Applied and Environmental Microbiology

Microbial engineering offers potential for improving the sustainability of agriculture by providing greater control of desired microbial functions. However, successful control of engineered functions requires greater understanding of their robustness under diverse conditions including those used for plant hydroponics. Here, we studied biomass accumulation and surfactin biosynthesis by an engineered derivative of Bacillus subtilis PY79 in common plant culture media as a model system for interrogating metabolic robustness. We report the observation that PY79 and all other B. subtilis strains that we tested, including natural isolates, exhibited difficulty growing under shaking incubation in defined media where the only nitrogen sources were inorganic. In contrast, assimilation of inorganic nitrogen sources functioned relatively robustly under static incubation in these same media. Our findings may offer some guidance for use of B. subtilis in controlled environment agriculture and could aid future efforts to identify the molecular basis for the agitation-dependent effect on nitrogen assimilation.

Insect gut microbiomes have important roles in overall host health and how hosts function in the environment. In laboratory and mass-reared insects, gut microbiomes can differ in composition and function compared to wild conspecifics. For fruit flies, like the Mediterranean fruit fly (medfly; Ceratitis capitata), these changes can influence male performance and behavior. Overall, understanding factors that influence the ability of bacteria to colonize hosts is an important for the establishment of lost or novel microbiota into mass-reared insects. The goal of this study was to evaluate how host age and diet inoculation method influenced bacterial establishment in laboratory and mass-reared medfly. We used an Enterobacter strain with antibiotic resistance and coupled it with full-length PacBio 16S rRNA sequencing to track the establishment of a specific isolates under different adult dietary conditions. We also used two longstanding reared lines of medfly in our study. Our results identified that diet had a strong interaction with age. Host medfly fed a liquid diet with the target bacteria were able to be colonized regardless of age, but those fed a slurry-based diet and separate water source were more resilient. This was consistent for both fly rearing lines used in the study. 16S rRNA sequencing corroborated the establishment of the specific strain, but also revealed some species/strain-level variation of Enterobacter sequences associated with the flies. Additionally, our study illustrates that long-read 16S rRNA sequencing may afford improved characterization of species- and strain-level distribution of Enterobacteriaceae in insects. ImportanceInsects form intimate relationships with gut microorganisms that can help facilitate several important roles. The goals of our study were to evaluate factors that influence microbial establishment in lines of the Mediterranean fruit fly (medfly), an important pest species throughout the world. Mass-reared insects for sterile insect technique often possess gut microbiomes that substantially differ from wild flies, which can impact their performance in pest control contexts. Here, we show that liquid-based formulations can be utilized to manipulate the gut microbiota of mass-reared medfly. Furthermore, using near full-length 16S rRNA metabarcoding sequencing, we uncovered strain-level diversity of that was not immediately obvious using other approaches. This is a notable finding, as it suggests that full-length 16S rRNA approaches can have marked improvements for some taxa compared to fewer hypervariable regions at approximately the same cost. Our results provide new avenues for exploring and interrogating medfly-microbiome interactions.

Desulfovibrio vulgaris has been the primary pure culture sulfate reducer for developing microbial corrosion concepts. Multiple mechanisms for how it accepts electrons from Fe0 have been proposed. We investigated Fe0 oxidation with a mutant of D. vulgaris in which hydrogenase genes were deleted. The hydrogenase mutant grew as well as the parental strain with lactate as the electron donor, but unlike the parental strain was not able to grow on H2. The parental strain reduced sulfate with Fe0 as the sole electron donor, but the hydrogenase mutant did not. H2 accumulated over time in Fe0 cultures of the hydrogenase mutant and sterile controls, but not in parental strain cultures. Sulfide stimulated H2 production in uninoculated controls apparently by both reacting with Fe0 to generate H2 and facilitating electron transfer from Fe0 to H+. Parental strain supernatants did not accelerate H2 production from Fe0, ruling out a role for extracellular hydrogenases. Previously proposed electron transfer between Fe0 and D. vulgaris via soluble electron shuttles was not evident. The hydrogenase mutant did not reduce sulfate in the presence of Fe0 and either riboflavin or anthraquinone-2,6-disulfonate and these potential electron shuttles did not stimulate parental strain sulfate reduction with Fe0 as the electron donor. The results demonstrate that D. vulgaris primarily accepts electrons from Fe0 via H2 as an intermediary electron carrier. These findings clarify the interpretation of previous D. vulgaris corrosion studies and suggest that H2-mediated electron transfer is an important mechanism for iron corrosion under sulfate-reducing conditions. ImportanceMicrobial corrosion of iron in the presence of sulfate-reducing microorganisms is economically significant. There is substantial debate over how microbes accelerate iron corrosion. Tools for genetic manipulation have only been developed for a few Fe(III)-reducing and methanogenic microorganisms known to corrode iron and in each case those microbes were found to accept electrons from Fe0 via direct electron transfer. However, iron corrosion is often most intense in the presence of sulfate-reducing microbes. The finding that Desulfovibrio vulgaris relies on H2 to shuttle electrons between Fe0 and cells revives the concept, developed in some of the earliest studies on microbial corrosion, that sulfate reducers consumption of H2 is a major microbial corrosion mechanism. The results further emphasize that direct Fe0-to-microbe electron transfer has yet to be rigorously demonstrated in sulfate-reducing microbes.

Spore-forming Bacillus species, including pathogenic Bacillus cereus and spoilage-associated Bacillus subtilis, are major contributors to foodborne illness and product degradation. Understanding their metabolic behaviour in diverse food matrices is essential for improving risk assessment, spoilage prediction, and fermentation control. This study integrates isothermal microcalorimetry and targeted metabolomics to characterize the metabolic activity of B. cereus and B. subtilis in five nutrient sources: Brain Heart Infusion (BHI) medium, oat drink, milk, pea hydrolysate, and a combined oat-pea matrix. Metabolic heat production was monitored for 24 hours at 30{degrees}C. In BHI, B. cereus exhibited a shorter lag phase (mean {+/-} sd: 4.3 hours {+/-} 0.8) than B. subtilis (7.9 hours {+/-} 1.0) but produced less total heat. Across all food matrices, B. subtilis consistently generated more heat. The oat-pea matrix supported the highest calorimetric growth rates, surpassing oat or pea alone, and showed sugar depletion and accumulation of organic acids, indicating enhanced carbohydrate metabolism. Free amino acid release was matrix- and species-specific: B. subtilis had increased levels in oat, while B. cereus did so in pea. While B. cereus was metabolically active in all matrices, cereulide levels were matrix-dependent: 47.3 {+/-} 1.7 ng/mL in oat, 3.0 {+/-} 0.1 ng/mL in oat-pea, and undetectable in pea. These findings reveal clade-specific and matrix-driven metabolic strategies. This is the first study to combine calorimetry and metabolomics to evaluate Bacillus activity in plant-based and dairy matrices. This approach enhances our understanding of microbial physiology in complex food systems and provides a foundation for developing targeted strategies to improve food safety, stability, and product design.

The plant-beneficial bacterium Pseudomonas protegens CHA0 (CHA0) is widely studied for the biological control of soil-borne plant diseases. Beyond its root-colonising capabilities, CHA0 can also infect and kill insect larvae and thus exhibits a multi-host lifestyle shared with other plant- and insect-colonising bacteria. To better understand the robustness of this multi-host lifestyle, we subjected CHA0 to ten consecutive passages through larvae of the pest insect Plutella xylostella via repeated cycles of insect colonisation and killing forcing it into an insect-only lifestyle. Overall, serial passaging did not result in consistent changes in insect killing speed, larval or root colonisation, plant protection efficiency, microbial antagonism or in vitro growth. This suggests that its multi-host lifestyle was conserved following serial passage. Nonetheless, a few independently passaged lines showed an increase in larval killing speed, which in one case might be linked to choline uptake. To disentangle changes specific to the insect host from those arising due to the experimental system itself, we conducted parallel serial passages through the same system while omitting the insect host. In some of these lines, exposure to the background of the system led to changes in microbial antagonism and in in vitro growth, which likely are associated with mutations in regions encoding for regulatory systems. Our findings indicate that P. protegens CHA0 remains phenotypically stable in complex environments such as an insect host, suggesting that the multi-host lifestyle might also be conserved when applied in the field and supporting CHA0s potential for reliable biocontrol performance against both plant diseases and insect pests. Author summaryControlling insect pests with living organisms, known as biological control, offers an environmentally friendly alternative to chemical pesticides. The plant-beneficial bacterium Pseudomonas protegens CHA0 is a promising biocontrol candidate that not only colonizes plant roots but also infects and kills certain insect larvae. This ability to colonize different hosts appears to be a conserved trait also observed in other bacteria. To better understand the robustness of this multi-host lifestyle, we repeatedly exposed CHA0 to larvae of the insect pest Plutella xylostella and assessed the resulting physiological and genetic changes. Surprisingly, after ten cycles, CHA0 largely retained its insect-killing and plant-protective traits. Although a few populations showed minor changes, including slightly faster insect killing and traits associated with aspects of the experimental system, these changes were limited in scope. Overall, our findings suggest that P. protegens CHA0 does not change rapidly in complex environments such as an insect host, supporting its potential for reliable biocontrol performance in the field.

Bacteriophages are gaining increased research attention as alternatives to antibiotics and microbiome manipulation tools to enhance feed efficiency and animal health in cattle. However, challenges associated with phage specificity, microbial ecosystem variations, and the absence of effective screening methods have hindered harnessing the power of phage application in cattle. The objectives of this study were to (i) optimize phage screening method for microbial samples obtained from different cattle body sites, (ii) isolate lytic phages against key bovine pathogens and commensal bacteria, and (iii) characterize the isolated phages and their bacterial hosts. A total of 1,214 samples from different cattle body sites (n = 1194) and environmental sources were screened using 13 phage detection methods, including one high-throughput approach. Eighty-three phages were isolated, primarily from ruminal fluid (59), feces (15), vaginal (7) and nasopharyngeal swabs (1), and fetal ruminal fluid (1). The bacterial hosts inhibited by these phages were from 29 genera, with Bacillus (34), Escherichia/Shigella (8), Shouchella (5), Corynebacterium (4), and Lysinibacillus (4) being the most common. No phages were identified against bovine pathogens including Trueperella pyogenes, Mannheimia haemolytica, Pasteurella multocida, or Moraxella bovis. Method 12 demonstrated the highest efficiency in phage recovery, particularly from ruminal samples. The isolation of phages against commensal bacteria from the gastrointestinal, reproductive, and respiratory tracts, and fetal gut highlights their potential for microbiome modulation to improve cattle health and feed efficiency. These findings underscore the need for further research into pathogen-targeting phage isolation in cattle. ImportanceBacteriophage application holds a promising potential for improving feed efficiency and reducing enteric methane emissions and fighting against antimicrobial resistant bacterial pathogens in cattle. In the present study, we optimized a bacteriophage screening and isolation method that could be used to isolate bacteriophages against commensal bacteria from the ruminal, respiratory and reproductive tract microbiota in cattle. We isolated 83 phages that are lytic to bacterial species originating from rumen, vagina, nasopharynx of healthy adult cattle, as well as the intestinal tract of 6-month-old calf fetus. Overall, our study provides an important basis for the development of bacteriophage-based interventions to modulate ruminal, reproductive and respiratory tract microbiomes and thereby improve animal health and feed efficiency while reducing methane emissions in cattle.

Salmonella enterica serovar Infantis (S. Infantis) is an important emerging pathogen, associated with poultry and poultry products and related to an increasing number of human infections in many countries. A concerning trend among S. Infantis isolates is the presence of plasmid-mediated multi-drug resistance. In many instances, the genes responsible for this resistance are carried on a megaplasmid known as the plasmid of emerging S. Infantis (pESI) or pESI like plasmids. Plasmids can be remarkably stable due to the presence of multiple replicons and post-segregational killing systems (PSKs), which contribute to their maintenance within bacterial populations. To enhance our understanding of S. Infantis and its multidrug resistance determinants toward the development of new vaccination strategies, we have devised a method for targeted plasmid curing. This approach effectively overcomes plasmid addiction by leveraging the temporal overproduction of specific antitoxins coupled with the deletion of the partition region. By employing this strategy, we successfully generated a plasmid-free strain from a field isolate derived from S. Infantis 119944. This method provides valuable tools for studying S. Infantis and its plasmid-borne multidrug resistance mechanisms.

Saccharolobus islandicus REY15A represents one of the very few archaeal models with versatile genetic tools, including efficient genome editing, gene silencing and robust protein expression systems. However, plasmid vectors constructed for this crenarchaeon thus far are solely based on the pRN2 cryptic plasmid. Although this plasmid co-exists with pRN1 in their original host, early attempts to test pRN1-based vectors consistently failed to yield any stable host-vector system for Sa. islandicus. Herein we identified a putative target sequence in orf904 encoding a putative replicase on pRN1 (TargetN1). Mutated targets were then designed (N1a, N1b, N1c) and tested for their capability of escaping from the host CRISPR immunity by using plasmid interference assay. This revealed that the original target triggers the CRISPR immunity in this archaeon whereas all three mutated targets do not, indicating that all designed target mutations evade the host immunity. These mutated targets were then incorporated into orf904 individually, yielding corresponding mutated pRN1 backbones with which shuttle plasmids were constructed (pN1aSD, pN1bSD and pN1cSD). Sa. islandicus transformation revealed that pN1aSD and pN1bSD were functional shuttle vectors, but pN1cSD lost the capability of replication. In addition, pRN1-based and pRN2-based vectors were stably maintained in the archaeal cells either alone or in combination, and this yielded a dual plasmid system for genetic study with this important archaeal model. Impact statementWhen pRN1 was employed for vector construction in Saccharolobus islandicus REY15A, pRN1-derived vectors are not stable in this archaeon. Here we show that pRN1 orf904 encoding a putative replicase on pRN1 carries a DNA segment to be targeted by the host I-A CRISPR system. By designing mutated target sequences that evade the CRISPR immunity, efficient plasmid vectors were obtained with mutated pRN1 backbones. This strategy could be applied in developing host-vector systems for other microorganisms with plasmids or viruses carrying CRISPR target sequences. Moreover, the resulting dual vector system would facilitate genetic studies with this crenarchaeal model.

Bifidobacterium breve, one of the main bifidobacterial species colonizing the human gastrointestinal tract in early life, has received extensive attention for its purported beneficial effects on human health. However, exploration of the mode of action of such beneficial effects exerted by B. breve is cumbersome due to the lack of effective genetic tools, which limits its synthetic biology application. Given the widespread presence of endogenous CRISPR-Cas systems in B. breve, the current study developed an endogenous CRISPR-based gene editing toolkit for genetic manipulation of B. breve. Deletion of the gene coding uracil phosphoribosyl-transferase (upp) was achieved in two different B. breve strains using this system. In addition, translational termination of uracil phosphoribosyl-transferase was successfully achieved in B. breve FJSWX38M7 by single-base substitution of the upp gene and insertion of three stop codons. The gene encoding linoleic acid isomerase (bbi) in B. breve, being a characteristic trait, was deleted after plasmid curing, which rendered it unable to convert linoleic acid into conjugated linoleic acid, demonstrating the feasibility of successive editing. This study expanded the gene manipulation toolkit of B. breve and provides a reference for functional genome editing and analysis using an endogenous CRISPR-Cas system in Bifidobacterium. ImportanceThe lack of effective genetic tools for Bifidobacterium breve is an obstacle to studying the molecular mechanisms of its health-promoting effects, hindering the development of next-generation probiotics. Here, we introduce a gene editing method based on the endogenous CRISPR-Cas system, which can achieve gene deletion, single base substitution, gene insertion and continuous gene editing in B. breve. This study will promote the excavation of functional genes and elucidation of molecular mechanisms of B. breve.

Mosquitoes develop in a wide range of aquatic habitats containing highly diverse and variable bacterial communities that shape both larval and adult traits, including the capacity of adult females of some mosquito species to vector disease-causing organisms to humans. However, while most mosquito studies control for host genotype and environmental conditions, the impact of microbiota variation on phenotypic outcomes of mosquitoes is often unaccounted for. The inability to conduct reproducible intra- and inter-laboratory studies of mosquito-microbiota interactions has also greatly limited our ability to identify microbial targets for mosquito-borne disease control. Here, we developed an approach to isolate and cryopreserve microbial communities derived from mosquito larval rearing environments in the lab and field. We then validated the use of our approach to generate experimental microcosms colonized by standardized lab- and field-derived microbial communities. Our results overall reveal minimal effects of cryopreservation on the recovery of bacteria when directly compared with isolation from non-cryopreserved fresh material. Our results also reveal improved reproducibility of microbial communities in replicate microcosms generated using cryopreserved stocks over fresh material. Altogether, these results provide a critical next step toward the standardization of mosquito studies to include larval rearing environments colonized by defined microbial communities. They also lay the foundation for long-term studies of mosquito-microbe interactions and the identification and manipulation of taxa with potential to reduce mosquito vectorial capacity.

Studies investigating surface microbiota in food facilities often include estimates of relative abundance. However, obtaining accurate relative abundance values can be challenging. Differences among microbes can lead to different degrees of cell recovery from the surface and different DNA extraction yields that skew downstream relative abundance estimates. Here, we evaluated (1) the impact of cell recovery from surfaces using sponge swabs and (2) DNA extraction protocol on the relative abundance estimates of relative abundance on artificially inoculated surfaces. Our results showed that Escherichia coli (Gram-negative cell), Listeria monocytogenes (Gram-positive cell), Bacillus cereus (bacterial spore), Alicyclobacillus suci (bacterial spore), Exophiala phaeomuriformis (fungal cell), Aspergillus fischeri (fungal spore) differed significantly (p<0.05) in their recovery rates from stainless steel surfaces, ranging from 2.9%{+/-}3.0% recovery to 94.9%{+/-}3.0% recovery. Modification of the DNA extraction protocol by extending the bead-beating step by 10 min generally improved DNA yields, though the impact varied by organism. For example, DNA yields of E. coli increased from 70 to 84 ng/mL while that of L. monocytogenes increased only from 23.2 to 29.2 ng/mL. Amplicon sequencing results indicated that the differences in cell recovery and DNA extraction among microbial species skewed the relative abundance estimates from inoculated surfaces. For example, the estimated relative abundance of L. monocytogenes was 9-17%, which was lower than its actual relative abundance (25%). These results underscore the limitations of surface microbiota characterization in food facilities and highlighted the need to improve current recovery and DNA extraction methods. ImportanceAmplicon sequencing has been used to characterize microbial communities on facility surfaces. However, few studies have evaluated the accuracy of the amplicon sequencing workflow for quantifying spoilage and pathogenic organisms in these microbial communities. Here, we assessed the accuracy for amplicon sequencing to evaluate the relative abundance of spoilage and pathogenic organisms commonly found in food processing environments. The results revealed biases in relative abundances due to limitations in cell recover and DNA extraction methods. These findings revealed the potential biases in surface microbiota characterization in food facilities, and the need to refine current recovery and extraction methods to enhance the accuracy of microbiota characterization.

In our era of rising antibiotic resistance, Stenotrophomonas maltophilia (STM) is an understudied, gram-negative, aerobic bacterium widespread in the environment and increasingly causing opportunistic infections. Treating STM infections remains difficult, leading to an increase in disease severity and higher hospitalization rates in people with Cystic Fibrosis (pwCF), cancer, and other immunocompromised health conditions. The lack of effective antibiotics has led to renewed interest in phage therapy; however, there is a need for well-characterized phages. In response to an oncology patient with a respiratory infection, we collected 18 phages from Southern California wastewater influent that exhibit different plaque morphology against STM host strain B28B, cultivated from a blood sample. Here, we characterize the genomes and life cycle kinetics of our STM phage collection. We hypothesize that genetically distinct phages give rise to unique lytic life cycles that can enhance bacterial killing when combined into a phage cocktail compared to the individual phages alone. We identified three genetically distinct clusters of phages, and a representative from each group was screened for potential therapeutic use and investigated for infection kinetics. The results demonstrated that the three-phage cocktail significantly suppressed bacterial growth compared to individual phages when observed for 48 hours. We also assessed the lytic impacts of our three-phage cocktail against a collection of 46 STM strains to determine if a multi-phage cocktail can expand the host range of individual phages. Our phages remained strain-specific and infect >50% of tested strains. The multi-phage cocktail maintains bacterial growth suppression and prevents the emergence of phage-resistant strains throughout our 40-hour assay. These findings suggest specialized phage cocktails may be an effective avenue of treatment for recalcitrant STM infections resistant to current antibiotics. IMPORTANCEPhage therapy could provide a vital strategy in the fight against antimicrobial resistance (AMR) bacterial infections; however, significant knowledge gaps remain. This study investigates phage cocktail development for the opportunistic pathogen Stenotrophomonas maltophilia (STM). Our findings contribute novel phages, their lytic characteristics, and limitations when exposed to an array of clinically relevant STM strains. Eighteen bacteriophages were isolated from wastewater influent from Escondido, California, and subjected to genomic analysis. We investigated genetically distinct phages to establish their infection kinetics and developed them into a phage cocktail. Our findings suggest that a genetically distinct STM phage cocktail provides an effective strategy for bacterial suppression of host strain B28B and five other clinically relevant STM strains. Phage therapy against STM remains poorly understood, as only 39 phages have been previously isolated. Future research into the underlying mechanism of how phage cocktails overwhelm the host bacteria will provide essential information that could aid in optimizing phage applications and impact alternative treatment options.

Intensive broiler production practices impair the transmission of commensal microbes from hens to offspring, resulting in a lower abundance of non-spore-forming strict anaerobic bacteria. We evaluated the effects of colonization by a defined community (DC) of bacteria including and excluding Megamonas hypermegale in chicks challenged with Salmonella. Inoculation with DC resulted in higher phylogenetic diversity and the dominance of Bacteroidetes species in the cecal microbiota, with a decrease in the relative abundance of Salmonella and Escherichia/Shigella, as well as a lower Enterobacteriaceae load. Substantial shifts in microbiota composition were coupled with subtle changes in metabolites and host responses, including changes in interferon-{gamma}, macrophage colony-stimulating factor, propionate, valerate, and isovalerate concentrations in the ceca. We identified bacterial species that were able to establish and persist after a single exposure, many of which were members of Bacteroidetes. Although co-culture with M. hypermegale reduced Salmonella counts by 99.3% in vitro, in vivo inoculation of M. hypermegale increased splenic Salmonella counts in inoculated chicks. The use of DC containing bacteria isolates harvested from the cecal contents of mature chickens can recapitulate the changes in volatile fatty acid concentrations observed in birds colonized with complex communities, and the presence of M. hypermegale specifically enhances the production of propionate. Our findings suggest that the use of DC can be explored as a strategy to control disease occurrence in broiler production; however, further research is warranted to properly understand the role of individual species in the broiler cecal community aiding the formulation of appropriate DCs. ImportanceIntensive production practices can reduce beneficial gut bacteria in broiler chickens, potentially leading to higher disease risk. We investigated whether introducing a defined community (DC) of beneficial bacteria, along with M. hypermegale, could improve gut health and resistance to Salmonella in broiler chicks. Our findings show that DC increases microbial diversity and reduces the relative abundance of potential pathogens, like Salmonella and Escherichia/Shigella, which was coupled with subtle changes in the immune responses of the birds and higher concentration of volatile fatty acids in the ceca. This study suggests that using DC can enhance poultry health and reduce disease, providing a potential strategy to improve broiler production. However, further research is needed to understand the roles of individual bacteria and refine these bacterial communities for practical use in farming. This work holds promise for developing natural methods to enhance poultry health and safety.

Cross-contamination of low-moisture foods (LMFs) with pathogens from equipment and environmental surfaces during production is a food safety concern. Wet sanitation is sometimes employed to mitigate cross-contamination in LMF facilities, but the introduction of moisture to otherwise dry environments can inadvertently promote pathogen growth. This study evaluated the risks associated with wet sanitation in LMF facilities by characterizing evaporation kinetics on powdered infant formula (PIF)-soiled surfaces, monitoring relative humidity (RH) in a LMF facility during and after wet sanitation, and assessing growth of Salmonella, Listeria monocytogenes, Cronobacter sakazakii, and Enterococcus faecium spot inoculated on PIF-soiled stainless steel coupons under dynamic RH conditions. As expected, higher RH slowed drying of PIF-soiled surfaces, prolonging periods when the soils water activity (aw) was high enough to support microbial growth. Correspondingly, all four organisms grew significantly at 81 and 97% RH over 120 h (p<0.05), while only E. faecium grew significantly below 81% RH (p<0.05). Monitoring of RH during and after wet sanitation in a commercial facility revealed spikes up to 100% RH during sanitation and sustained RH above 75% for more than 7 h in poorly ventilated areas. When those facility RH conditions were simulated in the laboratory, Salmonella populations on PIF-soiled coupons increased by more than 3.5 Log CFU/coupon within 66 h. These findings demonstrate the potential for wet sanitation to unintentionally enable environmental pathogen growth and highlight the importance of moisture and RH control in LMF facilities. ImportanceWet sanitation is commonly employed by LMF manufacturers for allergen changeovers and to prevent cross-contamination from surfaces, but regulators, manufacturers, and researchers have all expressed concerns that wet sanitation may promote the growth of pathogens in otherwise dry production environments. Despite these concerns, research on the impact of wet sanitation on facility RH and its influence on microbial proliferation in low moisture production environments remains limited. This study provides evidence that wet sanitation substantially increases facility RH, leading to persistent hydration of soiled surfaces, creating conditions that enable microbial growth. These findings reinforce concerns over the use of wet sanitation in LMF production. This also demonstrates the need for reducing water use in LMF production facilities, implementing RH control strategies, as well as the adoption of alternative or supplemental dry sanitation strategies to mitigate microbial risks.

Hanseniaspora uvarum is consistently observed as the dominant non-Saccharomyces species in spontaneous grape juice fermentations. However, the physiological mechanisms and physicochemical variables influencing the prevalence of H. uvarum over other non-Saccharomyces species remain unclear. We tested the physicochemical parameters contributing to H. uvarum dominance by inoculating a chemically diverse set of grape juices with a mock community whose composition was defined following a comprehensive microbial survey of spontaneous fermentations. Our findings indicated that the chemical composition of grape juice had minimal impact on the microbial dynamics of fermentation, with H. uvarum emerging as the dominant non-Saccharomyces species in nearly all conditions tested. Grape juice composition primarily influenced the total yeast abundance of the mock community. Flow cytometry analysis confirmed that H. uvarum has a faster growth rate than Saccharomyces cerevisiae and several other Hanseniaspora spp.. Moreover, its growth was not affected by the presence of S. cerevisiae, explaining its rapid dominance in spontaneous fermentations. The rapid growth of H. uvarum negatively impacted the growth of S. cerevisiae, with significant implications for fermentation performance and sugar consumption. The results of this study suggest that the fast growth rate of H. uvarum enables it to quickly dominate the grape juice environment during the early stages of fermentation. This physiological advantage indicates that the initial abundance of H. uvarum may be critical to the outcome of spontaneous fermentations, as evidenced by its direct impact on the growth of S. cerevisiae and fermentation performance.

Non-Typhoidal Salmonella (NTS) continues to be a leading cause of foodborne illness worldwide. Food manufacturers implement hurdle technology by combining more than one approach to control food safety and quality, including preservatives such as organic acids, refrigeration, and heating. We assessed the variation in survival in stresses of genotypically diverse isolates of Salmonella enterica to identify genotypes with potential elevated risk to sub-optimal processing or cooking. Sub-lethal heat treatment, survival in desiccated conditions and growth in the presence of NaCl or organic acids were investigated. S. Gallinarum strain 287/91 was most sensitive to all stress conditions. While none of the strains replicated in a food matrix at 4{degrees}C, S. Infantis strain S1326/28 retained the greatest viability, and six strains exhibited a significantly reduced viability. A S. Kedougou strain exhibited the greatest resistance to incubation at 60{degrees}C in a food matrix that was significantly greater than S. Typhimurium U288, S Heidelberg, S. Kentucky, S. Schwarzengrund and S. Gallinarum strains. Two isolates of monophasic S. Typhimurium, S04698-09 and B54 Col9 exhibited the greatest tolerance to desiccation that was significantly more than for the S. Kentucky and S. Typhimurium U288 strains. In general, the presence of 12mM acetic acid or 14mM citric acid resulted in a similar pattern of decreased growth in broth, but this was not observed for S. Enteritidis, and S. Typhimurium strains ST4/74 and U288 S01960-05. Acetic acid had a moderately greater effect on growth despite the lower concentration tested. A similar pattern of decreased growth was observed in the presence of 6% NaCl, with the notable exception that S. Typhimurium strain U288 S01960-05 exhibited enhanced growth in elevated NaCl concentrations. An understanding of the molecular basis of phenotypic variation in response to stress has the potential to improve process validation during food challenge tests, improve processing, and result in more reliable risk assessments in the food industry.

The emergence of multidrug-resistant Vibrio parahaemolyticus poses a severe threat to mariculture sustainability, highlighting the urgent need for eco-friendly antimicrobial agents. In this study, we demonstrated that L-Cysteine (L-Cys) functions as an inhibitor of V. parahaemolyticus, with a minimum inhibitory concentration of 7.5 mM. Microscopic observation and viability assays revealed that L-Cys compromises bacterial membrane integrity, ultimately leading to cell death. Further investigation indicated that the antibacterial effect is primarily attributed to the intracellular production of hydrogen sulfide (H2S) generated by L-Cys metabolism. Transcriptomic and biochemical analyses showed that L-Cys induced metabolic reprogramming by suppressing fatty acid {beta}-oxidation, one-carbon metabolism, and antioxidant enzymes. This disruption of redox homeostasis results in accelerated accumulation of reactive oxygen species (ROS). In addition to its antibacterial effect, L-Cys also effectively reduced the virulence factor of bacterial motility. Finally, L-Cys demonstrated broad-spectrum antimicrobial activity against other pathogenic Vibrio species, including V. alginolyticus and V. anguillarum. Our findings suggest that L-Cys is a promising antimicrobial agent inducing ROS to mediate membrane disruption, with the advantages of cost-effectiveness and environmental safety for controlling vibriosis. ImportanceVibrio parahaemolyticus is a significant pathogen in aquaculture and a common cause of seafood-borne gastroenteritis worldwide. The increasing prevalence of antibiotic-resistant strains of this bacterium highlights the need for alternative control agents. This study shows that L-cysteine (L-Cys) inhibits the growth of V. parahaemolyticus. Our data indicate that L-Cys is metabolized to produce hydrogen sulfide, which contributes to the accumulation of ROS and disrupts bacterial membrane integrity. Additionally, L-Cys reduces bacterial motility and shows inhibitory effects against other Vibrio species. These findings suggest that L-Cys may represent a useful agent for managing Vibrio infections in aquaculture settings.

The production of traditional agave spirits in Mexico is a deeply rooted traditional process that relies on environmental microorganisms to ferment the cooked must from agave plants. Analysis of these microorganisms provides the opportunity to understand the dynamics of the microbial communities in the interface of natural and human-associated environments in a biologically and culturally rich region of the world. Here, we performed 16S and ITS amplicon sequencing of close to 100 fermentation tanks from 42 distilleries throughout Mexico. The Agave species used, production practices, climatic conditions, and biogeographic characteristics varied considerably among sites. Yet, we did find taxa present in most fermentations suggesting that there is a core of microorganisms that are hallmarks of these communities. These core taxa are represented by hundreds of OTUs showing large intra-specific variation. The only variable that was consistently associated with the composition of both bacterial and fungal communities was the distillery, suggesting that microbial composition is determined by the local production practices and unique attributes of each site. Fermentation stage, climate and producing region were also associated with the community composition, but only for prokaryotes. Analysis of microbial composition in several tanks within three distilleries also revealed taxa that were enriched in specific fermentation stages or agave species. Our work provides a comprehensive analysis of the microbiome of agave fermentations, contributing key knowledge for its management and conservation.

Since the 1980s, chromosome-integration vectors have been used as a core method of engineering Bacillus subtilis. One of the most frequently used vector backbones contains chromosomally derived regions that direct homologous recombination into the amyE locus. Here, we report a gap in the homology region inherited from the original amyE integration vector, leading to erroneous recombination in a subset of transformants and a loss-of-function mutation in the downstream gene. Internal to the homology arm that spans the 3' portion of amyE and the downstream gene ldh, an unintentional 227-bp deletion generates two crossover events. The major event yields the intended genotype, but the minor event, occurring in [~]10% of colonies, results in a truncation of ldh, which encodes lactate dehydrogenase. Although both types of colonies test positive for amyE disruption by starch plating, the potential defect in fermentative metabolism may be left undetected and confound the results of subsequent experiments.

Symbiotic microbes play pivotal roles in insect ecology, including the detoxification of insecticides, which reduces target host mortality and diminishes the efficacy of chemical pest control agents. Quantifying the prevalence of symbiont-mediated insecticide detoxification across the microbiome is necessary to understand its contributions to pesticide resistance, and to understand how pesticides alter gut microbial communities. Here, we investigated the prevalence and mechanisms of pesticide degradation within the gut microbiota of the Colorado potato beetle (Leptinotarsa decemlineata), a significant agricultural pest that has driven ongoing insecticide innovation for decades. Beetles were collected from an organic farm in Tyler, TX, and 18 bacterial isolates representing the diversity of their gut microbiota were screened for their ability to degrade three common insecticides in vitro: imidacloprid, fenitrothion, and malathion. Among these, Acinetobacter calcoaceticus, Pseudomonas protegens, and an unnamed Microbacterium species degraded malathion as a sole carbon source, with distinct enantiomer-specific preferences. Untargeted GC-MS analysis revealed breakdown products, providing initial insights into the metabolic pathways utilized by these microbes. These findings suggest that microbial association with resistant insect hosts may select for microbial insecticide utilization, potentially enhancing resistance development in agricultural pests and influencing the surrounding soil microbiome.