Surveillance of pathogenic bacteria on a food matrix using machine-learning-enabled paper chromogenic arrays.

Global food systems can benefit significantly from continuous monitoring of microbial food safety, a task for which tedious operations, destructive sampling, and the inability to monitor multiple pathogens remain challenging. This study reports significant improvements to a paper chromogenic array sensor - machine learning (PCA-ML) methodology sensing concentrations of volatile organic compounds (VOCs) emitted on a species-specific basis by pathogens by streamlining dye selection, sensor fabrication, database construction, and machine learning and validation. This approach enables noncontact, time-dependent, simultaneous monitoring of multiple pathogens (Listeria monocytogenes, Salmonella, and E. coli O157:H7) at levels as low as 1 log CFU/g with over 90% accuracy. The report provides theoretical and practical frameworks demonstrating that chromogenic response, including limits of detection, depends on time integrals of VOC concentrations. The paper also discusses the potential for implementing PCA-ML in the food supply chain for different food matrices and pathogens, with species- and strain-specific identification.

Adaptive Laboratory Evolution of Probiotics toward Oxidative Stress Using a Microfluidic-Based Platform.

Adaptive laboratory evolution (ALE) can be used to make bacteria less susceptible to oxidative stress. An alternative to large batch scale ALE cultures is to use microfluidic platforms, which are often more economical and more efficient. Microfluidic ALE platforms have shown promise, but many have suffered from subpar cell passaging mechanisms and poor spatial definition. A new approach is presented using a microfluidic Evolution on a Chip (EVoc) design which progressively drives microbial cells from areas of lower H2 O2 concentration to areas of higher concentration. Prolonged exposure, up to 72 h, revealed the survival of adaptive strains of Lacticaseibacillus rhamnosus GG, a beneficial probiotic often included in food products. After performing ALE on this microfluidic platform, the bacteria persisted under high H2 O2 concentrations in repeated trials. After two progressive exposures, the ability of L. rhamnosus to grow in the presence of H2 O2 increased from 1 mm H2 O2 after a lag time of 31 h to 1 mm after 21 h, 2 mm after 28 h, and 3 mm after 42 h. The adaptive strains have different morphology, and gene expression compared to wild type, and genome sequencing revealed a potentially meaningful single nucleotide mutation in the protein omega-amidase.

Survivability of Clostridioides difficile spores in fermented pork summer sausage during refrigerated storage.

Background and Aim: Clostridioides difficile is a spore-forming pathogen that causes serious enteric disease in humans. Strains have been isolated from food animals and meat, including pork, which suggest a potential for foodborne transmission. Pork summer sausage is a popular fermented meat product, which is consumed cooked or cooked to a lower internal temperature due to acidification of the product. The effect of acidity and cooking on the viability of C. difficile spores in a fermented meat product has not been determined. Therefore, the aim was to study the survivability of C. difficile spores in fermented pork summer sausage. Materials and Methods: Fermented pork sausages were prepared according to a commercial recipe with or without starter culture and C. difficile spores followed by fermentation at 37°C for ~12 h under 85% relative humidity until pH 5.0 was reached and further processed as cooked (>57°C) or uncooked (≤57°C) and stored at 4°C. C. difficile spores in sausages were enumerated at 1 h following inoculation and on days 0, 1, 7, 14, 21, 30, 60, and 90 of storage. Results: It was observed that C. difficile spore viability in control unfermented treatment was significantly different on day 0 from the fermented, fermented cooked, and control unfermented cooked treatments (p<0.05); however, there was no significant difference among the latter three treatment groups throughout 90 days of storage (p>0.05). On day 90 of storage, the unfermented control sausages yielded ~4.0 log colony-forming unit (CFU)/g of C. difficile spores compared to ~3.5 log CFU/g recovered from fermented samples and the unfermented cooked control samples identifying spore viability in all treatment groups. Conclusion: C. difficile spores were found to survive the acidity and cooking of fermented pork summer sausage and storage at 4°C for 3 months, thereby highlighting the need for effective intervention strategies to reduce the risk of C. difficile contamination in pork products.

Survival of Salmonella enterica in Military Low-Moisture Food Products during Long-Term Storage at 4, 25, and 40°C.

ABSTRACT: Salmonella enterica has been increasingly implicated in foodborne outbreaks involving low-moisture foods (LMF) during the recent decade. This study aimed to investigate the potential for persistence of S. enterica in a range of LMF during storage at three temperatures. LMF products, boil-in-bag eggs (freeze-dried product), chocolate protein drink, cran-raspberry First Strike bars, mocha dessert bar, and peanut butter, were inoculated with a five-strain cocktail of S. enterica and stored at 4, 25, or 40°C for 36 months. Salmonella populations remained above 7 log CFU/g in all products stored at 4°C and above 6 log CFU/g in products stored at 25°C, excluding the cran-raspberry First Strike bars. Storage at 40°C resulted in Salmonella populations above 5.5 log CFU/g in boil-in-bag eggs after 36 months and demonstrated survivability for 12 months or less in the other five products. Additionally, a mocha bar production temperature profile study identified rapid cooling of bars in which the temperatures reached would have no measurable impact on Salmonella populations. The results indicate the ability of Salmonella to survive in a variety of LMF category foods, even under adverse storage conditions and identifies how the food matrix may affect Salmonella survivability. The data indicate the importance of establishing food processing procedures that adequately mitigate the presence of Salmonella throughout food processing systems, while also increasing comprehensive understanding of Salmonella survivability mechanisms.

Carvacrol reduces Clostridium difficile sporulation and spore outgrowth in vitro.

PURPOSE: Clostridium difficile is an anaerobic spore-forming pathogen that causes a serious toxin-mediated enteric disease in humans. Therapeutic agents that are capable of reducing C. difficile spore production could significantly minimize the transmission and relapse of C. difficile infections. This study investigated the efficacy of a food-grade, plant-derived compound, carvacrol (CR), in reducing C. difficile spore production, germination and spore outgrowth. METHODOLOGY: Two hyper-virulent C. difficile isolates (ATCC BAA 1870 and 1805) were grown with or without a sub-inhibitory concentration (SIC) of CR. Total viable counts and heat-resistant spore counts were determined at different time intervals. Moreover, spores and vegetative cells were visualized using phase-contrast microscopy. To determine the effect of CR on C. difficile germination and spore outgrowth, C. difficile spores were seeded in germination medium with or without the SIC and MIC of CR, and spore germination and spore outgrowth were measured by recording optical density at 600 nm. The effect of CR on C. difficile sporulation genes was also investigated using real-time qPCR. RESULTS: Carvacrol significantly reduced sporulation in C. difficile and down-regulated critical genes involved in spore production (P<0.05). The SIC or MIC of CR did not inhibit C. difficile spore germination; however, the MIC of CR completely inhibited spore outgrowth. CONCLUSION: The results suggest that CR could potentially be used to control C. difficile by reducing spore production and outgrowth.

Protective Effect of Carvacrol against Gut Dysbiosis and Clostridium difficile Associated Disease in a Mouse Model.

This study investigated the effect of carvacrol (CR), a phytophenolic compound on antibiotic-associated gut dysbiosis and C. difficile infection in a mouse model. Five to six-week-old C57BL/6 mice were randomly divided into seven treatment groups (challenge and control) of eight mice each. Mice were fed with irradiated feed supplemented with CR (0, 0.05, and 0.1%); the challenge groups were made susceptible to C. difficile by orally administering an antibiotic cocktail in water and an intra-peritoneal injection of clindamycin. Both challenge and control groups were infected with 105CFU/ml of hypervirulent C. difficile (ATCC 1870) spores or PBS, and observed for clinical signs for 10 days. Respective control groups for CR, antibiotics, and their combination were included for investigating their effect on mouse enteric microflora. Mouse body weight and clinical and diarrhea scores were recorded daily post infection. Fecal samples were collected for microbiome analysis using rRNA sequencing in MiSeq platform. Carvacrol supplementation significantly reduced the incidence of diarrhea and improved the clinical and diarrhea scores in mice (p < 0.05). Microbiome analysis revealed a significant increase in Proteobacteria and reduction in the abundance of protective bacterial flora in antibiotic-treated and C. difficile-infected mice compared to controls (p < 0.05). However, CR supplementation positively altered the microbiome composition, as revealed by an increased abundance of beneficial bacteria, including Firmicutes, and significantly reduced the proportion of detrimental flora such as Proteobacteria, without significantly affecting the gut microbiome diversity compared to control. Results suggest that CR could potentially be used to control gut dysbiosis and reduce C. difficile infection.

Characterization of a multidrug resistant C. difficile meat isolate.

Clostridium difficile is a pathogen of significant public health concern causing a life-threatening, toxin-mediated enteric disease in humans. The incidence and severity of the disease associated with C. difficile have increased in the US with the emergence of hypervirulent strains and community associated outbreaks. The detection of genotypically similar and identical C. difficile strains implicated from human infections in foods and food animals indicates the potential role of food as a source of community associated C. difficile disease. One hundred samples each of ground beef, pork and chicken obtained from geographically distant grocery stores in Connecticut were tested for C. difficile. Positive isolates were characterized by ribotyping, antibiotic susceptibility, toxin production and whole genome sequencing. Of the 300 meat samples, only two pork samples tested positive for C. difficile indicating a very low prevalence of C. difficile in meat. The isolates were non toxigenic; however, genome characterization revealed the presence of several antibiotic resistance genes and mobile elements that can potentially contribute to generation of multidrug resistant toxigenic C. difficile by horizontal gene transfer. Further studies are warranted to investigate potential food-borne transmission of the meat isolates and development of multi-drug resistance in these strains.

Carvacrol and trans-cinnamaldehyde reduce Clostridium difficile toxin production and cytotoxicity in vitro.

Clostridium difficile is a nosocomial pathogen that causes a serious toxin-mediated enteric disease in humans. Reducing C. difficile toxin production could significantly minimize its pathogenicity and improve disease outcomes in humans. This study investigated the efficacy of two, food-grade, plant-derived compounds, namely trans-cinnamaldehyde (TC) and carvacrol (CR) in reducing C. difficile toxin production and cytotoxicity in vitro. Three hypervirulent C. difficile isolates were grown with or without the sub-inhibitory concentrations of TC or CR, and the culture supernatant and the bacterial pellet were collected for total toxin quantitation, Vero cell cytotoxicity assay and RT-qPCR analysis of toxin-encoding genes. The effect of CR and TC on a codY mutant and wild type C. difficile was also investigated. Carvacrol and TC substantially reduced C. difficile toxin production and cytotoxicity on Vero cells. The plant compounds also significantly down-regulated toxin production genes. Carvacrol and TC did not inhibit toxin production in the codY mutant of C. difficile, suggesting a potential codY-mediated anti-toxigenic mechanism of the plant compounds. The antitoxigenic concentrations of CR and TC did not inhibit the growth of beneficial gut bacteria. Our results suggest that CR and TC could potentially be used to control C. difficile, and warrant future studies in vivo.