Scanning mutagenesis of RNA-binding protein ProQ reveals a quality control role for the Lon protease.

The FinO-domain protein ProQ belongs to a widespread family of RNA-binding proteins (RBPs) involved in gene regulation in bacterial chromosomes and mobile elements. While the cellular RNA targets of ProQ have been established in diverse bacteria, the functionally crucial ProQ residues remain to be identified under physiological conditions. Following our discovery that ProQ deficiency alleviates growth suppression of Salmonella with succinate as the sole carbon source, an experimental evolution approach was devised to exploit this phenotype. By coupling mutational scanning with loss-of-function selection, we identified multiple ProQ residues in both the amino-terminal FinO domain and the variable carboxy-terminal region that are required for ProQ activity. Two carboxy-terminal mutations abrogated ProQ function and mildly impaired binding of a model RNA target. In contrast, several mutations in the FinO domain rendered ProQ both functionally inactive and unable to interact with target RNA in vivo. Alteration of the FinO domain stimulated the rapid turnover of ProQ by Lon-mediated proteolysis, suggesting a quality control mechanism that prevents the accumulation of nonfunctional ProQ molecules. We extend this observation to Hfq, the other major sRNA chaperone of enteric bacteria. The Hfq Y55A mutant protein, defective in RNA-binding and oligomerization, proved to be labile and susceptible to degradation by Lon. Taken together, our findings connect the major AAA+ family protease Lon with RNA-dependent quality control of Hfq and ProQ, the two major sRNA chaperones of Gram-negative bacteria.

Metabolites from Marine-Derived Fungi as Potential Antimicrobial Adjuvants.

Marine-derived fungi constitute an interesting source of bioactive compounds, several of which exhibit antibacterial activity. These acquire special importance, considering that antimicrobial resistance is becoming more widespread. The overexpression of efflux pumps, capable of expelling antimicrobials out of bacterial cells, is one of the most worrisome mechanisms. There has been an ongoing effort to find not only new antimicrobials, but also compounds that can block resistance mechanisms which can be used in combination with approved antimicrobial drugs. In this work, a library of nineteen marine natural products, isolated from marine-derived fungi of the genera Neosartorya and Aspergillus, was evaluated for their potential as bacterial efflux pump inhibitors as well as the antimicrobial-related mechanisms, such as inhibition of biofilm formation and quorum-sensing. Docking studies were performed to predict their efflux pump action. These compounds were also tested for their cytotoxicity in mouse fibroblast cell line NIH/3T3. The results obtained suggest that the marine-derived fungal metabolites are a promising source of compounds with potential to revert antimicrobial resistance and serve as an inspiration for the synthesis of new antimicrobial drugs.

Microbiological analysis of frozen profiteroles and mini chocolate eclairs implicated in a national salmonellosis outbreak.

Between November 2018 and May 2019, Canada experienced a nationwide salmonellosis outbreak linked to the presence of Salmonella enterica ser. Enteritidis in frozen profiteroles. Analysis of the implicated food products revealed low levels of Salmonella ranging from 0.2 to 0.7 MPN/100g. Water activity and pH of the food samples ranged from 0.9479 to 0.9867 and 4.6-6.8 respectively indicating conditions conducive to bacterial growth. Higher levels of the hygiene indicators Enterobacteriaceae and coliforms were associated with Salmonella positive samples compared to Salmonella negative samples. Investigation of the relationship between storage conditions, temperature, and pathogen levels during thawing revealed that the profiteroles reached temperatures permissive to pathogen growth (≥5 °C) much sooner than pathogen growth was observed and that the composition of the food matrix can influence bacterial levels upon thawing. Collectively these data can be used to inform guidance to minimize the risk of infection from the consumption of contaminated cream-filled frozen desserts.

Variability of Amyloid Propensity in Imperfect Repeats of CsgA Protein of Salmonella enterica and Escherichia coli.

CsgA is an aggregating protein from bacterial biofilms, representing a class of functional amyloids. Its amyloid propensity is defined by five fragments (R1-R5) of the sequence, representing non-perfect repeats. Gate-keeper amino acid residues, specific to each fragment, define the fragment's propensity for self-aggregation and aggregating characteristics of the whole protein. We study the self-aggregation and secondary structures of the repeat fragments of Salmonella enterica and Escherichia coli and comparatively analyze their potential effects on these proteins in a bacterial biofilm. Using bioinformatics predictors, ATR-FTIR and FT-Raman spectroscopy techniques, circular dichroism, and transmission electron microscopy, we confirmed self-aggregation of R1, R3, R5 fragments, as previously reported for Escherichia coli, however, with different temporal characteristics for each species. We also observed aggregation propensities of R4 fragment of Salmonella enterica that is different than that of Escherichia coli. Our studies showed that amyloid structures of CsgA repeats are more easily formed and more durable in Salmonella enterica than those in Escherichia coli.

Air-ozonolysis activation of polyolefins versus use of laden finishing to form contact-active nonwoven materials.

Two synthetic approaches were explored for modification of the polyolefins polyethylene/polypropylene (PE/PP) to form contact-active nonwoven materials. In the first approach, polymer surfaces were activated by O2-free air-ozonolysis, and then the active agent (trimethoxysilyl) propyl-octadecyl-dimethyl-ammonium chloride (C18-TSA) was covalently bound. In the second approach, the active agent was directly conjugated to the commercial 'finishing' that was then applied to the polymer. The chemical, physical and microscopic properties of the modified polymers were comprehensively studied, and their active site density was quantified by fluorescein sodium salt-cetyltrimethylammonium chloride reaction. The antimicrobial activity of the prepared nonwovens against Bacillus subtilis (Gram-positive) and Salmonella enterica (Gram-negative), and their stability at various pHs and temperatures were examined. The two approaches conferred antimicrobial properties to the modified polymers and demonstrated stable linkage of C18-TSA. However, the performance of the nonwovens formed by the first approach was superior. The study suggests two feasible and safe pathways for the modification of polyolefins to form contact-active nonwoven materials that can be further applied in various fields, such as hygiene products, medical fabrics, sanitizing wipes, and more.

The highly dynamic nature of bacterial heteroresistance impairs its clinical detection.

Many bacterial species and antibiotic classes exhibit heteroresistance, a phenomenon in which a susceptible bacterial isolate harbors a resistant subpopulation that can grow in the presence of an antibiotic and cause treatment failure. The resistant phenotype is often unstable and without antibiotic selection it reverts back to susceptibility. Here we studied the dynamics by which these resistant subpopulations are enriched in the presence of antibiotic and recede back to their baseline frequency in the absence of selection. An increasing understanding of this instability will allow more effective diagnostics and treatment of infections caused by heteroresistant bacteria. We show for clinical isolates of Escherichia coli and Salmonella enterica that different antibiotics at levels below the MIC of the susceptible main population can cause rapid enrichment of resistant subpopulations with increased copy number of genes that cause resistance. Modelling and growth rate measurements of bacteria with increased gene copy number in cultures and by microscopy of single-cells in a microfluidic chip show that the fitness cost of gene amplifications and their intrinsic instability drives their rapid loss in the absence of selection. Using a common antibiotic susceptibility test, we demonstrate that this test strongly underestimates the occurrence of heteroresistance in clinical isolates.

The protected physiological state of intracellular Salmonella enterica persisters reduces host cell-imposed stress.

During infectious diseases, small subpopulations of bacterial pathogens enter a non-replicating (NR) state tolerant to antibiotics. After phagocytosis, intracellular Salmonella enterica serovar Typhimurium (STM) forms persisters able to subvert immune defenses of the host. Physiological state and sensing properties of persisters are difficult to analyze, thus poorly understood. Here we deploy fluorescent protein reporters to detect intracellular NR persister cells, and to monitor their stress response on single cell level. We determined metabolic properties of NR STM during infection and demonstrate that NR STM persisters sense their environment and respond to stressors. Since persisters showed a lower stress response compared to replicating (R) STM, which was not consequence of lower metabolic capacity, the persistent state of STM serves as protective niche. Up to 95% of NR STM were metabolically active at beginning of infection, very similar to metabolic capacity of R STM. Sensing and reacting to stress with constant metabolic activity supports STM to create a more permissive environment for recurrent infections. Stress sensing and response of persister may be targeted by new antimicrobial approaches.

Survival and transcriptomic response of Salmonella enterica on fresh-cut fruits.

Salmonella enterica is frequently implicated in foodborne disease outbreaks associated with fresh-cut fruits. In the U.S., more than one third of fruit-related outbreaks have been linked to two S. enterica serotypes Newport and Typhimurium. Approximately 80% of fruit-related human salmonellosis cases were associated with tomatoes, cantaloupes and cucumbers. In this study, we investigated the population dynamics of S. Newport and S. Typhimurium on fresh-cut tomato, cantaloupe, cucumber and apple under short-term storage conditions. We further compared the transcriptomic profiles of a S. Newport strain on fresh-cut tomato and cantaloupe using high-throughput RNA-seq. We demonstrated that both S. enterica Newport and Typhimurium survived well on various fresh-cut fruit items under refrigeration storage conditions, independent of inoculation levels. However, S. enterica displayed variable survival behaviors on different types of fruits. For example, at 7 d storage, the population of S. enterica reduced less than 0.2 log (p > 0.05) on fresh-cut tomato and cantaloupe, in contrast to ~0.5 log (p < 0.05) on cucumber and apple. RNA-seq analysis suggested that S. enterica mediates its survival on fresh-cut fruits through differentially regulating genes involved in specific carbon utilization and metabolic pathways. Several known bacterial virulence factors (e.g., pag gene) were found to be differentially regulated on fresh-cut tomato and cantaloupe, suggesting a link between the events of food contamination and subsequent human infection. Findings from this study contribute to a better understanding of S. enterica survival mechanisms on fresh-cut produce.

Limitation by a shared mutualist promotes coexistence of multiple competing partners.

Although mutualisms are often studied as simple pairwise interactions, they typically involve complex networks of interacting species. How multiple mutualistic partners that provide the same service and compete for resources are maintained in mutualistic networks is an open question. We use a model bacterial community in which multiple 'partner strains' of Escherichia coli compete for a carbon source and exchange resources with a 'shared mutualist' strain of Salmonella enterica. In laboratory experiments, competing E. coli strains readily coexist in the presence of S. enterica, despite differences in their competitive abilities. We use ecological modeling to demonstrate that a shared mutualist can create temporary resource niche partitioning by limiting growth rates, even if yield is set by a resource external to a mutualism. This mechanism can extend to maintain multiple competing partner species. Our results improve our understanding of complex mutualistic communities and aid efforts to design stable microbial communities.

Salmonella enterica Serovar Typhimurium Exploits Cycling through Epithelial Cells To Colonize Human and Murine Enteroids.

Enterobacterial pathogens infect the gut by a multistep process, resulting in colonization of both the lumen and the mucosal epithelium. Due to experimental constraints, it remains challenging to address how luminal and epithelium-lodged pathogen populations cross-feed each other in vivo Enteroids are cultured three-dimensional miniature intestinal organs with a single layer of primary intestinal epithelial cells (IECs) surrounding a central lumen. They offer new opportunities to study enterobacterial infection under near-physiological conditions, at a temporal and spatial resolution not attainable in animal models, but remain poorly explored in this context. We employed microinjection, time-lapse microscopy, bacterial genetics, and barcoded consortium infections to describe the complete infection cycle of Salmonella enterica serovar Typhimurium in both human and murine enteroids. Flagellar motility and type III secretion system 1 (TTSS-1) promoted Salmonella Typhimurium targeting of the intraepithelial compartment and breaching of the epithelial barrier. Strikingly, however, TTSS-1 also potently boosted colonization of the enteroid lumen. By tracing the infection over time, we identified a cycle(s) of TTSS-1-driven IEC invasion, intraepithelial replication, and reemergence through infected IEC expulsion as a key mechanism for Salmonella Typhimurium luminal colonization. These findings suggest a positive feed-forward loop, through which IEC invasion by planktonic bacteria fuels further luminal population expansion, thereby ensuring efficient colonization of both the intraepithelial and luminal niches.IMPORTANCE Pathogenic gut bacteria are common causes of intestinal disease. Enteroids-cultured three-dimensional replicas of the mammalian gut-offer an emerging model system to study disease mechanisms under conditions that recapitulate key features of the intestinal tract. In this study, we describe the full life cycle of the prototype gut pathogen Salmonella enterica serovar Typhimurium within human and mouse enteroids. We map the consecutive steps and define the bacterial virulence factors that drive colonization of luminal and epithelial compartments, as well as breaching of the epithelial barrier. Strikingly, our work reveals how bacterial colonization of the epithelium potently fuels expansion also in the luminal compartment, through a mechanism involving the death and expulsion of bacterium-infected epithelial cells. These findings have repercussions for our understanding of the Salmonella infection cycle. Moreover, our work provides a comprehensive foundation for the use of microinjected enteroids to model gut bacterial diseases.

Nature versus Nurture: Assessing the Impact of Strain Diversity and Pregrowth Conditions on Salmonella enterica, Escherichia coli, and Listeria Species Growth and Survival on Selected Produce Items.

Inoculation studies are important when assessing microbial survival and growth in food products. These studies typically involve the pregrowth of multiple strains of a target pathogen under a single condition; this emphasizes strain diversity. To gain a better understanding of the impacts of strain diversity ("nature") and pregrowth conditions ("nurture") on subsequent bacterial growth in foods, we assessed the growth and survival of Salmonella enterica (n = 5), Escherichia coli (n = 6), and Listeria (n = 5) inoculated onto tomatoes, precut lettuce, and cantaloupe rind, respectively. Pregrowth conditions included (i) 37°C to stationary phase (baseline), (ii) low pH, (iii) high salt, (iv) reduced water activity, (v) log phase, (vi) minimal medium, and (vii) 21°C. Inoculated tomatoes were incubated at 21°C; lettuce and cantaloupe were incubated at 7°C. Bacterial counts were assessed over three phases, including initial reduction (phase 1), change in bacterial numbers over the first 24 h of incubation (phase 2), and change over the 7-day incubation (phase 3). E. coli showed overall decline in counts (<1 log) over the 7-day period, except for a <1-log increase after pregrowth in high salt and to mid-log phase. In contrast, S. enterica and Listeria showed regrowth after an initial reduction. Pregrowth conditions had a substantial and significant effect on all three phases of S. enterica and E. coli population dynamics on inoculated produce, whereas strain did not show a significant effect. For Listeria, both pregrowth conditions and strain affected changes in phase 2 but not phases 1 and 3.IMPORTANCE Our findings suggest that inclusion of multiple pregrowth conditions in inoculation studies can best capture the range of growth and survival patterns expected for Salmonella enterica and Escherichia coli present on produce. This is particularly important for fresh and fresh-cut produce, where stress conditions encountered by pathogens prior to contamination can vary widely, making selection of a typical pregrowth condition virtually impossible. Pathogen growth and survival data generated using multiple pregrowth conditions will allow for more robust microbial risk assessments that account more accurately for uncertainty.

Salmonella enterica colonization and fitness in pre-harvest cantaloupe production.

Cantaloupes have emerged as significant vehicles of widespread foodborne illness outbreaks caused by bacterial pathogens, including Salmonella. The purpose of this study was to investigate the efficiency of Salmonella colonization and internalization in cantaloupes by relevant routes of contamination. Cantaloupe plants (Cucumis melo 'reticulatus') from two cultivars 'Athena' (Eastern) and 'Primo' (Western) were grown from commercial seed. Plants were maintained in the NCSU BSL-3P phytotron greenhouse. Salmonella enterica (a cocktail of cantaloupe-associated outbreak serovars Javiana, Newport, Panama, Poona and Typhimurium) contamination was introduced via blossoms or soil at ca. 4.4 log10 CFU/blossom or 8.4 log10 CFU/root zone, respectively. Cantaloupes were analyzed for Salmonella by enrichment in accordance with modified FDA-BAM methods. Five randomly chosen colonies from each Salmonella-positive sample were typed using the Agilent 2100 bioanalyzer following multiplex PCR. Data were analyzed for prevalence of contamination and serovar predominance in fruit, stems and soil. Of the total cantaloupe fruit harvested from Salmonella-inoculated blossoms (n = 63), 89% (56/63) were externally contaminated and 73% (46/63) had Salmonella internalized into the fruit. Serovar Panama was the most commonly isolated from the surface of fruit while S. Panama and S. Poona were the most prevalent inside the fruit. When soil was inoculated with Salmonella at one day post-transplant, 13% (8/60) of the plants were shown to translocate the organism to the lower stem (ca. 4 cm) by 7 days post-inoculation (dpi). We observed Salmonella persistence in the soil up to 60 dpi with S. Newport being the predominant serovar at 10 and 20 dpi. These data demonstrate that contaminated soil and blossoms can lead to Salmonella internalization into the plant or fruit at a relatively high frequency.

Inactivation of Salmonella enterica and Enterococcus faecium NRRL B2354 on cumin seeds using gaseous ethylene oxide.

The objectives of this study were to investigate the effects of processing parameters (relative humidity (RH), temperature, and exposure time) on the ethylene oxide (EtO) microbial inactivation of Salmonella spp. and to evaluate Enterococcus faecium NRRL B2354 as a suitable surrogate for Salmonella inactivation on cumin seeds. Five grams of cumin seeds inoculated with either Salmonella or E. faecium were treated with EtO at different temperatures (46, 53, and 60 °C) and RH (30, 40, and 50%) levels for different exposure time to investigate the effects of process parameters on the microbial inactivation. The Weibull model fit the survival data of both bacteria with a shape parameter p < 1, which showed a tailing effect with concave shape indicating that the sensitive cells were inactivated first, and the sturdy ones survived at low RH treatment conditions. In general, the log reductions of both bacteria on cumin seeds increased with the increasing RH and temperature for EtO treatment. RH is a critical factor for successful EtO inactivation treatment. RH must be higher than 40% to implement a successful and efficient EtO decontamination of cumin seeds. E. faecium consistently showed lower log reductions than those of Salmonella under all EtO treatment conditions investigated in this study, demonstrating that E. faecium is a suitable surrogate for Salmonella. Twenty minutes of EtO treatment at 50% RH achieved ~5 log reductions of both bacteria at all three temperatures. A response surface model was developed to predict the log reductions of both bacteria under different treatment conditions and the contour plots representing log reductions were created. Inactivation is positively correlated to temperature and RH. Therefore, a higher temperature is required to achieve the desired log reduction at lower RH and vice versa. The developed response surface model is a valuable tool for the spice industry in identifying the possible combinations of EtO process parameters (temperature, RH, and exposure time) required to achieve a desired microbial reduction of Salmonella for ensuring microbial food safety of spices.

In Vitro Assessment of Antimicrobial Resistance Dissemination Dynamics during Multidrug-Resistant-Bacterium Invasion Events by Using a Continuous-Culture Device.

Antimicrobial-resistant pathogens display significant public health threats by causing difficulties in clinical treatment of bacterial infection. Antimicrobial resistance (AMR) is transmissible between bacteria, significantly increasing the appearance of antimicrobial-resistant pathogens and aggravating the AMR problem. In this work, the dissemination dynamics of AMR from invading multidrug-resistant (MDR) Escherichia coli to a community of pathogenic Salmonella enterica was investigated using a continuous-culture device, and the behaviors of dissemination dynamics under different levels of antibiotic stress were investigated. Three MDR E. coli invasion events were analyzed in this work: MDR E. coli-S. enterica cocolonization, MDR E. coli invasion after antibiotic treatment of S. enterica, and MDR E. coli invasion before antibiotic treatment of S. enterica It was found that both horizontal gene transfer (HGT) and vertical gene transfer (VGT) play significant roles in AMR dissemination, although different processes contribute differently under different circumstances, that environmental levels of antibiotics promote AMR dissemination by enhancing HGT rather than leading to selective advantage for resistant bacteria, and that early invasion of MDR E. coli completely and quickly sabotages the effectiveness of antibiotic treatment. These findings contribute to understanding the drivers of AMR dissemination under different antibiotic stresses, the detrimental impact of environmental tetracycline contamination, and the danger of nosocomial presence and dissemination of MDR nonpathogens.IMPORTANCE Antimicrobial resistance poses a grave threat to public health and reduces the effectiveness of antimicrobial drugs in treating bacterial infections. Antimicrobial resistance is transmissible, either by horizontal gene transfer between bacteria or by vertical gene transfer following inheritance of genetic traits. The dissemination dynamics and behaviors of this threat, however, have not been rigorously investigated. In this work, with a continuous-culture device, we studied antimicrobial resistance dissemination processes by simulating antimicrobial-resistant Escherichia coli invasion to a pathogenic Salmonella enterica community. Using this novel tool, we provide evidence on the drivers of antimicrobial resistance dissemination, on the detrimental impact of environmental antibiotic contamination, and on the danger of antimicrobial resistance in hospitals, even if what harbors the antimicrobial resistance is not a pathogen. This work furthers our understanding of antimicrobial resistance and its dissemination between bacteria and of antibiotic therapy, our most powerful tool against bacterial infection.

Interferon-gamma promotes iron export in human macrophages to limit intracellular bacterial replication.

Salmonellosis and listeriosis together accounted for more than one third of foodborne illnesses in the United States and almost half the hospitalizations for gastrointestinal diseases in 2018 while tuberculosis afflicted over 10 million people worldwide causing almost 2 million deaths. Regardless of the intrinsic virulence differences among Listeria monocytogenes, Salmonella enterica and Mycobacterium tuberculosis, these intracellular pathogens share the ability to survive and persist inside the macrophage and other cells and thrive in iron rich environments. Interferon-gamma (IFN-γ) is a central cytokine in host defense against intracellular pathogens and has been shown to promote iron export in macrophages. We hypothesize that IFN-γ decreases iron availability to intracellular pathogens consequently limiting replication in these cells. In this study, we show that IFN-γ regulates the expression of iron-related proteins hepcidin, ferroportin, and ferritin to induce iron export from macrophages. Listeria monocytogenes, S. enterica, and M. tuberculosis infections significantly induce iron sequestration in human macrophages. In contrast, IFN-γ significantly reduces hepcidin secretion in S. enterica and M. tuberculosis infected macrophages. Similarly, IFN-γ-activated macrophages express higher ferroportin levels than untreated controls even after infection with L. monocytogenes bacilli; bacterial infection greatly down-regulates ferroportin expression. Collectively, IFN-γ significantly inhibits pathogen-associated intracellular iron sequestration in macrophages and consequently retards the growth of intracellular bacterial pathogens by decreasing iron availability.

The use of embryonic chicken eggs as an alternative model to evaluate the virulence of Salmonella enterica serovar Gallinarum.

Salmonella enterica serovar Gallinarum (S. Gallinarum) can cause fowl typhoid, a severe systemic disease responsible for considerable economic losses. Chicken pathogenicity test is the traditional method for assessing the virulence of S. Gallinarum. However, this method is limited by several factors, including ethical considerations, costs, and the need for specialized facilities. Hence, we established a chicken embryo lethality assay (ELA) model to determine the virulence of S. Gallinarum. Three virulent and three avirulent representative strains, which were confirmed by the chicken pathogenicity test, were used to perform the ELA. The most significant difference between the virulent and avirulent strains could be observed when 13-day-old embryos were inoculated via the AC route and incubated for 5 days. Based on a 50% embryo lethal dose (ELD50), isolates considered to be virulent had a Log10ELD50 of ≤ 4.0, moderately virulent strains had a Log10ELD50 of 4.0-6.1, and avirulent isolates had a Log10ELD50 of ≥ 6.1. Different abilities to invade the liver of embryos were found between the virulent and avirulent strains by a growth curve experiment in vitro. The maximum colony-forming units (CFU) of the virulent strain was about 10,000 times higher than that of the avirulent strain in the liver at 5 days post infection. The ELA results of 42 field strains showed that thirty-two strains (76.2%) were virulent, nine were moderately virulent (21.4%), and one strain was avirulent (2.4%). In conclusion, these results suggest that the ELA can be used as an alternative method to assess the virulence of S. Gallinarum, which will contribute to the study of virulence genes, virulence evolution, pathogenic mechanisms and vaccine development.

Antimicrobial activities of organic acid vapors against Acidovorax citrulli, Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes on Cucurbitaceae seeds.

This study investigated the antimicrobial activities of organic acid vapors against a phytopathogen (Acidovorax citrulli) and foodborne pathogens (Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes) on the surface of Cucurbitaceae seeds. Germination percentages of cucumber, honeydew melon and watermelon seeds treated with acetic and propionic acid vapors (100 mg/L) at 50 °C and 43% or 85% relative humidity (RH) for up to 2 h did not significantly (P > 0.05) decrease. Treatment with formic acid significantly (P ≤ 0.05) decreased the germination percentage. The antimicrobial activities of acetic and propionic acid vapors (100 mg/L; 50 °C; 43% or 85% RH) were determined. A. citrulli was inactivated within 1 h on cucumber and watermelon seeds, regardless of type of organic acid or RH. The phytopathogen was reduced to levels below the detection limit (-0.5 log CFU/g) for enrichment on honeydew melon seeds treated with acetic acid vapor. S. enterica and L. monocytogenes were inactivated within 2 h at 85% RH on honeydew melon and watermelon seeds treated with acetic acid and propionic acid vapors. E. coli O157: H7 was inactivated by treatment with acetic acid vapor at 85% RH. This study provides useful information for developing a method to decontaminate Curcurbitaceae seeds using organic acid vapors as lethal agents.

A quasimetagenomics method for concerted detection and subtyping of Salmonella enterica and E. coli O157:H7 from romaine lettuce.

Quasimetagenomics refers to the sequencing of a modified food microbiome to facilitate combined detection and subtyping of targeted pathogens in a single workflow. Through quasimetagenomic sequencing, pathogens are detected and subtyped in a shortened time frame compared to traditional culture enrichment and whole genome sequencing-based analyses. While this method was previously used to detect and subtype Salmonella enterica from chicken, iceberg lettuce, and black pepper, it has not been applied to investigate multiple pathogens in one workflow. A quasimetagenomic method to concertedly detect and subtype Salmonella enterica and Escherichia coli O157:H7 from artificially contaminated romaine lettuce in a single workflow was developed. All quasimetagenomic samples with initial target pathogen inoculum levels of ~1 CFU/g were detected and serotyped after co-enrichment of the two pathogens for 12 h. Single nucleotide polymorphism typing was achievable for some initial pathogen inoculum levels as low as ~0.1 CFU/g. Our results suggest that this method can be used for concerted detection and subtyping of multiple bacterial pathogens from romaine lettuce even at low contamination levels.

Gaseous antimicrobial treatments to control foodborne pathogens on almond kernels and whole black peppercorns.

Gaseous treatments with ClO2 and O3 on low-moisture foods (LMFs) have been reported for their efficient bacterial reduction without affecting the external quality of food. However, these studies were conducted on a small scale, which limits their application to LMF industries. We aimed to evaluate the effectiveness of gaseous antimicrobial intervention with ClO2 or O3 to reduce foodborne pathogens (Shiga toxin-producing Escherichia coli, serovars of Salmonella enterica, and Listeria monocytogenes) inoculated on almonds and peppercorns maintained under various conditions. Almonds were treated for over 4 or 6 h. Peppercorns were treated for over 2.5 or 4 h. Gaseous O3 treatment was used for 6 h on almonds and 2 or 4 h on peppercorns. Additionally, the effects of relative humidity (RH) during the treatment of peppercorns and post-treatment heating on almonds were evaluated. Heating at 65 °C post-ClO2 treatment yielded the highest bacterial log reduction of 4.6 CFU/g on almonds, while 80% RH resulted in 3.7-log bacterial reduction on peppercorns. Gaseous O3 resulted in maximum log reductions of 1.3 and 2.5 CFU/g on almonds and peppercorns, respectively. No visual damage was observed. In conclusion, ClO2 was more efficient than O3 and the treatment can be incorporated into industrial practices.

Comparing serotyping with whole-genome sequencing for subtyping of non-typhoidal Salmonella enterica: a large-scale analysis of 37 serotypes with a public health impact in the USA.

Serotyping has traditionally been used for subtyping of non-typhoidal Salmonella (NTS) isolates. However, its discriminatory power is limited, which impairs its use for epidemiological investigations of source attribution. Whole-genome sequencing (WGS) analysis allows more accurate subtyping of strains. However, because of the relative newness and cost of routine WGS, large-scale studies involving NTS WGS are still rare. We aimed to revisit the big picture of subtyping NTS with a public health impact by using traditional serotyping (i.e. reaction between antisera and surface antigens) and comparing the results with those obtained using WGS. For this purpose, we analysed 18 282 sequences of isolates belonging to 37 serotypes with a public health impact that were recovered in the USA between 2006 and 2017 from multiple sources, and were available at the National Center for Biotechnology Information (NCBI). Phylogenetic trees were reconstructed for each serotype using the core genome for the identification of genetic subpopulations. We demonstrated that WGS-based subtyping allows better identification of sources potentially linked with human infection and emerging subpopulations, along with providing information on the risk of dissemination of plasmids and acquired antimicrobial resistance genes (AARGs). In addition, by reconstructing a phylogenetic tree with representative isolates from all serotypes (n=370), we demonstrated genetic variability within and between serotypes, which formed monophyletic, polyphyletic and paraphyletic clades. Moreover, we found (in the entire data set) an increased detection rate for AARGs linked to key antimicrobials (such as quinolones and extended-spectrum cephalosporins) over time. The outputs of this large-scale analysis reveal new insights into the genetic diversity within and between serotypes; the polyphyly and paraphyly of certain serotypes may suggest that the subtyping of NTS to serotypes may not be sufficient. Moreover, the results and the methods presented here, leading to differentiation between genetic subpopulations based on their potential risk to public health, as well as narrowing down the possible sources of these infections, may be used as a baseline for subtyping of future NTS infections and help efforts to mitigate and prevent infections in the USA and globally.