Multi-functional nanozyme-based colorimetric, fluorescence dual-mode assay for Salmonella typhimurium detection in milk.

Rapid and high-sensitive Salmonella detection in milk is important for preventing foodborne disease eruption. To overcome the influence of the complex ingredients in milk on the sensitive detection of Salmonella, a dual-signal reporter red fluorescence nanosphere (RNs)-Pt was designed by combining RNs and Pt nanoparticles. After being equipped with antibodies, the immune RNs-Pt (IRNs-Pt) provide an ultra-strong fluorescence signal when excited by UV light. With the assistance of the H2O2/TMB system, a visible color change appeared that was attributed to the strong peroxidase-like catalytic activity derived from Pt nanoparticles. The IRNs-Pt in conjunction with immune magnetic beads can realize that Salmonella typhimurium (S. typhi) was captured, labeled, and separated effectively from untreated reduced-fat pure milk samples. Under the optimal experimental conditions, with the assay, as low as 50 CFU S. typhi can be converted to detectable fluorescence and absorbance signals within 2 h, suggesting the feasibility of practical application of the assay. Meanwhile, dual-signal modes of quantitative detection were realized. For fluorescence signal detection (emission at 615 nm), the linear correlation between signal intensity and the concentration of S. typhi was Y = 83C-3321 (R2 = 0.9941), ranging from 103 to 105 CFU/mL, while for colorimetric detection (absorbamce at 450 nm), the relationship between signal intensity and the concentration of S. typhi was Y = 2.9logC-10.2 (R2 = 0.9875), ranging from 5 × 103 to 105 CFU/mL. For suspect food contamination by foodborne pathogens, this dual-mode signal readout assay is promising for achieving the aim of convenient preliminary screening and accurate quantification simultaneously.

Red cabbage extract immobilized in bacterial cellulose film as an eco-friendly sensor to monitor microbial contamination and gamma irradiation of stored cucumbers.

The aim of the present study is to develop a pH-sensing biopolymer film based on the immobilization of red cabbage extract (RCE) within bacterial cellulose (BC) to detect contamination and gamma radiation exposure in cucumbers. The results obtained show a sensitivity to pH changes for RCE in its aqueous form and that incorporated within BC films (RCE-BC), both showed color change correlated to bacterial growth (R2 = 0.91), this was supported with increase in pH values from 2 to 12 (R2 = 0.98). RCE and RCE-BC exposure to gamma radiation (0, 2.5, 5, 10, 15, 20, 25 kGy) resulted in gradual decrease in color that was more evident in RCE aqueous samples. To sense bacterial contamination of cucumbers, the total count was followed at 0, 5, 10 and 15 days in cold storage conditions and was found to reach 9.13 and 5.47 log cfu/mL for non-irradiated and 2 kGy irradiated samples, respectively. The main isolates detected throughout this storage period were identified as Pseudomonas fluorescens, Erwinia sp. Pantoea agglomerans using matrix assisted laser desorption ionization-time of flight-ms (MALDI-TOF-MS). Bacterial growth in stored irradiated cucumbers was detected by color change within 5 and 10 days of storage, after which there was no evident change. This is very useful since contamination within the early days of storage cannot be sensed with the naked eye. This study is the first to highlight utilizing RCE and RCE-BC as eco-friendly pH-sensing indicator films for intelligent food packaging to detect both food contamination and gamma preservation for refrigerator stored cucumbers.

Reported Incidence of Infections Caused by Pathogens Transmitted Commonly Through Food: Impact of Increased Use of Culture-Independent Diagnostic Tests - Foodborne Diseases Active Surveillance Network, 1996-2023.

Reducing foodborne disease incidence is a public health priority. This report summarizes preliminary 2023 Foodborne Diseases Active Surveillance Network (FoodNet) data and highlights efforts to increase the representativeness of FoodNet. During 2023, incidences of domestically acquired campylobacteriosis, Shiga toxin-producing Escherichia coli infection, yersiniosis, vibriosis, and cyclosporiasis increased, whereas those of listeriosis, salmonellosis, and shigellosis remained stable compared with incidences during 2016-2018, the baseline used for tracking progress towards federal disease reduction goals. During 2023, the incidence and percentage of infections diagnosed by culture-independent diagnostic tests (CIDTs) reported to FoodNet continued to increase, and the percentage of cases that yielded an isolate decreased, affecting observed trends in incidence. Because CIDTs allow for diagnosis of infections that previously would have gone undetected, lack of progress toward disease reduction goals might reflect changing diagnostic practices rather than an actual increase in incidence. Continued surveillance is needed to monitor the impact of changing diagnostic practices on disease trends, and targeted prevention efforts are needed to meet disease reduction goals. During 2023, FoodNet expanded its catchment area for the first time since 2004. This expansion improved the representativeness of the FoodNet catchment area, the ability of FoodNet to monitor trends in disease incidence, and the generalizability of FoodNet data.

Prospective modeling and estimating the epidemiologically informative match rate within large foodborne pathogen genomic databases.

OBJECTIVES: Much has been written about the utility of genomic databases to public health. Within food safety these databases contain data from two types of isolates-those from patients (i.e., clinical) and those from non-clinical sources (e.g., a food manufacturing environment). A genetic match between isolates from these sources represents a signal of interest. We investigate the match rate within three large genomic databases (Listeria monocytogenes, Escherichia coli, and Salmonella) and the smaller Cronobacter database; the databases are part of the Pathogen Detection project at NCBI (National Center for Biotechnology Information). RESULTS: Currently, the match rate of clinical isolates to non-clinical isolates is 33% for L. monocytogenes, 46% for Salmonella, and 7% for E. coli. These match rates are associated with several database features including the diversity of the organism, the database size, and the proportion of non-clinical BioSamples. Modeling match rate via logistic regression showed relatively good performance. Our prediction model illustrates the importance of populating databases with non-clinical isolates to better identify a match for clinical samples. Such information should help public health officials prioritize surveillance strategies and show the critical need to populate fledgling databases (e.g., Cronobacter sakazakii).

Inhibition and Mechanism of Citral on Bacillus cereus Vegetative Cells, Spores, and Biofilms.

Bacillus cereus causes food poisoning by producing toxins that cause diarrhea and vomiting and, in severe cases, endocarditis, meningitis, and other diseases. It also tends to form biofilms and spores that lead to contamination of the food production environment. Citral is a potent natural antibacterial agent, but its antibacterial activity against B. cereus has not been extensively studied. In this study, we first determined the minimum inhibitory concentrations and minimum bactericidal concentrations, growth curves, killing effect in different media, membrane potential, intracellular adenosine triphosphate (ATP), reactive oxygen species levels, and morphology of vegetative cells, followed by germination rate, morphology, germination state of spores, and finally biofilm clearance effect. The results showed that the minimum inhibitory concentrations and minimum bactericidal concentrations of citral against bacteria ranged from 100 to 800 µg/mL. The lag phase of bacteria was effectively prolonged by citral, and the growth rate of bacteria was slowed down. Bacteria in Luria-Bertani broth were reduced to below the detection limit by citral at 800 µg/mL within 0.5 h. Bacteria in rice were reduced to 3 log CFU/g by citral at 4000 µg/mL within 0.5 h. After treatment with citral, intracellular ATP concentration was reduced, membrane potential was altered, intracellular reactive oxygen species concentration was increased, and normal cell morphology was altered. After treatment with citral at 400 µg/mL, spore germination rate was reduced to 16.71%, spore morphology was affected, and spore germination state was altered. It also had a good effect on biofilm removal. The present study showed that citral had good bacteriostatic activity against B. cereus vegetative cells and its spores and also had a good clearance effect on its biofilm. Citral has the potential to be used as a bacteriostatic substance for the control of B. cereus in food industry production.

Assessment of the Sensitivity of Foodborne Cronobacter spp. Strains and Other Enterobacteria to Temperature Stresses and Chlorine-Containing Biocides.

We examined the effects of elevated temperatures and biocides on survivability of food isolates of Cronobacter spp. (C. sakazakii) and concomitant enterobacteriaceae obtained in microbiological control of infant nutrition products. Increased resistance of certain strains of Cronobacter, Enterobacter cloacae, and Pantoea spp. to thermal processing was revealed. Salmonella, Pantoea, and Cronobacter bacteria were least sensitive to antimicrobial action of chlorine-containing agents. The above properties varied in the strains of the same species. Specifically, only two of three examined isolates of Cronobacter spp. demonstrated lower sensitivity to heat in comparison with the enterobacterial test-cultures of other species.

Phenotypic characterization and genomic analysis of a Salmonella phage L223 for biocontrol of Salmonella spp. in poultry.

The escalating incidence of foodborne salmonellosis poses a significant global threat to food safety and public health. As antibiotic resistance in Salmonella continues to rise, there is growing interest in bacteriophages as potential alternatives. In this study, we isolated, characterized, and evaluated the biocontrol efficacy of lytic phage L223 in chicken meat. Phage L223 demonstrated robust stability across a broad range of temperatures (20-70 °C) and pH levels (2-11) and exhibited a restricted host range targeting Salmonella spp., notably Salmonella Typhimurium and Salmonella Enteritidis. Characterization of L223 revealed a short latent period of 30 min and a substantial burst size of 515 PFU/cell. Genomic analysis classified L223 within the Caudoviricetes class, Guernseyvirinae subfamily and Jerseyvirus genus, with a dsDNA genome size of 44,321 bp and 47.9% GC content, featuring 72 coding sequences devoid of antimicrobial resistance, virulence factors, toxins, and tRNA genes. Application of L223 significantly (p < 0.005) reduced Salmonella Typhimurium ATCC 14,028 counts by 1.24, 2.17, and 1.55 log CFU/piece after 2, 4, and 6 h of incubation, respectively, in experimentally contaminated chicken breast samples. These findings highlight the potential of Salmonella phage L223 as a promising biocontrol agent for mitigating Salmonella contamination in food products, emphasizing its relevance for enhancing food safety protocols.

Isolation, whole genome sequencing and application of a broad-spectrum Salmonella phage.

Salmonella is considered as one of the most common zoonotic /foodborne pathogens in the world. The application of bacteriophages as novel antibacterial agents in food substrates has become an emerging strategy. Bacteriophages have the potential to control Salmonella contamination.We have isolated and characterized a broad-spectrum Salmonella phage, SP154, which can lyse 9 serotypes, including S. Enteritidis, S. Typhimurium, S. Pullorum, S. Arizonae, S. Dublin, S. Cholerasuis, S. Chester, S. 1, 4, [5], 12: i: -, and S. Derby, accounting for 81.9% of 144 isolates. SP154 showed a short latent period (40 min) and a high burst size (with the first rapid burst size at 107 PFUs/cell and the second rapid burst size at approximately 40 PFUs/cell). Furthermore, SP154 activity has higher survival rates across various environmental conditions, including pH 4.0-12.0 and temperatures ranging from 4 to 50 °C for 60 min, making it suitable for diverse food processing and storage applications. Significant reductions in live Salmonella were observed in different foods matrices such as milk and chicken meat, with a decrease of up to 1.9 log10 CFU/mL in milk contamination and a 1 log10 CFU/mL reduction in chicken meat. Whole genome sequencing analysis revealed that SP154 belongs to the genus Ithacavirus, subfamily Humphriesvirinae, within the family Schitoviridae. Phylogenetic analysis based on the terminase large subunit supported this classification, although an alternate tree using the tail spike protein gene suggested affiliation with the genus Kuttervirus, underscoring the limitations of relying on a single gene for phylogenetic inference. Importantly, no virulence or antibiotic resistance genes were detected in SP154. Our research highlights the potential of using SP154 for biocontrol of Salmonella in the food industry.

An integrated magnetic separation enzyme-linked colorimetric sensing platform for field detection of Escherichia coli O157: H7 in food.

An intelligent colorimetric sensing platform integrated with in situ immunomagnetic separation function was developed for ultrasensitive detection of Escherichia coli O157: H7 (E. coli O157: H7) in food. Captured antibody modified magnetic nanoparticles (cMNPs) and detection antibody/horseradish peroxidase (HRP) co-functionalized AuNPs (dHAuNPs) were firstly synthesized for targeted enrichment and colorimetric assay of E. coli O157: H7, in which remarkable signal amplification was realized by loading large amounts of HRP on the surface of AuNPs. Coupling with the optical collimation attachments and embedded magnetic separation module, a highly integrated optical device was constructed, by which in situ magnetic separation and high-quality imaging of 96-well microplates containing E. coli O157: H7 was achieved with a smartphone. The concentration of E. coli O157: H7 could be achieved in one-step by performing digital image colorimetric analysis of the obtained image with a custom-designed app. This biosensor possesses high sensitivity (1.63 CFU/mL), short detecting time (3 h), and good anti-interference performance even in real-sample testing. Overall, the developed method is expected to be a novel field detection platform for foodborne pathogens in water and food as well as for the diagnosis of infections due to its portability, ease of operation, and high feasibility.

One-pot electrochemical detection of foodborne pathogen based on in situ nucleic acid amplification and wash-free assay.

A signal amplification electrochemical biosensor chip was developed to integrate loop-mediated isothermal amplification (LAMP) based on in situ nucleic acid amplification and methyl blue (MB) serving as the hybridization redox indicator for sensitive and selective foodborne pathogen detection without a washing step. The electrochemical biosensor chip was designed by a screen-printed carbon electrode modified with gold nanoparticles (Au NPs) and covered with polydimethylsiloxane membrane to form a microcell. The primers of the target were immobilized on the Au NPs by covalent attachment for in situ amplification. The electroactive MB was used as the electrochemical signal reporter and embedded into the double-stranded DNA (dsDNA) amplicons generated by LAMP. Differential pulse voltammetry was introduced to survey the dsDNA hybridization with MB, which differentiates the specifically electrode-unbound and -bound labels without a washing step. Pyrene as the back-filling agent can further improve response signaling by reducing non-specific adsorption. This method is operationally simple, specific, and effective. The biosensor showed a detection linear range of 102-107 CFU mL-1 with the limit of detection of 17.7 CFU mL-1 within 40 min. This method showed promise for on-site testing of foodborne pathogens and could be integrated into an all-in-one device.

The Similarities in Microbial and Chemical Patterns of Fermentation in Two Open Environments were Promoted by Using 150-Year-Old Nukadoko as Starters.

Nukadoko, a fermented rice bran employed in traditional Japanese pickling, uses lactic acid bacteria to ferment vegetables. Here, we report the microbial and chemical data of a mixture of matured 150-year-old nukadoko and commercially available rice bran placed in two open environments over 29 days. Across the two environments, Loigolactobacillus was identified as the dominant microbial genera in the later stages of fermentation in nukadoko. The period of increase in the relative abundance of Loigolactobacillus correlated with a decrease in pH and Oxidation-Reduction Potential (ORP) values. While the two environments showed a difference in the rate of change in microbial diversity, they shared the common process through which Loigolactobacillus outcompeted adventitious bacteria in nukadoko, as indicated by the alpha and beta diversity index. Thus, the similarities in microbial and chemical data across two open environments during fermentation using starters indicate that starters contribute to the stability of fermentation in open environments.

Effects of different marination conditions on the physico-chemical and microbiological quality of anchovy (Engraulis encrasicolus) fillets inoculated with Morganella psychrotolerans during cold storage.

This study is aimed to determine the effects of different marination conditions (1, 2, 3, 4% acetic and 6, 8, 10% NaCl) on the anchovy fillets inoculated with Morganella psychrotolerans during refrigerated storage (4±1°C) for three months. According to the results of study, marination has great inhibitory effects on the growth of M. psychrotolerans. Total psychrophilic bacteria, total lactic acid bacteria, total yeast and mold, Total Enterobacteriaceae and M. psychrotolerans growth were not observed in the groups treated with 3 and 4% acetic acid. Control groups and fillets marinated with 1% acetic acid showed lower sensory scores. Those groups were rejected on 30th, 45th and 60th days of the storage, respectively, while the groups marinated with 2%, 3%, and 4% acetic acid had higher sensory scores and they were still acceptable until at the end of the study. According to peroxide value (PV) and thiobarbituric acid reactive substances (TBARS) assessment, lipid oxidation was delayed in the fillets marinated with high acetic acid concentrations (3 and 4%) comparing with the control and other inoculated fillets. From this research it can be revealed that high acetic acid and salt concentrations suppress the bacteria growth in the anchovy fillets. Thus, marination process can be recommended to be used as a preservation method to inhibit bacterial growth in anchovy fillets for a safe consumption.

Evaluating the effects of temperature and agitation on biofilm formation of bacterial pathogens isolated from raw cow milk.

OBJECTIVES: To study the effect of agitation and temperature on biofilm formation (cell aggregates embedded within a self-produced matrix) by pathogenic bacteria isolated from Raw cow milk (RCM). METHODS: A 40 RCM samples were gathered from eight dairy farms in Riyadh, Saudi Arabia. After bacterial culturing and isolation, gram staining was performed, and all pathogenic, identified using standard criteria established by Food Standards Australia New Zealand (FSANZ), and non-pathogenic bacteria were identified using VITEK-2 and biochemical assays. To evaluate the effects of temperature and agitation on biofilm formation, isolated pathogenic bacteria were incubated for 24 h under the following conditions: 4 °C with no agitation (0 rpm), 15 °C with no agitation, 30 °C with no agitation, 30 °C with 60 rpm agitation, and 30 °C with 120 rpm agitation. Then, biofilms were measured using a crystal violet assay. RESULTS: Of the eight farm sites, three exhibited non-pathogenic bacterial contamination in their raw milk samples. Of the total of 40 raw milk samples, 15/40 (37.5%; from five farms) were contaminated with pathogenic bacteria. Overall, 346 bacteria were isolated from the 40 samples, with 329/346 (95.1%) considered as non-pathogenic and 17/346 (4.9%) as pathogenic. Most of the isolated pathogenic bacteria exhibited a significant (p < 0.01) increase in biofilm formation when grown at 30 °C compared to 4 °C and when grown with 120 rpm agitation compared to 0 rpm. CONCLUSION: Herein, we highlight the practices of consumers in terms of transporting and storing (temperature and agitation) can significantly impact on the growth of pathogens and biofilm formation in RCM.

Enterococcus spp. in fish: Analysis of the presence and resistance in samples from Tri-City, Poland.

The growing concern over antibiotic resistance in foodborne pathogens necessitates comprehensive assessments of its prevalence and associated risks in various food products. The present study aimed to assess the occurrence of Enterococcus spp. in samples of fish purchased at various points of sale in the Tricity region. The selection of products (n = 74) was based on their availability and included both fish caught in the Baltic region and products imported from, Vietnam, China, Norway, and European Union (EU) countries. For bacterial isolation, samples were inoculated into selective broth, and the growth of enterococci was assessed based on turbidity. Positive cultures were confirmed by a change in color in bromocresol purple broth and were isolated on Slanetz-Bartley agar. Bacteria were present in all tested samples regardless of the degree of raw material processing as follows: frozen (F)- 55%, fresh/raw (FS)- 70.6%, thawed (DF)- 30%, smoked (S)- 50%, and the packaging methods, modified atmosphere packaging (MAP)- 34.4%, unit packaging (UP)- 75%, and sold in bulk (SB)- 76.9%, with an overall frequency of occurrence of 58.1%. The number of bacteria ranged from not detected to 4.28-log cfu/g, with the lowest mean values for thawed fish and those packed in MAP. Tests conducted on 24 strains isolated from samples showed their varied sensitivity to tetracyclines. Single cases of multidrug resistance of the tested strains were also observed. The conducted statistical analysis did not show statistically significant differences in the count of enterococci based on the origin, degree of processing, or packaging (p < 0.05). Moreover, differences in strain sensitivity to ampicillin were observed. Detected cases of resistance, especially to tetracycline, require careful monitoring and action to limit the health risks associated with resistant bacterial strains in food products.

History repeats itself: consumption of a local traditional roast pork meat recipe leads to Salmonella ser. Give cases in Greece.

INTRODUCTION: An outbreak of gastroenteritis due to Salmonella Give, a very rarely identified serotype in human isolates in Greece, occurred in participants of a religious festival in a rural area of southern Greece, in September 2022. The objectives of this study were to describe the outbreak in terms of epidemiology, identify the vehicle of transmission of the foodborne pathogen and recommend prevention measures. METHODS: The outbreak was linked to the consumption of a local traditional recipe of roasted pork meat served by a street food vendor. In 2018, the same food item, served in a restaurant in the same region, was implicated in another S. Give outbreak. RESULTS: Outbreak investigations revealed that outbreak-associated isolates, of food and human origin, belonged to the same S. Give strain. Significant deficiencies regarding food safety practices were identified. CONCLUSION: Technical knowledge about pathogen transmission paths is important in order for both food handlers and consumers to follow hygiene and sanitary measures, mainly in cases of mass gatherings, where large quantities of food are prepared, handled, cooked and served. Efficient official supervision, mainly during summer festivals, is required in order to avoid recurrence of foodborne infections by different combinations of pathogens/food commodities.

Prevalence and antimicrobial resistance of salmonellae isolated from eggs of local chicken in selected towns of Ethiopia.

BACKGROUND: Salmonellosis is one of the most common food-borne diseases in industrialised and developing countries. In recent year, an increase in antimicrobial resistance among different Salmonella serotypes has been observed. OBJECTIVE: A cross-sectional study was conducted to assess the prevalence and antimicrobial susceptibility of Salmonella isolated from local chicken eggs in four selected towns in Ethiopia. METHODS: A total of 115 eggs were examined to detect Salmonella by using standard microbiological methods. The susceptibilities of the isolates to nine antimicrobials were tested by the Kirby-Bauer disk diffusion method. RESULT: The study revealed that of the 115 eggs examined, 22 (19.1%) were positive for Salmonella of which 14 (12.2%) and 8 (7%) of the isolates were from shells and contents, respectively. The occurrence of Salmonella in egg shells and content and between different altitudes did not differ significantly (p > 0.05). Most isolates were resistant to more than three antimicrobials with a high resistance to kanamycin, ampicillin, nalidixic acid, cotrimoxazole, oxytetracycline and chloramphenicol. CONCLUSION: The results indicate the potential importance of local chicken eggs as source of multiple antimicrobial-resistant salmonellae and the need for proper cooking before consumption. Further studies are required to describe the epidemiology of Salmonella in various agroclimatic zones of Ethiopia.

The effects of Lactiplantibacillus plantarum 3-19 and Pediococcus pentosaceus 18-1 on preventing the accumulation of biogenic amines and promoting the production of volatile organic compounds during sour meat fermentation.

Lactic acid bacteria (LAB) are frequently used in meat fermentation, and mixed stater cultures are reported to perform better than single ones. Lactiplantibacillus plantarum 3-19 and Pediococcus pentosaceus 18-1 were chosen from 28 sour-meat-origin strains to examine the effects of single and combined inoculation on sour meat quality. Natural fermentation was used as a control to investigate changes in pH, water activity (aw), amino acid nitrogen (AN), texture, microbial diversity, and volatile organic compounds (VOCs) during fermentation. The pH and aw of each inoculation group were significantly decreased, and AN content was significantly increased. The inoculation of P. pentosaceus 18-1 significantly reduced putrescine, cadaverine, and tryptamine content (p < 0.05), while the inoculation of Lpb. plantarum 3-19 significantly reduced cadaverine amounts (p < 0.05). At the fermentation endpoint, the total biogenic amines content in the C group was 992.96 ± 14.07, which was 1.65, 2.57, and 3.07 times higher than that in the Lp, Pe, and M groups, respectively. The mixed inoculation group combined the advantages of both strains and decreased total biogenic amines most significantly. At the end of fermentation, the VOCs in C, Lp, Pe, and M groups were 10.11, 11.56, 12.45, and 13.39 times higher than those at the beginning of fermentation. Inoculation promoted the production of key VOCs (OAV > 2000) such as heptanal, octanal, and (E)-2-nonanal. The mixed inoculation group had the highest variety and content of VOCs and the highest content of the above key VOCs, significantly enhancing its fruity, floral, ester, and other aromas. Sensory evaluation indicated that the M group had the best overall acceptability. Finally, it was suggested that a combination of Lpb. plantarum 3-19 and P. pentosaceus 18-1 is a novel and efficient starter culture for processing sour meat since they lower the amounts of biogenic amines in the meat and promote the production of VOCs.

In vitro antimicrobial, antibiofilm photodynamic activity, and molecular dynamic simulations of tetra-cationic porphyrinmembrane interactions against foodborne microorganisms.

This manuscript presents a new report on the in vitro antimicrobial photo-inactivation of foodborne microorganisms (Salmonella spp. and Listeria monocytogenes) using tetra-cationic porphyrins. Isomeric tetra-cationic porphyrins (3MeTPyP, 4MeTPyP, 3PtTPyP, and 4PtTPyP) were tested, and antimicrobial activity assays were performed at specific photosensitizer concentrations under dark and white-light LED irradiation conditions. Among the tested bacterial strains, 4MeTPyP exhibited the highest efficiency, inhibiting bacterial growth within just 60 min at low concentrations (17.5 µM). The minimal inhibitory concentration of 4MeTPyP increased when reactive oxygen species scavengers were present, indicating the significant involvement of singlet oxygen species in the photooxidation mechanism. Furthermore, the checkerboard assay testing the association of 4MeTPyP showed an indifferent effect. Atomic force microscopy analyses and dynamic simulations were conducted to enhance our understanding of the interaction between this porphyrin and the strain's membrane.

Novel misos shape distinct microbial ecologies: opportunities for flavourful sustainable food innovation.

Fermentation is resurgent around the world as people seek healthier, more sustainable, and tasty food options. This study explores the microbial ecology of miso, a traditional Japanese fermented paste, made with novel regional substrates to develop new plant-based foods. Eight novel miso varieties were developed using different protein-rich substrates: yellow peas, Gotland lentils, and fava beans (each with two treatments: standard and nixtamalisation), as well as rye bread and soybeans. The misos were produced at Noma, a restaurant in Copenhagen, Denmark. Samples were analysed with biological and technical triplicates at the beginning and end of fermentation. We also incorporated in this study six samples of novel misos produced following the same recipe at Inua, a former affiliate restaurant of Noma in Tokyo, Japan. To analyse microbial community structure and diversity, metabarcoding (16S and ITS) and shotgun metagenomic analyses were performed. The misos contain a greater range of microbes than is currently described for miso in the literature. The composition of the novel yellow pea misos was notably similar to the traditional soybean ones, suggesting they are a good alternative, which supports our culinary collaborators' sensory conclusions. For bacteria, we found that overall substrate had the strongest effect, followed by time, treatment (nixtamalisation), and geography. For fungi, there was a slightly stronger effect of geography and a mild effect of substrate, and no significant effects for treatment or time. Based on an analysis of metagenome-assembled genomes (MAGs), strains of Staphylococccus epidermidis differentiated according to substrate. Carotenoid biosynthesis genes in these MAGs appeared in strains from Japan but not from Denmark, suggesting a possible gene-level geographical effect. The benign and possibly functional presence of S. epidermidis in these misos, a species typically associated with the human skin microbiome, suggests possible adaptation to the miso niche, and the flow of microbes between bodies and foods in certain fermentation as more common than is currently recognised. This study improves our understanding of miso ecology, highlights the potential for developing novel misos using diverse local ingredients, and suggests how fermentation innovation can contribute to studies of microbial ecology and evolution.

Activation of a silent lactose utilization pathway in an evolved Listeria monocytogenes F2365 outbreak isolate.

Listeria monocytogenes, a widespread food-borne pathogen, utilizes diverse growth substrates including mono- and di-saccharides via PEP-phosphotransferase (PTS) systems. We evaluated a collection of L. monocytogenes isolates of different origins for their ability to utilize lactose, a disaccharide composed of galactose and glucose and the main carbon source in milk and dairy products. Notably, the dairy-associated outbreak strain F2365 could not utilize lactose efficiently, conceivably due to a frameshift mutation (lacR887del) resulting in a truncated LacR. Transcriptional activator LacR is involved in the expression of two PTS systems, encoded by the lpo operon lmo1718-1720 in combination with lmo2708 and the lmo2683-2685 operon, and linked to lactose and/or cellobiose metabolism in L. monocytogenes. Via experimental evolution of the ancestral strain F2365, an evolved isolate F2365 EV was obtained which showed enhanced growth and metabolism of lactose. Using the lactose-positive model strain L. monocytogenes EGDe as a control, HPLC experiments showed that EGDe and F2365 EV could consume lactose and utilize the glucose moiety, while the galactose moiety was exported from the cells. Genome sequencing of F2365 EV found the original lacR887del mutation was still present but an additional point mutation lmo2766C415T had occurred, resulting in an amino acid substitution in the putative regulator Lmo2766. The lmo2766 gene is located next to operon lmo2761-2765 with putative PTS genes in the genome. Notably, comparative RNAseq analysis confirmed that the lmo2761-2765 operon was strongly upregulated in F2365 EV in the presence of lactose but not in EGDe and F2365. Conversely, the LacR-regulated lpo operon, lmo2708, and lmo2683-2685 operon were only upregulated in EGDe. Additional growth and HPLC experiments, using mutants constructed in lactose-positive L. monocytogenes EGDe, showed reduced growth of the EGDe lacR887del mutant with no utilization of lactose, while the double mutant EGDe lacR887dellmo2766C415T showed enhanced growth and lactose utilization. Hence, these results demonstrate that an amino acid substitution in the Lmo2766 regulator activates a previously silent lactose utilization pathway encoded by PTS operon lmo2761-2765, facilitating the growth and metabolism of L. monocytogenes with lactose as a substrate. This finding enhances our understanding of the metabolic capabilities and adaptability of L. monocytogenes, offering a broader view of the lactose utilization capacity of this pathogen.