use in which only a limited effect of contact time was observed. There is a need for further investigation into which factors have the greatest impact on disinfectant towelette efficacy when applied in clinical settings.

Contact time and disinfectant formulation significantly impact the efficacies of disinfectant towelettes against Candida auris on hard, non-porous surfaces.

There has been an increase in Candida auris healthcare-associated infections, which result from cross-contamination from surfaces and equipment. In this study, we tested the efficacies of EPA-registered disinfectant towelettes products that are increasingly used for infection control against C. auris at a range of contact times following modifications to standard EPA protocol MB-33-00. Hydrogen peroxide (HP)-based disinfectant towelettes were more efficacious against C. auris than the quaternary ammonium chloride (QAC)-alcohol-based disinfectant towelettes irrespective of tested contact times. Thirty s contact time was significantly less effective in reducing C. auris compared to 1-, 2-, 3-, and 10-min contact times. However, there were no statistically significant differences in the level of disinfection among 1-min and longer contact times regardless of product chemistry. None of the products achieved a standard six-log10 reduction at any tested contact times. Overall, the HP-based disinfectant towelette was significantly more fungicidal than the QAC-alcohol-based disinfectant towelette. For all product types, 30 s contact time did not achieve the same level of disinfection as 1-min or longer contact times. Overall, disinfectant towelette efficacy is dependent upon product formulation and contact time.

Evaluation of automated floor cleaning, disinfection, and application methods against Staphylococcus aureus.

BACKGROUND: Optimization of automated floor disinfection practices using different application methods and product types is important to ensure that pathogens such as Staphylococcus aureus do not transfer from contaminated floors to other high contact areas resulting in healthcare-associated infections (HAIs). We hypothesized that there would be significant differences among the disinfectants and a cleaner under different application methods. Also, performance of application methods would be dependent upon type of product used. METHODS: We tested and compared efficacies of 5 EPA registered disinfectants and one cleaner using an automated Taski 455B floor cleaner against S aureus ATCC 6538 on 2 meters of contaminated vinyl flooring using 3 application methods. RESULTS: Hydrogen peroxide and quaternary ammonium compounds were more efficacious against S aureus than the neutral cleaner. There were no significant differences among the sampling areas tested and application methods regardless of product type. Mean log10 densities recovered from different machine parts and wastewater collected were statistically higher for the cleaner than disinfectants. CONCLUSIONS: All disinfectants had more bactericidal efficacy than the cleaner for all sampling zones on the tested floor. Overall, performance of the floor machine is dependent upon the type of product used.

Integrative Assessment of Reduced Listeria monocytogenes Susceptibility to Benzalkonium Chloride in Produce Processing Environments.

For decades, quaternary ammonium compounds (QAC)-based sanitizers have been broadly used in food processing environments to control foodborne pathogens such as Listeria monocytogenes. Still, there is a lack of consensus on the likelihood and implication of reduced Listeria susceptibility to benzalkonium chloride (BC) that may emerge due to sublethal exposure to the sanitizers in food processing environments. With a focus on fresh produce processing, we attempted to fill multiple data and evidence gaps surrounding the debate. We determined a strong correlation between tolerance phenotypes and known genetic determinants of BC tolerance with an extensive set of fresh produce isolates. We assessed BC selection on L. monocytogenes through a large-scale and source-structured genomic survey of 25,083 publicly available L. monocytogenes genomes from diverse sources in the United States. With the consideration of processing environment constraints, we monitored the temporal onset and duration of adaptive BC tolerance in both tolerant and sensitive isolates. Finally, we examined residual BC concentrations throughout a fresh produce processing facility at different time points during daily operation. While genomic evidence supports elevated BC selection and the recommendation for sanitizer rotation in the general context of food processing environments, it also suggests a marked variation in the occurrence and potential impact of the selection among different commodities and sectors. For the processing of fresh fruits and vegetables, we conclude that properly sanitized and cleaned facilities are less affected by BC selection and unlikely to provide conditions that are conducive for the emergence of adaptive BC tolerance in L. monocytogenes. IMPORTANCE Our study demonstrates an integrative approach to improve food safety assessment and control strategies in food processing environments through the collective leveraging of genomic surveys, laboratory assays, and processing facility sampling. In the example of assessing reduced Listeria susceptibility to a widely used sanitizer, this approach yielded multifaceted evidence that incorporates population genetic signals, experimental findings, and real-world constraints to help address a lasting debate of policy and practical importance.

Hydrogen peroxide, sodium dichloro-s-triazinetriones and quaternary alcohols significantly inactivate the dry-surface biofilms of Staphylococcus aureus and Pseudomonas aeruginosa more than quaternary ammoniums.

Globally, healthcare-associated infections (HAI) are the most frequent adverse outcome in healthcare delivery. Although bacterial biofilms contribute significantly to the incidence of HAI, few studies have investigated the efficacy of common disinfectants against dry-surface biofilms (DSB). The objective of this study was to evaluate the bactericidal efficacy of seven Environmental Protection Agency (EPA)-registered liquid disinfectants against DSB of Staphylococcus aureus and Pseudomonas aeruginosa. We hypothesized that overall, there will be significant differences among the bactericidal efficacies of tested disinfectants by product type and active ingredient class. We also hypothesized that depending on the species, higher bactericidal efficacies against DSB will be exhibited after 24 h of dehydration compared to 72 h. Wet-surface biofilms of S. aureus and P. aeruginosa were grown following EPA-MLB-SOP-MB-19 and dehydrated for 24 and 72 h to establish DSB. Seven EPA-registered disinfectants were tested against dehydrated DSB following EPA-MLB-SOP-MB-20. Overall, quaternary ammonium plus alcohol, sodium dichloro-s-triazinetrione and hydrogen peroxide products were more efficacious against DSB than quaternary ammoniums for both tested species. While there was no significant difference in the log10 reductions between 24 and 72 h S. aureus biofilms, significantly higher log10 reductions were observed when products were challenged with 24 h P. aeruginosa DSB compared to 72 h P. aeruginosa DSB. Species type, active ingredient class and dry time significantly impact disinfectant efficacy against DSB of S. aureus or P. aeruginosa.

Listeria monocytogenes 10403S Alternative Sigma-54 Factor σL Has a Negative Role on Survival Ability Under Bile Exposure.

Listeria monocytogenes is a Gram-positive bacterium causing listeriosis in animals and humans. To initiate a foodborne infection, L. monocytogenes has to pass through the host gastrointestinal tract (GIT). In this study, we evaluated survival abilities of L. monocytogenes 10403S wild type (WT) and its isogenic mutants in alternative sigma (σ) factor genes (i.e., sigB, sigC, sigH, and sigL) under simulated gastric, duodenal, and bile fluids. Within 10min of exposures, only bile fluid was able to significantly reduce survival ability of L. monocytogenes WT by 2 logs CFU/ml. Loss of sigL showed the greatest bile resistance among 16 strains tested, p<0.0001, (i.e., WT, four single alternative σ factor mutants, six double mutants, four triple mutants, and one quadruple mutant). To further investigate the role of σL in bile response, RNA-seq was conducted to compare the transcriptional profiles among L. monocytogenes 10403S ΔBCH triple mutant (lacking sigB, sigC, and sigH genes; expressing housekeeping σA and σL) and ΔBCHL quadruple mutant (lacking all alternative sigma factor genes; expressing only σA) strains under BHI and 1% bile conditions. A total of 216 and 176 differentially expressed genes (DEGs) were identified in BHI and bile, respectively. We confirmed that mpt operon was shown to be strongly activated by σL. Interestingly, more than 80% of DEGs were found to be negatively regulated in the presence of σL. This includes PrfA regulon and its mediated genes (i.e., hly, hpt, inlB, clpP, clpE, groL, and inlC) which were downregulated in response to bile in the presence of σL. This result suggests the potential negative role of σL on bile survival, and the roles of σL and σB might be in a seesaw model prior to host cell invasion.

Enterobacteriaceae, coliform, yeast, and mold contamination patterns in peanuts compared to production, storage, use practices, and knowledge of food safety among growers in Senegal.

Peanuts and peanut products are significant revenue sources for smallholder farmers in the Senegalese peanut basin. However, microbial contamination during production and storage can greatly affect market access for producers. Peanut products have emerged as possible sources of foodborne illness, encouraging discussions on international standards for peanuts. In this study, we interviewed 198 households throughout the Senegalese peanut basin to assess current production practices, storage methods, and producers' prior knowledge of microbial contamination using a 162-question survey. A member of each household orally completed the survey with a trained enumerator and the results were compared to microbiological results obtained from peanut samples collected at the time of the interview using linear regression and an analysis of variance model. Samples were collected from stored peanuts at each household; peanuts were shelled and total Enterobacteriaceae, coliform, and yeast and mold populations were enumerated. Of the 198 samples analyzed, 13.0% and 13.6% were greater than the upper detection limits for Enterobacteriaceae and coliforms, respectively. A total of 21.2% of samples were above the detection limit for yeast and mold populations. Only 22.7% and 18.7% of producers were aware of pathogenic bacteria or aflatoxins, respectively; there were no significant differences in observed microbial populations between household who took preventative measures against microbial contamination and those who did not. Additionally, four households reported washing their kitchen utensils before using them to eat and 60.1% reported always washing their hands before eating. Enumerators were asked to report peanut storage container type and if the containers were stored off the ground at the time of collection. While the interaction between storage container type and if the container was stored off the ground was significant for Enterobacteriaceae and coliforms, it was not significant for yeast and mold. Additionally, when storage container type and if peanuts were stored off the ground were included in the regression model, these methods were predictive of contamination levels for Enterobacteriaceae and coliforms. To our knowledge, this is the first study to analyze the relationship among Enterobacteriaceae, coliforms, and yeast and mold contamination and producer knowledge of Senegalese peanuts. These results provide preliminary data to inform future studies to determine pathogen prevalence and impactful preventative measures to minimize microbial contamination of peanuts produced in Senegal.

Occurrence of Chemical Contaminants in Peruvian Produce: A Food-Safety Perspective.

The presence of chemical contaminants in agricultural products is a continued food-safety challenge in Peru. This country has robust agriculture potential, but its output of fruits and vegetables is severely impacted by massive mining activities, as well as poor farming practices, including the use of polluted irrigation water, misuse of pesticides, and inadequate postharvest conditions. This review examines the current scientific knowledge on the levels of pesticide residues, heavy metals, and mycotoxins on crops produced in Peru. The available data shows that several crop varieties are contaminated with these classes of chemical contaminants, and at levels that exceed the national and international permissible limits. The abundance of chemical contaminants in produce indicates a relevant food-safety issue, which increases the risks of chronic human diseases, like cancer-a leading cause of death in Peru. Finally, this review presents recommendations to address these contamination problems in produce grown in the Andean country.

Alternative σ Factors Regulate Overlapping as Well as Distinct Stress Response and Metabolic Functions in Listeria monocytogenes under Stationary Phase Stress Condition.

Listeria monocytogenes can regulate and fine-tune gene expression, to adapt to diverse stress conditions encountered during foodborne transmission. To further understand the contributions of alternative sigma (σ) factors to the regulation of L. monocytogenes gene expression, RNA-Seq was performed on L. monocytogenes strain 10403S and five isogenic mutants (four strains bearing in-frame null mutations in three out of four alternative σ factor genes, ΔCHL, ΔBHL, ΔBCL, and ΔBCH, and one strain bearing null mutations in all four genes, ΔBCHL), grown to stationary phase. Our data showed that 184, 35, 34, and 20 genes were positively regulated by σB, σL, σH, and σC (posterior probability > 0.9 and Fold Change (FC) > 5.0), respectively. Moreover, σB-dependent genes showed the highest FC (based on comparisons between the ΔCHL and the ΔBCHL strain), with 44 genes showing an FC > 100; only four σL-dependent, and no σH- or σC-dependent genes showed FC >100. While σB-regulated genes identified in this study are involved in stress-associated functions and metabolic pathways, σL appears to largely regulate genes involved in a few specific metabolic pathways, including positive regulation of operons encoding phosphoenolpyruvate (PEP)-dependent phosphotransferase systems (PTSs). Overall, our data show that (i) σB and σL directly and indirectly regulate genes involved in several energy metabolism-related functions; (ii) alternative σ factors are involved in complex regulatory networks and appear to have epistatic effects in stationary phase cells; and (iii) σB regulates multiple stress response pathways, while σL and σH positively regulate a smaller number of specific pathways.

Lytic Capacity Survey of Commercial Listeria Phage Against Listeria spp. with Varied Genotypic and Phenotypic Characteristics.

Listeria monocytogenes is regularly isolated from food processing environments and is endemic in some facilities. Bacteriophage have potential as biocontrol strategies for L. monocytogenes. In this study, the lytic capacity of a commercial Listeria phage cocktail was evaluated against a library of 475 Listeria spp. isolates (426 L. monocytogenes and 49 other Listeria spp.) with varied genotypic and phenotypic characteristics. The lytic capacity of the Listeria phages was measured by spot assays where lysis was scored on a scale of 0-3 (0 = no lysis; 1 = slight lysis; 2 = moderate lysis; 3 = confluent lysis). Only 5% of all tested Listeria spp. isolates, including L. monocytogenes, were either moderately or highly susceptible (score 2 or 3) to lysis by Listeria phage when scores were averaged across temperature and phage concentration; 155 of 5700 treatment (multiplicity of infection [MOI] and temperature) and characteristic (genotype, sanitizer tolerance, and attachment capacity) combinations resulted in confluent lysis (score = 3). Odds ratios for susceptibility to lysis were calculated using multinomial logistic regression. The odds of susceptibility to lysis by phage decreased (p < 0.05) if the L. monocytogenes isolate was previously found to persist or if the phage-bacteria culture was incubated at 30°C; neither isolate persistence or temperature was significant (p ≥ 0.05) when all factors were considered. In addition, lytic efficacy varied (p < 0.05) among pulse field gel electrophoresis (PFGE) pulsotypes and may be affected by host MOI (p < 0.05). There was no effect (p > 0.05) of attachment capacity or sanitizer tolerance on phage susceptibility. This study underscores the complexity of using Listeria phage as a biocontrol for Listeria spp. in food processing facilities and highlights that phage susceptibility is most greatly impacted by genotype. Further studies are needed to evaluate these findings within a processing environment.

Genomic and Transcriptomic Analysis of Biofilm Formation in Persistent and Transient Listeria monocytogenes Isolates from the Retail Deli Environment Does Not Yield Insight into Persistence Mechanisms.

Persistence of Listeria monocytogenes in retail deli environments is a serious food safety issue, potentially leading to cross-contamination of ready-to-eat foods such as deli meats, salads, and cheeses. We previously discovered strong evidence of L. monocytogenes persistence in delis across multiple states. We hypothesized that this was correlated with isolates' innate characteristics, such as biofilm-forming capacity or gene differences. To test this hypothesis, we sequenced the genomes of 21 L. monocytogenes isolates previously collected longitudinally from the retail deli environment. Isolates were chosen to represent varying attachment capacity and sanitizer tolerance as well as persistence or transience. We used single-nucleotide polymorphism analysis to characterize the isolates' genetic relationships and used BLAST to search the isolates' genomes for antibiotic resistance elements, quaternary ammonium tolerance genes, and stress survival islets. We further chose four isolates for RNA-sequencing analysis and compared their global biofilm transcriptome with their global planktonic transcriptome. We did not find genetic content that explained persistence. The presence of stress survival islet-1 correlated to increased attachment capacity (p < 0.05), but not persistence. Further, the presence of sanitizer tolerance elements was not significantly correlated with phenotypic sanitizer tolerance. Analysis of biofilm versus planktonic gene expression did not show the expected differences in gene expression patterns. Overall, L. monocytogenes persistence in the deli environment is likely a matter of poor sanitation and/or facility design, rather than isolates' biofilm-forming capacity, sanitizer tolerance, or genomic content.

Food safety in Peru: A review of fresh produce production and challenges in the public health system.

Peru has a commodities-based economy where agriculture plays an essential role in the nation's development. Among agricultural products, fruits and vegetables are foundational to Peruvian culture and a healthy and nutritious diet. Produce is also the primary income source for thousands of small-scale farmers and producers throughout the country. Peru has significant potential to export agricultural and value-added products. Nevertheless, the Peruvian food chain has weak food safety and quality standards, limiting access to international markets. The inherent lack of food safety surveillance and management systems negatively affects public health. In the past decade, fresh and raw produce has been associated with several foodborne outbreaks worldwide, resulting in significant health and economic losses. This alarming situation for public health officials and regulators has called for the strengthening of produce safety standards and food safety risk management for safer food and to reduce the incidence of foodborne illnesses. This review summarizes the current status of produce safety in Peru and explores opportunities (e.g., policy, university capacity development) toward a safer food system.

Disinfectant wipes transfer Clostridioides difficile spores from contaminated surfaces to uncontaminated surfaces during the disinfection process.

BACKGROUND: Pre-wetted disinfectant wipes are increasingly being used in healthcare facilities to help address the risk of healthcare associated infections (HAIs). However, HAIs are still a major problem in the US with Clostridioides difficile being the most common cause, leading to approximately 12,800 deaths annually in the US. An underexplored risk when using disinfectant wipes is that they may cross-contaminate uncontaminated surfaces during the wiping process. The objective of this study was to determine the cross-contamination risk that pre-wetted disinfectant towelettes may pose when challenged with C. difficile spores. We hypothesized that although the tested disinfectant wipes had no sporicidal claims, they will reduce spore loads. We also hypothesized that hydrogen peroxide disinfectant towelettes would present a lower cross-contamination risk than quaternary ammonium products. METHODS: We evaluated the risk of cross-contamination when disinfectant wipes are challenged with C. difficile ATCC 43598 spores on Formica surfaces. A disinfectant wipe was used to wipe a Formica sheet inoculated with C. difficile. After the wiping process, we determined log10 CFU on previously uncontaminated pre-determined distances from the inoculation point and on the used wipes. RESULTS: We found that the disinfectant wipes transferred C. difficile spores from inoculated surfaces to previously uncontaminated surfaces. We also found that wipes physically removed C. difficile spores and that hydrogen peroxide disinfectants were more sporicidal than the quaternary ammonium disinfectants. CONCLUSION: Regardless of the product type, all disinfectant wipes had some sporicidal effect but transferred C. difficile spores from contaminated to otherwise previously uncontaminated surfaces. Disinfectant wipes retain C. difficile spores during and after the wiping process.

A rapid model for developing dry surface biofilms of Staphylococcus aureus and Pseudomonas aeruginosa for in vitro disinfectant efficacy testing.

BACKGROUND: Bacterial biofilms persistent on dry environmental surfaces in healthcare facilities play an important role in the occurrence of healthcare associated infections (HAI). Compared to wet surface biofilms and planktonic bacteria, dry surface biofilms (DSB) are more tolerant to disinfection. However, there is no official method for developing DSB for in vitro disinfectant efficacy testing. The objectives of this study were to (i) develop an in vitro model of DSB of S. aureus and P. aeruginosa for disinfectant efficacy testing and (ii) investigate the effect of drying times and temperatures on DSB development. We hypothesized that a minimum six log10 density of DSB could be achieved on glass coupons by desiccating wet surface biofilms near room temperatures. We also hypothesized that a DSB produced by the model in this study will be encased in extracellular polymeric substances (EPS). METHODS: S. aureus ATCC-6538 and P. aeruginosa ATCC-15442 wet surface biofilms were grown on glass coupons following EPA MLB SOP MB-19. A DSB model was developed by drying coupons in an incubator and viable bacteria were recovered following a modified version of EPA MLB SOP MB-20. Scanning electron microscopy was used to confirm the EPS presence on DSB. RESULTS: Overall, a minimum of six mean log10 densities of DSB for disinfectant efficacy were recovered per coupon after drying at different temperatures and drying times. Regardless of strain, temperature and dry time, 86% of coupons with DSB were confirmed to have EPS. CONCLUSION: A rapid model for developing DSB with characteristic EPS was developed for disinfectant efficacy testing against DSB.

Cross-contamination by disinfectant towelettes varies by product chemistry and strain.

BACKGROUND: Disinfectant products are used frequently on environmental surfaces (e.g. medical equipment, countertops, patient beds) and patient care equipment within healthcare facilities. The purpose of this study was to assess the risk of cross-contamination of Staphylococcus aureus and Pseudomonas aeruginosa during and after disinfection of predetermined surface areas with ready-to-use (RTU) pre-wetted disinfectant towelettes. METHODS: This study tested six disinfectant towelette products against S. aureus ATCC CRM-6538 and P. aeruginosa strain ATCC-15442 on Formica surfaces. Each disinfectant was evaluated on a hard nonporous surface and efficacy was measured every 0.5 m2 using a modified version of EPA MLB SOP-MB-33 to study the risk of cross-contamination. RESULTS: We found that all of the wipes used in this study transferred S. aureus and P. aeruginosa from an inoculated surface to previously uncontaminated surfaces. Disinfectant towelettes with certain chemistries also retained a high level of viable bacteria after disinfection of the surface area. The cross-contamination risk also varied by product chemistry and bacterial strain. CONCLUSION: Disinfectant wipes can cross-contaminate hard nonporous surfaces and retain viable bacterial cells post-disinfection, especially over larger surface areas. This highlights a need to further investigate the risk disinfectant wipes pose during and post-disinfection and guidance on maximum surface areas treated with a single towelette.

Prevalence and Antimicrobial Resistance of Salmonella from Antibiotic-Free Broilers During Organic and Conventional Processing.

ABSTRACT: Salmonella is one of the top causes for bacterial foodborne infections in the United States, emphasizing the importance of controlling this pathogen for protecting public health. Poultry and poultry products are commonly associated with Salmonella, and interventions during production and processing are necessary to manage the risk of infection due to consumption of poultry products. In recent times, the demand for organic and antibiotic-free poultry has increased owing to consumer perceptions and concerns of increasing prevalence of antimicrobial-resistant (AMR) pathogens. However, the microbiological effect of these management practices is not clear. This study was conducted to determine the difference in the AMR of Salmonella isolated from poultry processed conventionally and organically. Fecal samples, carcass rinses, and environmental samples were collected over 1 year and analyzed for the prevalence of Salmonella and AMR. Results of this experiment showed that organic chickens were associated with statistically higher levels of Salmonella during early processing steps. However, no difference in Salmonella prevalence was observed between organic and conventional carcasses postchill. In addition, for most antimicrobial agents tested, prevalence of AMR Salmonella in conventional processing was lower in this study than was reported by the National Antimicrobial Resistance Monitoring System for chickens at slaughter. These observations indicate that organic methods may introduce greater risk of Salmonella contamination; however, proper interventions during processing can abate this risk. In addition, this study supports the assertion that raising chickens without the use of antibiotics may result in lower prevalence of AMR Salmonella.

Listeria monocytogenes σA Is Sufficient to Survive Gallbladder Bile Exposure.

Listeria monocytogenes is a foodborne Gram-positive bacterium causing listeriosis in both animals and humans. It can persist and grow in various environments including conditions countered during saprophytic or intra-host lifestyles. Sigma (σ) subunit of RNA polymerase is a transcriptional factor responsible for guiding the core RNA polymerase and initiating gene expression under normal growth or physiological changes. In L. monocytogenes, there is one housekeeping sigma factor, σA, and four alternative sigma factors σB, σC, σH, and σL. Generally, σA directs expression of genes required for normal growth while alternative σ factors alter gene expression in response to specific conditions (e.g., stress). In this study, we aimed to determine the exclusive role of σA in L. monocytogenes by comparing a wild type strain with its isogenic mutant lacking genes encoding all alternative sigma factors (i.e., sigB, sigC, sigH, and sigL). We further investigated their survival abilities in 6% porcine bile (pH 8.2) mimicking gallbladder bile and their transcriptomics profiles in rich medium (i.e., BHI) and 1% porcine bile. Surprisingly, the results showed that survival abilities of wild type and ΔsigBΔsigCΔsigHΔsigL (or ΔsigBCHL) quadruple mutant strains in 6% bile were similar suggesting a compensatory role for σA. RNA-seq results revealed that bile stimulon of L. monocytogenes wild type contained 66 genes (43 and 23 genes were up- and down-regulated, respectively); however, only 29 genes (five up- and 24 down-regulated by bile) were differentially expressed in ΔsigBCHL. We have shown that bile exposure mediates increased transcription levels of dlt and ilv operons and decreased transcription levels of prfA and heat shock genes in wild type. Furthermore, we identified σA-dependent bile inducible genes that are involved in phosphotransferase systems, chaperones, and transporter systems; these genes appear to contribute to L. monocytogenes cellular homeostasis. As a result, σA seemingly plays a compensatory role in the absence of alternative sigma factors under bile exposure. Our data support that the bile stimulon is prone to facilitate resistance to bile prior to initiated infection.

Salmonella enterica subsp. enterica Serovar Heidelberg Food Isolates Associated with a Salmonellosis Outbreak Have Enhanced Stress Tolerance Capabilities.

Salmonella enterica serovar Heidelberg is currently the 12th most common serovar of Salmonella enterica causing salmonellosis in the United States and results in twice the average incidence of blood infections caused by nontyphoidal salmonellae. Multiple outbreaks of salmonellosis caused by Salmonella Heidelberg resulted from the same poultry processor, which infected 634 people during 2013 and 2014. The hospitalization and invasive illness rates were 38% and 15%, respectively. We hypothesized that the outbreak strains of Salmonella Heidelberg had enhanced stress tolerance and virulence capabilities. We sourced nine food isolates collected during the outbreak investigation and three reference isolates to assess their tolerance to heat and sanitizers, ability to attach to abiotic surfaces, and invasiveness in vitro We performed RNA sequencing on three isolates (two outbreak-associated isolates and a reference Salmonella Heidelberg strain) with various levels of heat tolerance to gain insight into the mechanism behind the isolates' enhanced heat tolerance. We also performed genomic analyses to determine the genetic relationships among the outbreak isolates. Ultimately, we determined that (i) six Salmonella Heidelberg isolates associated with the foodborne outbreak had enhanced heat tolerance, (ii) one outbreak isolate with enhanced heat tolerance also had an enhanced biofilm-forming ability under stressful conditions, (iii) exposure to heat stress increased the expression of Salmonella Heidelberg multidrug efflux and virulence genes, and (iv) outbreak-associated isolates were likely transcriptionally primed to better survive processing stresses and, potentially, to cause illness.IMPORTANCE This study provides a deep analysis of the intrinsic stress tolerance and virulence capabilities of Salmonella Heidelberg that may have contributed to the length and severity of a recent salmonellosis outbreak. Additionally, this study provides a comprehensive analysis of the transcriptomic response of S. enterica strains to heat stress conditions and compares baseline stationary-phase gene expression among outbreak- and non-outbreak-associated Salmonella Heidelberg isolates. These data can be used in assay development to screen isolates for stress tolerance and subsequent survival. This study adds to our understanding of the strains associated with the outbreak and informs ongoing regulatory discussions on Salmonella in poultry.

Assessment of early onset surface damage from accelerated disinfection protocol.

Background: The objective of this study was to evaluate the extent and potential mechanisms of early onset surface damage from simulated wiping typical of six-months of routine disinfection and to assess the subsequent microbial risk of surfaces damaged by disinfectants. Methods: Eight common material surfaces were exposed to three disinfectants and a neutral cleaner (neutral cleaner, quaternary ammonium, hydrogen peroxide, sodium hypochlorite) in accelerated aging tests to simulate a long-term disinfection routine. Materials were also immersed in dilute and concentrated chemical solutions to induce surface damage. Surfaces were chemically and physically characterized to determine extent of surface damage. Bactericidal efficacy testing was performed on the Quat-based disinfectant using a modified version of EPA standard operating procedure MB-25-02. Results: The wiping protocol increased surface roughness for some material surfaces due to mechanical abrasion of the wiping cloth. The increased roughness did not correlate with changes in bactericidal efficacy. Chemical damage was observed for some surface-disinfectant combinations. The greatest observed effects from disinfectant exposure was in changes in wettability or water contact angle. Conclusions: Early onset surface damage was observed in chemical and physical characterization methods. These high-throughput material measurement methods were effective at assessing nanoscale disinfectant-surface compatibility which may go undetected though routine macroscale testing.

There is no additional bactericidal efficacy of Environmental Protection Agency-registered disinfectant towelettes after surface drying or beyond label contact time.

BACKGROUND: Disinfectant towelettes are commonly used for surface disinfection to prevent health care-associated infections; however, there is limited consensus as to whether a surface needs to remain wet for the full label contact time after the disinfectant towelette has been used in order for complete efficacy to be achieved. The purpose of this study was to quantify the effect of contact time, including times before and after a product dries, on bactericidal efficacy of 6 towelette products registered by the Environmental Protection Agency . METHODS: Six disinfectant towelette products were tested at varying contact times, including defined label contact times. Quantitative Environmental Protection Agency MB-33-00 was used to measure disinfectant efficacy against Staphylococcus aureus on Formica. Complete dry time for each disinfectant was measured gravimetrically. RESULTS: There were significant differences in dry times among the towelette products; contact time did not have a significant effect on bactericidal efficacy. There was no longitudinal effect when a disinfectant's contact time was greater than defined label contact time, irrespective of whether the product was wet or dry on the surface. DISCUSSION: Overall, bactericidal efficacy varied by towelette product tested and surface area wiped. Wiping larger surface areas may lead to decreased bactericidal efficacy but is product dependent. CONCLUSIONS: There was no additional bactericidal effect after a product dried, indicating that extended contact times beyond when the product dries will not enhance disinfection.