Exploring native arsenic (As)-resistant bacteria: unveiling multifaceted mechanisms for plant growth promotion under As stress.

AIMS: This study explores the plant growth-promoting effect (PGPE) and potential mechanisms of the arsenic (As)-resistant bacterium Flavobacterium sp. A9 (A9 hereafter). METHODS AND RESULTS: The influences of A9 on the growth of Arabidopsis thaliana, lettuce, and Brassica napus under As(V) stress were investigated. Additionally, a metabolome analysis was conducted to unravel the underlying mechanisms that facilitate PGPE. Results revealed that A9 significantly enhanced the fresh weight of Arabidopsis seedlings by 62.6%-135.4% under As(V) stress. A9 significantly increased root length (19.4%), phosphorus (25.28%), chlorophyll content (59%), pod number (24.42%), and weight (18.88%), while decreasing As content (48.33%, P ≤ .05) and oxidative stress of Arabidopsis. It also significantly promoted the growth of lettuce and B. napus under As(V) stress. A9 demonstrated the capability to produce ≥31 beneficial substances contributing to plant growth promotion (e.g. gibberellic acid), stress tolerance (e.g. thiamine), and reduced As accumulation (e.g. siderophores). CONCLUSIONS: A9 significantly promoted the plant growth under As stress and decreased As accumulation by decreasing oxidative stress and releasing beneficial compounds.

An outbreak of Shiga toxin-producing Escherichia coli (STEC) O157:H7 associated with contaminated lettuce and the cascading risks from climate change, the United Kingdom, August to September 2022.

Shiga-toxin producing Escherichia coli (STEC) O157 is a food-borne pathogen which causes gastrointestinal illness in humans. Ruminants are considered the main reservoir of infection, and STEC exceedance has been associated with heavy rainfall. In September 2022, a large outbreak of STEC O157:H7 was identified in the United Kingdom (UK). A national-level investigation was undertaken to identify the source of the outbreak and inform risk mitigation strategies. Whole genome sequencing (WGS) was used to identify outbreak cases. Overall, 259 cases with illness onset dates between 5 August and 12 October 2022, were confirmed across the UK. Epidemiological investigations supported a UK grown, nationally distributed, short shelf-life food item as the source of the outbreak. Analytical epidemiology and food chain analysis suggested lettuce as the likely vehicle of infection. Food supply chain tracing identified Grower X as the likely implicated producer. Independent of the food chain investigations, a novel geospatial analysis triangulating meteorological, flood risk, animal density and land use data was developed, also identifying Grower X as the likely source. Novel geospatial analysis and One Health approaches are potential tools for upstream data analysis to predict and prevent contamination events before they occur and to support evidence generation in outbreak investigations.

Agricultural Biotechnological Potential of Bacillus velezensis C3-3 and Cytobacillus sp. T106 from Resource Islands of a Semi-arid Zone of La Guajira-Colombia.

Resource islands are vegetative formations in arid and semi-arid ecosystems that harbor microorganisms facing extreme conditions. However, there is a limitation in the knowledge of the agricultural biotechnological potential of microorganisms present in these islands. This study aimed to determine the capacity of Bacillus velezensis C3-3 and Cytobacillus sp. T106 isolates from resource islands to promote plant growth and control the phytopathogen Rhizoctonia solani. The bacteria were sequenced, and both grew at 50 °C, resisted 5% NaCl, withstood UV exposure, and grew in extreme pH conditions. Sixty-six genes in C3-3 and 71 in T106 were identified associated with plant growth promotion, and C3-3 was shown to promote leaf growth in lettuce plants. This promotional effect was associated with the production of indole-3-acetic acid (IAA), phosphorus solubilization, and the presence of genes related to the assimilation of rhizosphere exudates. Both strains inhibited R. solani through the production of volatile compounds and antagonism. Forty-five and 40 of these genes in C3-3 and T106, respectively, were associated with the production of proteases, lipases, siderophores, antimicrobial compounds, degradation enzymes, and secretion systems. Notably, Cytobacillus sp. has not been previously reported as a biocontrol agent. This work contributes to the evidence of the biotechnological potential of semi-arid region bacteria, offering prospects for improving agricultural production in areas with limiting conditions.

Mobility and growth in confined spaces are important mechanisms for the establishment of Bacillus subtilis in the rhizosphere.

The rhizosphere hosts complex and abundant microbiomes whose structure and composition are now well described by metagenomic studies. However, the dynamic mechanisms that enable micro-organisms to establish along a growing plant root are poorly characterized. Here, we studied how a motile bacterium utilizes the microhabitats created by soil pore space to establish in the proximity of plant roots. We have established a model system consisting of Bacillus subtilis and lettuce seedlings co-inoculated in transparent soil microcosms. We carried out live imaging experiments and developed image analysis pipelines to quantify the abundance of the bacterium as a function of time and position in the pore space. Results showed that the establishment of the bacterium in the rhizosphere follows a precise sequence of events where small islands of mobile bacteria were first seen forming near the root tip within the first 12-24 h of inoculation. Biofilm was then seen forming on the root epidermis at distances of about 700-1000 µm from the tip. Bacteria accumulated predominantly in confined pore spaces within 200 µm from the root or the surface of a particle. Using probabilistic models, we could map the complete sequence of events and propose a conceptual model of bacterial establishment in the pore space. This study therefore advances our understanding of the respective role of growth and mobility in the efficient colonization of bacteria in the rhizosphere.

Antibiotic-resistant bacteria contaminating leafy vegetables in Saudi Arabia's eastern region.

BACKGROUND: Food-associated antibiotic-resistant bacteria can cause infections that may critically impact human health. The objectives of this study were to determine the microbial contamination level of green leafy vegetables and their antibiotic resistance pattern. METHODS: Sixty-three samples of leafy vegetables were collected from Dammam Central Fruit and Vegetables Market from January to June 2023. The vegetables included lettuce (Lactuca sativa), parsley (Petroselinum crispum), and watercress (Nasturtium officinale). Samples were tested by standard microbiological techniques for identification and antibiotic susceptibility testing. RESULT: Eight types of bacteria belonging to six different genera were detected. Enterobacteriaceae family was represented by four genera: Klebsiella, Proteus, Morganella, and Enterobacter. The other two genera were Pseudomonas and Aeromonas. Enterobacter cloacae was the most abundant organism, followed by Pseudomonas putida and Aeromonas sobria. On the other hand, Morganella morganii, Aeromonas hydrophila, and Proteus mirabilis were the least abundant. The three vegetable types had different levels of bacterial contamination. All isolated organisms were sensitive to penicillin, cephalosporin, aminoglycoside, and fluoroquinolone. However, Klebsiella oxytoca, M. morganii, and K. pneumonia showed resistance to ampicillin. A. hydrophila, Morganella morganii, and E. cloacae showed resistance to amoxicillin. M. morganii and E. cloacae were found to be resistant to cefalotin. Moreover, A. hydrophila, M. morganii, and E. cloacae were resistant to cefoxitin. Again, A. hydrophila was found to be resistant to imipenem. Only M. morganii was resistant to Ciprofloxacin. Two isolates, P. mirabilis and M. morganii were resistant to tigecycline. Another two, M. morganii and P. mirabilis were resistant to Nitrofurantoin. Only M. morganii was found to be resistant to trimethoprim. CONCLUSION: This study aligns with the broad consensus in the literature about the significance of bacterial contamination in vegetables and the public health implications. The unique focus on antibiotic resistance patterns adds an essential dimension to the existing body of knowledge.

Formation and recovery of Listeria monocytogenes in viable but nonculturable state under different temperatures combined with low nutrition and high NaCl concentration.

The viable but nonculturable (VBNC) state occurs when bacteria lose their ability to grow and multiply on conventional media when stressed by adverse environmental factors, but they remain active and can revive under certain conditions, posing a food safety risk. In this study, the VBNC state of Listeria monocytogenes was induced with different temperatures combined with low nutrient conditions; the VBNC state of L. monocytogenes was confirmed in conjunction with the housekeeping gene abcZ using a molecular biology assay (PMA-qPCR) to calculate the viable bacterial count; The resuscitation conditions for the VBNC state of L. monocytogenes were investigated utilizing various nutrients in the culture medium and pasteurized milk. Four strains of L. monocytogenes reached the VBNC stage after 14, 21, 21, and 35 days at 20°C with 20% (or 30%) NaCl. Resuscitation studies indicate that Trypticase Soy Broth (TSB) combined with Tween 80 and sodium pyruvate is more effective for resuscitation. The Chinese national standard technology GB 4789.30-2016 was used to inoculate lettuce, chicken, and pasteurized milk with L. monocytogenes ATCC 19115 VBNC state. This research has significant implications for commercial food processing, long-term storage, disinfection, disease prevention, and control.

Scandinavium lactucae sp. nov. Isolated from Healthy Lettuce in South Korea.

Four Gram-negative, facultative anaerobic, oxidase-negative and catalase-positive strains were isolated from lettuce sample collected from test beds at the National Institute of Agricultural Sciences in Wanju, South Korea. The whole genome sequences of the strains ranged from 4,624,629 to 4,849,846 bp in size, with DNA G + C contents of 54.32 to 54.56 mol%. Phylogenetic analyses based on 16S rRNA gene and four housekeeping (atpD, gyrB, infB, and rpoB) gene sequences showed that the four strains clustered closely together with Scandinavium type strains within the Enterobacteriaceae family. Moreover, the average nucleotide identity and digital DNA-DNA hybridization value of the proposed type strain (V105_6T) with the closely related Scandinavium type strains were in the range of 85.71-86.16% and 30.2-31.2%, respectively, which were all below the species delineation threshold values. The major cellular fatty acid of V105_6T was C16:0. Growth was observed at 7, 10 and 35 °C, and in the presence of 7% NaCl concentration. Based on phenotypic and genotypic results, strain V105_6T represents a novel species of the genus Scandinavium, for which the name Scandinavium lactucae sp. nov. is proposed. The type strain is V105_6T (= LMG 33389T = DSM 117134T).

Optimization of the Method for Isolating Bacterial DNA from the Aboveground Part of Lettuce.

Developing an effective method for isolating bacterial genetic material from plants is a relatively challenging task and often does not yield adequately prepared material for further analyses. Previous studies often overlook connections, primarily focusing on laboratory investigations. With advancements in high-throughput sequencing techniques, we can now revisit and delve deeper into these interactions. Our study focuses on the initial phase of these investigations: genetic material isolation. Extracting bacterial DNA from aboveground plant parts, known as the phyllosphere, poses a significant challenge due to plant-derived contaminants. Existing isolation protocols frequently yield inconsistent results, necessitating continuous refinement and optimization. In our study, we developed an effective isolation protocol employing mechanical-chemical lysis, sonication, and membrane filtration. This approach yielded high-quality DNA at a concentration of 38.08 ng/µL, suitable for advanced sequencing applications. Our results underscore the effectiveness and necessity of these methods for conducting comprehensive microbiological analyses. Furthermore, our research not only lays the groundwork for further studies on lettuce microbiota, but also highlights the potential for utilizing our developed protocol in investigating other plants and their microbiomes.

Application of a dual-modality colorimetric analysis method to inkjet printing lateral flow detection of Salmonella typhimurium.

Lateral flow assay (LFA) color signal quantification methods were developed by utilizing both International Commission on Illumination (CIE) LAB (CIELAB) color space and grayscale intensity differences. The CIELAB image processing procedure included calibration, test, control band detection, and color difference calculation, which can minimize the noise from the background. The LFA platform showcases its ability to accurately discern relevant colorimetric signals. The rising occurrence of infectious outbreaks from foodborne pathogens like Salmonella typhimurium presents significant economic, healthcare, and public health risks. The study introduces an aptamer-based lateral flow (ABLF) platform by using inkjet printing for specially detecting S. typhimurium. The ABLF utilized gold-decorated polystyrene microparticles, functionalized with specific S. typhimurium aptamers (Ps-AuNPs-ssDNA). The platform demonstrates a detection limit of 102 CFU mL-1 in buffer solutions and 103 CFU mL-1 in romaine lettuce tests. Furthermore, it sustained performance for over 8 weeks at room temperature. The ABLF platform and analysis methods are expected to effectively resolve the low-sensitivity problems of the former LFA systems and to bridge the gap between lab-scale platforms to market-ready solutions by offering a simple, cost-effective, and consistent approach to detecting foodborne pathogens in real samples.

Migration and accumulation of microplastics in soil-plant systems mediated by symbiotic microorganisms and their ecological effects.

The coexistence of microorganisms in complex soil environments greatly affects the environmental behavior and ecological effects of microplastics (MPs). However, relevant studies are sparse, and internal mechanisms remain unclear. Herein, arbuscular mycorrhizal fungi (AMF), a common symbiotic microorganism in the soil-plant system, was proved to significantly affect MPs absorption and migration with a "size effect". Specifically, the existence of AMF accelerated small-sized MPs (0.5 µm) uptake but slowed large-sized MPs (2 µm) uptake in lettuce. The content of 0.5 µm MPs absorbed by plants with AMF was 1.26 times that of the non-AMF group, while the content of 2 µm MPs was only 77.62 % that of non-AMF group. Additionally, the different effects of microorganisms on the intake content of MPs with different particle sizes in plants also led to different toxic effects of MPs on lettuce, that is, AMF exacerbated small-size MPs toxicity in lettuce (e.g., reduced plant biomass, photosynthesis, etc), and it weakened large-sized MPs toxicity (e.g., increased plant height, antioxidant enzyme activity, etc). The above phenomenon mainly because of the change in AMF on the plant root structure, which can be visually observed through the intraradical and extraradical hyphae. The symbiotic structure (hyphae) formed by AMF and host plants root could enhance the absorption pathway for small-sized MPs in lettuce, although not for large-sized MPs. Additionally, the effects of AMF varied with the soil environment of differently sized MPs, which promoted the migration of small-particle MPs to plants but aggravated large-particle MPs fixation at the soil interface. These findings could deepen the understanding of MPs pollution in terrestrial systems and provide theoretical basis and technical support to accurately assess soil MPs pollution.

Time of arrival during plant disease progression and humidity additively influence Salmonella enterica colonization of lettuce.

The interplay between plant hosts, phytopathogenic bacteria, and enteric human pathogens in the phyllosphere has consequences for human health. Salmonella enterica has been known to take advantage of phytobacterial infection to increase its success on plants, but there is little knowledge of additional factors that may influence the relationship between enteric pathogens and plant disease. In this study, we investigated the role of humidity and the extent of plant disease progression on S. enterica colonization of plants. We found that high humidity was necessary for the replication of S. enterica on diseased lettuce, but not required for S. enterica ingress into the UV-protected apoplast. Additionally, the Xanthomonas hortorum pv. vitians (hereafter, X. vitians)-infected lettuce host was found to be a relatively hostile environment for S. enterica when it arrived prior to the development of watersoaking or following necrosis onset, supporting the existence of an ideal window during X. vitians infection progress that maximizes S. enterica survival. In vitro growth studies in sucrose media suggest that X. vitians may allow S. enterica to benefit from cross-feeding during plant infection. Overall, this study emphasizes the role of phytobacterial disease as a driver of S. enterica success in the phyllosphere, demonstrates how the time of arrival during disease progress can influence S. enterica's fate in the apoplast, and highlights the potential for humidity to transform an infected apoplast into a growth-promoting environment for bacterial colonizers. IMPORTANCE: Bacterial leaf spot of lettuce caused by Xanthomonas hortorum pv. vitians is a common threat to leafy green production. The global impact caused by phytopathogens, including X. vitians, is likely to increase with climate change. We found that even under a scenario where increased humidity did not enhance plant disease, high humidity had a substantial effect on facilitating Salmonella enterica growth on Xanthomonas-infected plants. High humidity climates may directly contribute to the survival of human enteric pathogens in crop fields or indirectly affect bacterial survival via changes to the phyllosphere brought on by phytopathogen disease.

Rapid and Sensitive Detection of Shiga Toxin-Producing Escherichia coli (STEC) from Food Matrices Using the CANARY Biosensor Assay.

Shiga toxin-producing Escherichia coli (STEC) causes a wide spectrum of diseases including hemorrhagic colitis and hemolytic uremic syndrome (HUS). Previously, we developed a rapid, sensitive, and potentially portable assay that identified STEC by detecting Shiga toxin (Stx) using a B-cell based biosensor platform. We applied this assay to detect Stx2 present in food samples that have been implicated in previous STEC foodborne outbreaks (milk, lettuce, and beef). The STEC enrichment medium, modified Tryptone Soy Broth (mTSB), inhibited the biosensor assay, but dilution with the assay buffer relieved this effect. Results with Stx2a toxoid-spiked food samples indicated an estimated limit of detection (LOD) of ≈4 ng/mL. When this assay was applied to food samples inoculated with STEC, it was able to detect 0.4 CFU/g or 0.4 CFU/mL of STEC at 16 h post incubation (hpi) in an enrichment medium containing mitomycin C. Importantly, this assay was even able to detect STEC strains that were high expressors of Stx2 at 8 hpi. These results indicate that the STEC CANARY biosensor assay is a rapid and sensitive assay applicable for detection of STEC contamination in food with minimal sample processing that can complement the current Food Safety Inspection Service (US) methodologies for STEC.

Host cell wall composition and localized microenvironment implicated in resistance to basal stem degradation by lettuce drop (Sclerotinia minor).

BACKGROUND: Sclerotinia spp. are generalist fungal pathogens, infecting over 700 plant hosts worldwide, including major crops. While host resistance is the most sustainable and cost-effective method for disease management, complete resistance to Sclerotinia diseases is rare. We recently identified soft basal stem as a potential susceptibility factor to Sclerotinia minor infection in lettuce (Lactuca sativa) under greenhouse conditions. RESULTS: Analysis of stem and root cell wall composition in five L. sativa and one L. serriola accessions with varying growth habits and S. minor resistance levels revealed strong association between hemicellulose constituents, lignin polymers, disease phenotypes, and basal stem mechanical strength. Accessions resistant to basal stem degradation consistently exhibited higher levels of syringyl, guaiacyl, and xylose, but lower levels of fucose in stems. These findings suggest that stem cell wall polymers recalcitrant to breakdown by lignocellulolytic enzymes may contribute to stem strength-mediated resistance against S. minor. CONCLUSIONS: The lignin content, particularly guaiacyl and syringyl, along with xylose could potentially serve as biomarkers for identifying more resistant lettuce accessions and breeding lines. Basal stem degradation by S. minor was influenced by localized microenvironment conditions around the stem base of the plants.

From kitchen to crop: The efficacy and safety of the microbial consortium treated dishwasher wastewater for the Zero Mile system.

A microbial consortium, based on the functional integration of photosynthetic and heterotrophic microorganisms, is the core of the Zero Mile System. This system is designed for reusing and upcycling household greywaters, a still untapped water resource. The previous challenges of dishwasher wastewater bioremediation demonstrated the capability of an ad hoc consortium (including a photosynthetic cyanobacterium and three heterotrophic bacterial isolates from dishwasher wastewater) to reclaim the wastewater at small/medium scale. In this study the wastewater treatment demonstrated to be effective in nutrient recycling and upcycling at a larger scale, i.e. 4 L (in three replicates to treat the total amount of wastewater discharged by the dishwasher), by removing high percentage of N and P from the wastewater (70% nitrogen, 50% phosphorous, respectively). Again, the reclaimed wastewater successfully fertilized lettuce plants both indoor (in the Zero Mile System demonstrator) and outdoor (in open field). Plants showed a significant higher biomass productivity in fresh weight compared to control plants and comparable or better values of the pigments and quality indices (e.g., soluble solids, total phenols, total flavonoids). Furthermore, the safety of the reclaimed wastewater is demonstrated by the analysis of the metabolic/ecologically relevant functions of the microbial communities in both untreated and treated wastewater. Colonizers were mainly organic matter degraders and bacteria involved in nitrogen cycling. The human related genera are quite few and no pathogens or potential microbiological contaminants of water bodies (as E. coli), were found. Hence, the utilization of treated dishwasher wastewater does not imply biological risks to agricultural products, soil, or groundwater.

γ-aminobutyric acid treatment inhibits browning and promotes storage quality by regulating reactive oxygen species and membrane lipid metabolism in fresh-cut stem lettuce.

The effects of γ-aminobutyric (GABA) on enzymatic browning, storage quality, membrane and reactive oxygen species (ROS) metabolism in fresh-cut stem lettuce were investigated. The results illustrated that GABA treatment delayed browning degree, polyphenol oxidase (PPO) activity and the expression of LsPPO. Meanwhile, higher chlorophyll and ascorbic acid contents were exhibited in GABA-treated stem lettuce, as well as the slower microbial propagation. Further investigation revealed that exogenous GABA application declined malondialdehyde content, electrolyte leakage and the enzyme activities of membrane metabolism, and the expression levels of related genes were also downregulated. In addition, GABA treatment scavenged ROS and strengthened the enzyme activities of ROS metabolism, as well as the expression levels of corresponding genes. Taken together, these findings implied that the repressed enzymatic browning and microbial propagation in GABA-treated stem lettuce were due to the inhibition of ROS accumulation, enhancement of membrane stability and increased resistance to oxidation.

Plant endophyte immobilization technology: A promising approach for chromium-contaminated water and soil remediation.

Microbial immobilization technology is considered an efficient bioremediation method for chromium (Cr) pollution. However, it is currently unclear which strain is more beneficial for the remediation of Cr-contaminated water and soil. Therefore, corn straw biochar was used as a carrier to prepare materials for fixing the endophytes Serratia sp. Y-13 (BSR1), Serratia nematodiphila (BSR2), Lysinibacillus sp. strain SePC-36 (BLB1), Lysinibacillus mangiferihumi strain WK63 (BLB2) and the commercial bacteria Shewanella oneidensis MR-1 (BSW). The results demonstrated that, compared with BSW, endophyte-loaded biochar (especially BSR1) was more effective at remediating Cr pollution in water and soil. Endophyte-loaded biochar reduced the abundance of soil pathogenic bacteria, enhanced the number of beneficial plant endophytes, reduced the soil Cr(VI) concentration, improved soil fertility, reduced the plant Cr concentration and improved the yield of lettuce. Redundancy analysis (RDA) and structural equation modelling (PLS-PM) suggested that soil microbes are closely related to soil Cr(VI), plant fresh weight and soil organic matter, whereas endophyte-loaded biochar directly influences plant cell motility pathways by altering plant microbes. This study represents a pioneering investigation into the efficacy of endophyte-loaded biochar as a remediation strategy for Cr pollution.

Assembly of root-associated bacterial community and soil health in cadmium-contaminated soil affected by nano/bulk-biochar compost associations.

Biochar (BC) has been proven effective in promoting the production of safety food in cadmium (Cd)-polluted soil and the impact can be further enhanced through interaction with compost (CM). However, there existed unclear impacts of biochar with varying particle sizes in conjunction with compost on microbiome composition, rhizosphere functions, and soil health. Hence, in this study, two bulk-biochar derived from wood chips and pig manure were fabricated into nano-biochar using a ball-milling method. Subsequently, in a field experiment, the root-associated bacterial community and microbial functions of lettuce were evaluated in respond to Cd-contaminated soil remediated with nano/bulk-BCCM. The results showed that compared to bulk-BCCM, nano-BCCM significantly reduced the Cd concentration in the edible part of lettuce and the available Cd in the soil. Both nano-BCCM and bulk-BCCM strongly influenced the composition of bacterial communities in the four root-associated niches, and enhanced rhizosphere functions involved in nitrogen, phosphorus, and carbon cycling, as well as the relative abundance and biodiversity of keystone modules in rhizosphere soil. Furthermore, soil quality index analysis indicated that nano-BCCM exhibited greater potential than bulk-BCCM in maintaining soil health. The data revealed that nano-BCCM could regulate the Cd concentration in lettuce shoot by promoting microbial biodiversity of keystone modules in soil-root continuum and rhizosphere bacterial functions. These findings suggest that nano-biochar compost associations can be a superior strategy for enhancing microbial functions, maintaining soil health, and ensuring crop production safety in the Cd-contaminated soil compared to the mix of bulk-biochar and compost.

Potential risk of biofilm-forming Bacillus cereus group in fresh-cut lettuce production chain.

Bacillus cereus and Bacillus thuringiensis, which belong to the B. cereus group, are widely distributed in nature and can cause food poisoning symptoms. In this study, we collected 131 isolates belonging to the B. cereus group, comprising 124B. cereus and seven B. thuringiensis isolates, from fresh-cut lettuce production chain and investigated their potential risk by analyzing genotypic (enterotoxin and emetic toxin gene profiles) and phenotypic (antibiotic susceptibility, sporulation, and biofilm formation) characteristics. Enterotoxin genes were present only in B. cereus, whereas the emetic toxin gene was not detected in any of the B. cereus isolates. All isolates were susceptible to vancomycin, which is a last resort for treating B. cereus group infection symptoms, but generally resistant to ß-lactam antimicrobials, and had the ability to form spores (at an average sporulation rate of 24.6 %) and biofilms at 30 °C. Isolates that formed strong biofilms at 30 °C had a superior possibility of forming a dense biofilm by proliferating at 10 °C compared to other isolates. Additionally, confocal laser scanning microscopy (CLSM) images revealed a notable presence of spores within the submerged biofilm formed at 10 °C, and the strengthened attachment of biofilm inner cells to the substrate was further revealed through biofilm structure parameters analysis. Collectively, our study revealed the prevalence and contamination levels of B. cereus and B. thuringiensis at fresh-cut lettuce production chain and investigated their genotypic and phenotypic characteristics, aiming to provide valuable insights for the development of potential risk management strategies to ensure food safety, especially along the cold chain.

Fitness Differences Between Listeria monocytogenes Serotypes 1/2a, 4b, and 4bv-1 in Competition for Growth on Lettuce Leaf Sections.

Listeria monocytogenes is a foodborne pathogen that lives in nature as a saprophyte. Two of the three most common serotypes that cause foodborne listeriosis are 1/2a and 4b. Within serotype 4b, there is a variant called 4bv-1. In the last decade, several produce-related outbreaks (linked to leafy salad, caramel apples, and stone fruit) were linked to 4bv-1 strains, specifically those of Sequence Type 382. This study assessed the fitness of ST 382 strains on lettuce leaf sections to determine if they are more fit on produce than strains of other serotypes. Strains of serotypes 1/2a, 4b, and ST 382 were inoculated as mixtures onto lettuce and incubated at 4 °C for 7 days or 25 °C for 24 h. Thirty L. monocytogenes colonies resulting from the growth on each lettuce piece were characterized for serotype by multiplex PCR, and the percentages of each serotype recovered were compared. In the individual mixtures with three strains, none of the ST 382 strains showed better fitness for growth on lettuce at either 4 °C or 25 °C. Overall, ST 382 strains showed better recovery from lettuce sections grown at 4 °C than at 25 °C. Statistical analysis of the recovery of twelve strains tested in competition experiments indicated that ST 382 strains were less fit for lettuce growth when competing against the other serotypes. The data indicate that ST 382 strains do not have a competitive fitness advantage on cut lettuce sections.

Lettuce Genotype-Dependent Effects of Temperature on Escherichia coli O157:H7 Persistence and Plant Head Growth.

Lettuce has been commonly associated with the contamination of human pathogens, such as Escherichia coli O157:H7 (hereafter O157:H7), which has resulted in serious foodborne illnesses. Contamination events may happen throughout the farm-to-fork chain, when O157:H7 colonizes edible tissues and closely interacts with the plant. Environmental conditions have a significant impact on many plant-microbe interactions; however, it is currently unknown whether temperature affects O157:H7 colonization of the lettuce phyllosphere. In this study, we investigated the relationship between elevated growth temperatures, O157:H7 persistence, and lettuce head growth using 25 lettuce genotypes. Plants were grown under optimal or elevated temperatures for 3.5 weeks before being inoculated with O157:H7. The bacterial population size in the phyllosphere and lettuce head area was estimated at 0- and 10-days postinoculation (DPI) to assess bacterial persistence and head growth during contamination. We found that growing temperature can have a positive, negative, or no effect on O157:H7 persistence depending on the lettuce genotype. Furthermore, temperature had a greater effect on head area size than the presence of O157:H7. The results suggested that the combination of plant genotype and temperature level is an important factor for O157:H7 colonization of lettuce and the possibility to combine desirable food safety traits with heat tolerance into the lettuce germplasm.