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.

Effects of gnotobiotic fermentation on global gene expression of germ-free vegetables.

Existing research has underscored the vital interplay between host organisms and their associated microbiomes, which affects health and function. In both plants and animals, host factors critically shape microbial communities and influence growth, health, and immunity. Post-harvest plants, such as those used in kimchi, a traditional Korean dish, offer a unique avenue for exploring host-microbe dynamics during fermentation. Despite the emphasis on lactic acid bacteria (LAB) in fermentation studies, the roles of host factors remain unclear. This study aimed to investigate the influence of these factors on plant transcriptomes during kimchi fermentation. We individually inoculated nine LAB strains into germ-free kimchi to generate LAB-mono-associated gnotobiotic kimchi and performed RNA-sequencing analysis for the host vegetables during fermentation. The transcriptomes of post-harvest vegetables in kimchi change over time, and microbes affect the transcriptome profiles of vegetables. Differentially expressed gene analyses revealed that microbes affected the temporal expression profiles of several genes in the plant transcriptomes in unique directions depending on the introduced LAB strains. Cluster analysis with other publicly available transcriptomes of post-harvest vegetables and fruits further revealed that the plant transcriptome is more profoundly influenced by the environment harboring the host than by host phylogeny. Our results bridge the gap in understanding the bidirectional relationship between host vegetables and microbes during food fermentation, illuminating the complex interplay between vegetable transcriptomes, fermentative microbes, and the fermentation process in food production. The different transcriptomic responses elicited by specific LAB strains suggest the possibility of microbial manipulation to achieve the desired fermentation outcomes.

Microbiological quality of irrigation water on highly diverse fresh produce smallholder farms: elucidating environmental routes of contamination.

AIM: To evaluate the microbiological safety, potential multidrug-resistant bacterial presence and genetic relatedness (DNA fingerprints) of Escherichia coli isolated from the water-soil-plant nexus on highly diverse fresh produce smallholder farms. METHODS AND RESULTS: Irrigation water (n = 44), soil (n = 85), and fresh produce (n = 95) samples from six smallholder farms with different production systems were analysed for hygiene indicator bacterial counts and the presence of shigatoxigenic E. coli and Salmonella spp. using standard microbiological methods. Identities of isolates were confirmed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), and the genetic relatedness of the E. coli isolates determined using enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) analysis. Irrigation water E. coli levels ranged between 0 and 3.45 log MPN/100 ml-1 with five farms having acceptable levels according to the World Health Organization limit (3 log MPN/100 ml-1). Fresh produce samples on four farms (n = 65) harboured E. coli at low levels (<1 log CFU/g-1) except for one sample from kale, spring onion, green pepper, onion, and two tomato samples, which exceeded international acceptable limits (100 CFU/g-1). Only one baby carrot fresh produce sample tested positive for Salmonella spp. Of the 224 samples, E. coli isolates were identified in 40% (n = 90) of all water, soil, and fresh produce types after enrichment. Additionally, the DNA fingerprints of E. coli isolates from the water-soil-plant nexus of each respective farm clustered together at high similarity values (>90%), with all phenotypically characterized as multidrug-resistant. CONCLUSIONS: The clustering of E. coli isolated throughout the water-soil-plant nexus, implicated irrigation water in fresh produce contamination. Highlighting the importance of complying with irrigation water microbiological quality guidelines to limit the spread of potential foodborne pathogens throughout the fresh produce supply chain.

Mitigating Emerging and Reemerging Diseases of Fruit and Vegetable Crops in a Changing Climate.

Fruit and vegetable crops are important sources of nutrition and income globally. Producing these high-value crops requires significant investment of often scarce resources, and, therefore, the risks associated with climate change and accompanying disease pressures are especially important. Climate change influences the occurrence and pressure of plant diseases, enabling new pathogens to emerge and old enemies to reemerge. Specific environmental changes attributed to climate change, particularly temperature fluctuations and intense rainfall events, greatly alter fruit and vegetable disease incidence and severity. In turn, fruit and vegetable microbiomes, and subsequently overall plant health, are also affected by climate change. Changing disease pressures cause growers and researchers to reassess disease management and climate change adaptation strategies. Approaches such as climate smart integrated pest management, smart sprayer technology, protected culture cultivation, advanced diagnostics, and new soilborne disease management strategies are providing new tools for specialty crops growers. Researchers and educators need to work closely with growers to establish fruit and vegetable production systems that are resilient and responsive to changing climates. This review explores the effects of climate change on specialty food crops, pathogens, insect vectors, and pathosystems, as well as adaptations needed to ensure optimal plant health and environmental and economic sustainability.

Association Between Biofilm Formation and Extended-Spectrum Beta-Lactamase Production in Klebsiella pneumoniae Isolated from Fresh Fruits and Vegetables.

The presence of extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae in fresh fruits and vegetables is a growing public health concern. The primary objective of this study was to investigate the relationship between biofilm formation and extended-spectrum ß-lactamase (ESBL) production in K. pneumoniae strains obtained from fresh fruits and vegetables. Out of 120 samples analysed, 94 samples (78%) were found to be positive for K. pneumoniae. Among the K. pneumoniae strains isolated, 74.5% were from vegetables, whereas the remaining (25.5%) were from fresh fruits. K. pneumoniae isolates were resistant to at least three different classes of antibiotics, with ceftazidime (90%) and cefotaxime (70%) showing the highest resistance rates. While the high occurrence of ESBL-producing and biofilm-forming K. pneumoniae strains were detected in vegetables (73.5% and 73.7%, respectively), considerable amounts of the same were also found in fresh fruits (26.5% and 26.3%, respectively). The results further showed a statistically significant (P < 0.001) association between biofilm formation and ESBL production in K. pneumoniae strains isolated from fresh fruits and vegetables. Furthermore, the majority (81%) of the ESBL-producing strains harbored the blaCTX-M gene, while a smaller proportion of strains carried the blaTEM gene (30%), blaSHV gene (11%) or blaOXA (8%). This study highlights the potential public health threat posed by K. pneumoniae in fresh fruits and vegetables and emphasizes the need for strict surveillance and control measures.

The Gamma concept approach as a tool to predict fresh produce supporting or not the growth of L. monocytogenes.

Challenge tests are commonly employed to evaluate the growth behavior of L. monocytogenes in food matrices; they are known for being expensive and time-consuming. An alternative could be the use of predictive models to forecast microbial behavior under different conditions. In this study, the growth behavior of L. monocytogenes in different fresh produce was evaluated using a predictive model based on the Gamma concept considering pH, water activity (aw), and temperature as input factors. An extensive literature search resulted in a total of 105 research articles selected to collect growth/no growth behavior data of L. monocytogenes. Up to 808 L. monocytogenes behavior values and physicochemical characteristics were extracted for different fruits and vegetables. The predictive performance of the model as a tool for identifying the produce commodities supporting the growth of L. monocytogenes was proved by comparing with the experimental data collected from the literature. The model provided satisfactory predictions on the behavior of L. monocytogenes in vegetables (>80% agreement with experimental observations). For leafy greens, a 90% agreement was achieved. In contrast, the performance of the Gamma model was less satisfactory for fruits, as it tends to overestimate the potential of acid commodities to inhibit the growth of L. monocytogenes.

Prevalence of Salmonella spp. in meat, seafood, and leafy green vegetables from local markets and vegetable farms in Phnom Penh, Cambodia.

Salmonella is a major bacterial concern for public health globally. Although there are limited documentation on the prevalence of Salmonella species in Cambodia's food chain, some reports indicate that salmonellosis is a severe gastrointestinal infection in its population and especially in children. To investigate the presence of Salmonella spp., 285 food samples (75 meat, 50 seafood, and 160 leafy green vegetable samples) were randomly collected from various local markets in Phnom Penh capital and nearby farms in Cambodia. Concurrently, field observations were conducted to collect data on food hygiene and practices among the relevant actors. All food samples were analyzed using bacterial culture and plate counts, and the findings were confirmed serially with biochemical, serological, and PCR tests. The observational data on food hygiene and practices from farm to market revealed that the spread of Salmonella in the food-value chain from farm to market could pose health risks to consumers. The overall prevalence of Salmonella spp. was 48.4% (138/285), while the prevalence in meat, seafood, and vegetables was 71% (53/75), 64% (32/50), and 33% (53/160), respectively. Mean Salmonella plate count ranged from 1.2 to 7.40 log10 CFU/g, and there was no significant difference in bacterial counts between meat, seafood, and vegetable samples (p > 0.05). The most common serogroups among the isolated Salmonella spp. were B and C. These results suggest that a large proportion of meat, seafood, and vegetable products sold at local markets in Phnom Penh are contaminated with Salmonella spp. This is likely linked to inadequate hygiene and sanitation practices, including handling, storage, and preservation conditions. Observations on farms suggested that the prevalence of Salmonella in vegetables sold at the market could be linked to contamination relating to agricultural practices. Thus, controlling the spread of foodborne salmonellosis through the food-value chain from farms and retailers to consumers is warranted to enhance food safety in Cambodia.

Analysis on Foodborne Pathogen Contamination of Food Samples in Longnan City from 2013 to 2022.

The objective was to determine the prevalence of foodborne pathogens in food in Longnan City, Gansu Province, China. In this research, we conducted tests on baked foods, catering foods, meat, and fruits and vegetables sold in supermarkets, farmers' markets, restaurants, retail stores, street stalls, and school canteens from 2013 to 2022. We analyzed the variety of foodborne pathogens (Salmonella, Staphylococcus aureus, Listeria monocytogenes, Bacillus cereus, and diarrheagenic Escherichia coli) in different sites and food types. Once foodborne pathogens were detected in the sample, it was deemed unqualified. The total detection rates of foodborne pathogens were 1.559%, 3.349%, 1.980%, 1.040%, 3.383%, and 1.303% in food from supermarkets, farmers' markets, restaurants, retail stores, street stalls, and school canteens, respectively. No pathogenic bacteria were detected in baked foods. Salmonella, S. aureus, L. monocytogenes, B. cereus, and diarrheagenic E. coli were detected in catering foods, among which B. cereus had the highest detection rate. Salmonella was the most common pathogenic bacteria detected in meat, while the detection rate of pathogenic bacteria in fruits and vegetables was low, with only one positive sample for diarrheagenic E. coli. Among the six sites, street stalls (3.382%) and farmers' markets (3.349%) had higher detection rates of pathogens. In general, the detection rate of pathogens from 2013 to 2022 was not high, but there were also some hidden dangers. Catering food is vulnerable to pathogen contamination, and street stalls and farmers' markets are the main sites of pollution. According to the above findings, the regulatory authorities should continue to strengthen supervision, guarantee food safety through early warning, and reduce the risk of food contamination.

Determination of Post-Fermentation Waste from Fermented Vegetables as Potential Substitutes for Preservatives in o/w Emulsion.

Post-fermentation wastes are rich sources of various biologically active compounds with antimicrobial activity, whose potential is not being fully exploited. One of the possible applications of post-fermentation waste may be its use as a natural preservative that effectively combats pathogens found in formulations. The study aims included the following: (1) compare the antimicrobial and antioxidant activity of fermented vegetable extracts (FVEs), (2) examine the inhibition of cosmetic-borne pathogens by FVEs, and (3) estimate the preservative effectiveness of FVEs in o/w emulsions. It was found that fermented white cabbage, cucumber, celery, and the mixture of fermented white cabbage, cucumber, and celery (1:1:1) showed antibacterial and antifungal activity against all the tested reference microbial strains. The addition of fermented cucumber, celery, and the mixture of fermented white cabbage, cucumber, and celery (1:1:1) to the o/w emulsion fulfilled criterion A of the preservative effectiveness test for S. aureus, E. coli, and A. brasiliensis, but did not fulfill the criterion for P. aeruginosa and C. albicans. The tested FVEs have comparable activity to inhibit pathogens in o/w emulsion as sodium benzoate. The results of our study prove that FVEs can be valuable raw materials supporting the preservative system, which, in turn, can significantly reduce the concentration of preservatives used in o/w emulsion.

A review on fermented vegetables: Microbial community and potential upgrading strategy via inoculated fermentation.

Fermentation is a traditional method utilized for vegetable preservation, with microorganisms playing a crucial role in the process. Nowadays, traditional spontaneous fermentation methods are widely employed, which excessively depend on the microorganisms attached to the surface of raw materials, resulting in great difficulties in ideal control over the fermentation process. To achieve standardized production and improve product quality, it is essential to promote inoculated fermentation. In this way, starter cultures can dominate the fermentation processes successfully. Unfortunately, inoculated fermentation has not been thoroughly studied and applied. Therefore, this paper provides a systematic review of the potential upgrading strategy of vegetable fermentation technology. First, we disclose the microbial community structures and succession rules in some typical spontaneously fermented vegetables to comprehend the microbial fermentation processes well. Then, internal and external factors affecting microorganisms are explored to provide references for the selection of fermented materials and conditions. Besides, we widely summarize the potential starter candidates with various characteristics isolated from spontaneously fermented products. Subsequently, we exhibited the inoculated fermentation strategies with those isolations. To optimize the product quality, not only lactic acid bacteria that lead the fermentation, but also yeasts that contribute to aroma formation should be combined for inoculation. The inoculation order of the starter cultures also affects the microbial fermentation. It is equally important to choose a proper processing method to guarantee the activity and convenience of starter cultures. Only in this way can we achieve the transition from traditional spontaneous fermentation to modern inoculated fermentation.

Growth of Listeria monocytogenes in fresh vegetables and vegetable salad products: An update on influencing intrinsic and extrinsic factors.

The ability of foodborne pathogens to grow in food products increases the associated food safety risks. Listeria monocytogenes (Lm) is a highly adaptable pathogen that can survive and grow under a wide range of environmental circumstances, including otherwise inhibitory conditions, such as restrictive cold temperatures. It can also survive long periods under adverse environmental conditions. This review examines the experimental evidence available for the survival and growth of Lm on fresh vegetables and ready-to-eat vegetable salads. Published data indicate that, depending on certain intrinsic (e.g., nutrient composition) and extrinsic factors (e.g., storage temperature, packaging atmosphere), Lm can survive on and in a wide variety of vegetables and fresh-cut minimally processed vegetable salads. Studies have shown that temperature, modified atmosphere packaging, relative humidity, pH, water activity, background microbiota of vegetables, microbial strain peculiarities, and nutrient type and availability can significantly impact the fate of Lm in vegetables and vegetable salads. The influence of these factors can either promote its growth or decline. For example, some studies have shown that background microbiota inhibit the growth of Lm in vegetables and minimally processed vegetable salads, but others have reported a promoting, neutral, or insignificant effect on the growth of Lm. A review of relevant literature also indicated that the impact of most influencing factors is related to or interacts with other intrinsic or extrinsic factors. This literature synthesis contributes to the body of knowledge on possible strategies for improving food safety measures to minimize the risk of Lm-associated foodborne outbreaks involving vegetables and vegetable salads.

A meta-analysis of factors influencing the inactivation of Shiga toxin-producing Escherichia coli O157:H7 in leafy greens.

Recent advancements in modeling suggest that microbial inactivation in leafy greens follows a nonlinear pattern, rather than the simple first-order kinetics. In this study, we evaluated 17 inactivation models commonly used to describe microbial decline and established the conditions that govern microbial survival on leafy greens. Through a systematic review of 65 articles, we extracted 530 datasets to model the fate of Shiga toxin-producing Escherichia coli O157:H7 on leafy greens. Various factor analysis methods were employed to evaluate the impact of identified conditions on survival metrics. A two-parameter model (jm2) provided the best fit to most of both natural and antimicrobial-induced persistence datasets, whereas the one-parameter exponential model provided the best fit to less than 20% of the datasets. The jm2 model (adjusted R2 = .89) also outperformed the exponential model (adjusted R2 = .58) in fitting the pooled microbial survival data. In the context of survival metrics, the model averaging approach generated higher values than the exponential model for >4 log reduction times (LRTs), suggesting that the exponential model may be overpredicting inactivation at later time points. The random forest technique revealed that temperature and inoculum size were common factors determining inactivation in both natural and antimicrobial-induced die-offs.. The findings show the limitations of relying on the first-order survival metric of 1 LRT and considering nonlinear inactivation in produce safety decision-making.

A review of conditions influencing fate of Shiga toxin-producing Escherichia coli O157:H7 in leafy greens.

The accuracy of predictive microbial models used in quantitative microbial risk assessment (QMRA) relies on the relevancy of conditions influencing growth or inactivation. The continued use of log-linear models in studies remains widespread, despite evidence that they fail to accurately account for biphasic kinetics or include parameters to account for the effect of environmental conditions within the model equation. Although many experimental studies detail conditions of interest, studies that do not do so lead to uncertainty in QMRA modeling because the applicability of the predictive microbial models to the conditions in the risk scenarios is questionable or must be extrapolated. The current study systematically reviewed 65 articles that provided quantitative data and documented the conditions influencing the inactivation or growth of Shiga toxin-producing Escherichia coli (STEC) O157:H7 in leafy greens. The conditions were identified and categorized as environmental, biological, chemical, and/or processing. Our study found that temperature (n = 37 studies) and sanitizing and washing procedures (n = 12 studies) were the most studied conditions in the farm-to-table continuum of leafy greens. In addition, relative humidity was also established to affect growth and inactivation in more than one stage in the continuum. This study proposes the evaluation of the interactive effects of multiple conditions in processing and storage stages from controlled experiments as they relate to the fate of STEC O157:H7 in leafy greens for future quantitative analysis.

Revisiting the current and emerging concepts of postharvest fresh fruit and vegetable pathology for next-generation antifungal technologies.

Fungal infections of fresh fruits and vegetables (FFVs) can lead to safety problems, including consumer poisoning by mycotoxins. Various strategies exist to control fungal infections of FFVs, but their effectiveness and sustainability are limited. Recently, new concepts based on the microbiome and pathobiome have emerged and offer a more holistic perspective for advancing postharvest pathogen control techniques. Understanding the role of the microbiome in FFV infections is essential for developing sustainable control strategies. This review examines current and emerging approaches to postharvest pathology. It reviews what is known about the initiation and development of infections in FFVs. As a promising concept, the pathobiome offers new insights into the basic mechanisms of microbial infections in FFVs. The underlying mechanisms uncovered by the pathobiome are being used to develop more relevant global antifungal strategies. This review will also focus on new technologies developed to target the microbiome and members of the pathobiome to control infections in FFVs and improve safety by limiting mycotoxin contamination. Specifically, this review stresses emerging technologies related to FFVs that are relevant for modifying the interaction between FFVs and the microbiome and include the use of microbial consortia, the use of genomic technology to manipulate host and microbial community genes, and the use of databases, deep learning, and artificial intelligence to identify pathobiome markers. Other approaches include programming the behavior of FFVs using synthetic biology, modifying the microbiome using sRNA technology, phages, quorum sensing, and quorum quenching strategies. Rapid adoption and commercialization of these technologies are recommended to further improve the overall safety of FFVs.

Prevalence, serotype, antimicrobial susceptibility, contamination factors, and control methods of Salmonella spp. in retail fresh fruits and vegetables: A systematic review and meta-analysis.

This research presents a comprehensive review of Salmonella presence in retail fresh fruits and vegetables from 2010 to 2023, utilizing data from recognized sources such as PubMed, Scopus, and Web of Science. The study incorporates a meta-analysis of prevalence, serovar distribution, antimicrobial susceptibility, and antimicrobial resistance genes (ARGs). Additionally, it scrutinizes the heterogeneous sources across various food categories and geographical regions The findings show a pooled prevalence of 2.90% (95% CI: 0.0180-0.0430), with an increase from 4.63% in 2010 to 5.32% in 2022. Dominant serovars include S. Typhimurium (29.14%, 95% CI: 0.0202-0.6571) and S. Enteritidis (21.06%, 95% CI: 0.0181-0.4872). High resistance rates were noted for antimicrobials like erythromycin (60.70%, 95% CI: 0.0000-1.0000) and amoxicillin (39.92%, 95% CI: 0.0589-0.8020). The most prevalent ARGs were blaTEM (80.23%, 95% CI: 0.5736-0.9692) and parC mutation (66.67%, 95% CI: 0.3213-0.9429). Factors such as pH, water activity, and nutrient content, along with external factors like the quality of irrigation water and prevailing climatic conditions, have significant implications on Salmonella contamination. Nonthermal sterilization technologies, encompassing chlorine dioxide, ozone, and ultraviolet light, are emphasized as efficacious measures to control Salmonella. This review stresses the imperative need to bolster prevention strategies and control measures against Salmonella in retail fresh fruits and vegetables to alleviate related food safety risks.

Mathematical modeling of nutritional, color, texture, and microbial activity changes in fruit and vegetables during drying: A critical review.

Retention of quality attributes during drying of fruit and vegetables is a prime concern since the product's acceptability depends on the overall quality; particularly on the nutritional, color, and physical attributes. However, these quality parameters deteriorate during drying. Food quality changes are strongly related to the drying conditions and researchers have attempted to develop mathematical models to understand these relationships. A better insight toward the degradation of quality attributes is crucial for making real predictions and minimizing the quality deterioration. The previous empirical quality models employed kinetic modeling approaches to describe the quality changes and therefore, lack the realistic understanding of fundamental transport mechanisms. In order to develop a physics based mathematical model for the prediction of quality changes during drying, an in-depth understanding of research progress made toward this direction is indispensable. Therefore, the main goal of this paper is to present a critical review of the mathematical models developed and applied to describe the degradation kinetics of nutritional, color, and texture attributes during drying of fruit and vegetables and microbial growth model during storage. This review also presents the advantages and drawbacks of the existing models along with their industrial relevance. Finally, future research propositions toward developing physics-based mathematical model are presented.

LysP53 activity against Salmonella and its application in decontamination of Salmonella on fresh romaine lettuce.

Salmonella is a zoonotic pathogen that is commonly associated with foodborne disease outbreaks. This study found that a newly identified Gram-negative lysin LysP53 had good activity against a wide range of Salmonella, including Salmonella Newington, Salmonella Typhimurium, and Salmonella Dublin. Without the help of an outer membrane permeabilizer, 4 µM LysP53 could reduce 97.6% of planktonic Salmonella Enteritidis and 90% of the bacteria in biofilms. Moreover, LysP53 was highly thermostable because it maintained >90% activity even after exposure to temperatures up to 95 °C. Although high concentrations of salts could reduce the activity, LysP53 was found safe for oral gavage of mice without affecting body weights and cytokines in sera and able to reduce 90% of Salmonella Enteritidis loads on fresh romaine lettuce after 30 min of treatment. Because of its good activity against a wide range of bacteria, thermal stability, safe for oral administration, LysP53 could be used as a biocontrol agent for reducing bacterial loads in fresh vegetable food. KEY POINTS: • Lysin LysP53 has high bactericidal activity against Salmonella. • LysP53 is thermostable even at high temperature of up to 95 °C. • LysP53 can be used for topical decontamination of Salmonella on vegetables.

Growth models for Salmonella, E. coli O157:H7 and L. monocytogenes give different predictions for pathogen growth in cut leafy greens transportation, but are consistent in identifying higher risk conditions.

Leafy greens are frequently implicated in foodborne disease outbreaks and cut-leafy greens are a food that requires time and temperature control for safety. Predictive microbiology uses mathematical models to predict the growth of bacteria based on environmental conditions. The objective of our study was to compare published square root growth models for Salmonella (n = 6), pathogenic E. coli (n = 6) and Listeria monocytogenes (n = 4) using real world transport temperature data. Data from trucks transporting fresh-cut leafy greens during cross-country shipments were used as temperature inputs to the models. Bacterial growth was computed using the temperatures from each probe in every truck over the duration of transit, which resulted in 12-18 growth predictions per truck for each model. Each model generally gave significantly different predictions than other models for the same organism. The exception was for the two Salmonella models predicting the least growth and the two Salmonella models predicting the most growth which gave predictions that were not significantly different. Although different models tended to give different predictions, their ability to rank risk by truck was generally consistent across models. While absolute risk might be dependent upon choice of model, relative risk is independent of model choice.

Internalization of Salmonella in Leafy Greens and Impact on Acid Tolerance.

Salmonella colonizes the surface or the inner part of leafy greens, while the ability of internalized bacteria to evade common disinfection practices may pose a considerable risk. Hereby, we aimed to assess how the colonization and internalization of Salmonella spp. (i) vary with the type of leafy green, the storage conditions (temperature, time), and Salmonella serovar at phenotypic and gene transcriptional level (regarding stress- and virulence- or type III secretion system [T3SS]-associated genes) and (ii) potentially impact the survival of the pathogen against subsequent exposure at lethal pH (2.7), mimicking the gastric acidity. Internalized Salmonella reached 3.0 to 5.0 log CFU/g depending on storage conditions and vegetable, with spinach and chicory allowing the highest (P < 0.05) internalization. Prolonged storage (48 h) at 20°C increased the recovery of internalized Salmonella in spinach and green amaranth by 1.0 to 1.5 log units. Colonization of Salmonella on/in leafy vegetables induced the transcription (maximum fold change [FCmax], ∼2,000) of T3SS-related genes. Interserovar variation regarding the internalization ability of Salmonella was observed only in lettuce and green amaranth in a time- and temperature-dependent manner. Attached cells exhibited higher survival rates against low pH than the internalized subpopulation; however, habituation at 20°C in lettuce and amaranth induced acid tolerance to internalized cells, manifested by the 1.5 to 2.0 log CFU/g survivors after 75 min at pH 2.7. Habituation of Salmonella in vegetable extracts sensitized it toward acid, while indigenous microbiota had limited impact on acid resistance of the organism. These findings reveal physiological aspects of Salmonella colonizing leafy vegetables that could be useful in fresh produce microbial risk assessment. IMPORTANCE Consumption of leafy greens has been increasingly associated with foodborne illnesses, and their contamination could occur at pre- and/or postharvest level. Human pathogens may become passively or actively internalized in plant tissues, thereby escaping decontamination procedures. Plant colonization may impact bacterial physiology such as stress resistance and virulence. In this study, it was demonstrated that internalization of Salmonella spp., at the postharvest level, varied with type of vegetable, serovar, and storage conditions. Attached and internalized subpopulations of Salmonella on/in leafy greens showed distinct physiological responses regarding transcriptional changes of stress- and virulence-associated genes, as well as survival capacity against subsequent exposure to lethal pH (2.7). These findings could contribute to a better understanding and potential (re)definition of the risk of enteric pathogens colonizing leafy greens, as well as to the design of intervention strategies aiming to improve the microbiological safety of fresh produce.

Impact of population density and stress adaptation on the internalization of Salmonella in leafy greens.

Salmonella enterica is capable of entering the interior of leafy greens and establishing in the apoplastic area, a phenomenon known as internalization. The ability of internalized bacteria to evade common disinfection practices poses a well-established risk. Our aim was to study the effect of: i) inoculum size and ii) prior adaptation of Salmonella to sublethal stresses, on the internalization of the pathogen in four leafy vegetables. Spinach, lettuce, arugula and chicory were inoculated, by immersion for 2 min at room temperature with: i) Salmonella Enteritidis at 3.0, 4.0, 5.0, 6.0, 7.0 log CFU/mL and ii) non-adapted or adapted S. Enteritidis to acid (in TSB with 1% glucose, incubated for 24 h at 37 °C), cold (in TSB for 7 days at 4 °C), starvation (0.85% NaCl of pH 6.6, 48 h at 37 °C) or desiccation (1.5 h at 42 °C, 4 days at 21 °C) stress at appx 3.5 log CFU/mL). Inoculated leafy greens were subsequently stored at 5 °C and 20 °C for 2 h and 48 h (n = 2 × 2). Population of internalized Salmonella, after surface decontamination with 1% w/v AgNO3, was assessed on selective media. Even the lowest initial bacterial inoculum was adequate for internalization of Salmonella to occur in leafy vegetables. Non-adapted Salmonella inoculum of 7.0 (maximum) and 3.0 log CFU/mL (lowest inoculation level tested) after short storage (2 h) resulted in 3.7-4.3 and 1.3-1.5 log CFU/g internalized bacterial population, respectively. Colonization (including both attachment and internalization processes), as well as internalization process, were positively correlated to initial inoculum level. These processes reached a different plateau beyond which, no further increase in internalization was observed. Adaptation of the pathogen to mild stresses enhanced internalization (P < 0.05), with desiccation- and acid-adapted Salmonella demonstrating the highest internalization capacity, regardless of the vegetable and storage temperature. These findings could contribute to further elucidation of colonization capacity of Salmonella in leafy vegetables and assist in selecting the proper conditions that contribute to the prevention of fresh produce contamination with Salmonella.