Listeria monocytogenes is a foodborne pathogen that can cause severe disease (listeriosis) in susceptible individuals. It is ubiquitous in the environment and often exhibits resistance to heavy metals. One of the determinants that enables Listeria to tolerate exposure to cadmium is the cadAC efflux system, with CadA being a P-type ATPase. Three different cadA genes (designated cadA1 to cadA3) were previously characterized in L. monocytogenes. A novel putative cadmium resistance gene (cadA4) was recently identified through whole-genome sequencing, but experimental confirmation for its involvement in cadmium resistance is lacking. In this study, we characterized cadA4 in L. monocytogenes strain F8027, a cadmium-resistant strain of serotype 4b. By screening a mariner-based transposon library of this strain, we identified a mutant with reduced tolerance to cadmium and that harbored a single transposon insertion in cadA4. The tolerance to cadmium was restored by genetic complementation with the cadmium resistance cassette (cadA4C), and enhanced cadmium tolerance was conferred to two unrelated cadmium-sensitive strains via heterologous complementation with cadA4C. Cadmium exposure induced cadA4 expression, even at noninhibitory levels. Virulence assessments in the Galleria mellonella model suggested that a functional cadA4 suppressed virulence, potentially promoting commensal colonization of the insect larvae. Biofilm assays suggested that cadA4 inactivation reduced biofilm formation. These data not only confirm cadA4 as a novel cadmium resistance determinant in L. monocytogenes but also provide evidence for roles in virulence and biofilm formation.IMPORTANCE Listeria monocytogenes is an intracellular foodborne pathogen causing the disease listeriosis, which is responsible for numerous hospitalizations and deaths every year. Among the adaptations that enable the survival of Listeria in the environment are the abilities to persist in biofilms, grow in the cold, and tolerate toxic compounds, such as heavy metals. Here, we characterized a novel determinant that was recently identified on a larger mobile genetic island through whole-genome sequencing. This gene (cadA4) was found to be responsible for cadmium detoxification and to be a divergent member of the Cad family of cadmium efflux pumps. Virulence assessments in a Galleria mellonella model suggested that cadA4 may suppress virulence. Additionally, cadA4 may be involved in the ability of Listeria to form biofilms. Beyond the role in cadmium detoxification, the involvement of cadA4 in other cellular functions potentially explains its retention and wide distribution in L. monocytogenes.KEYWORDS Listeria monocytogenes, cadmium resistance, cadA, biofilm, virulence L isteria monocytogenes is a Gram-positive rod-shaped bacterium widely distributed in nature (1). As a facultative intracellular foodborne pathogen causing the disease listeriosis, L. monocytogenes organisms are responsible for numerous hospitalizations and deaths in the United States every year (2). T...

Salmonella enterica is the leading foodborne pathogen associated with outbreaks involving low-moisture foods (LMFs). However, the genes involved in Salmonella's longterm survival on LMFs remain poorly characterized. In this study, in-shell pistachios were inoculated with Tn5-based mutant libraries of S. Enteritidis P125109, S. Typhimurium 14028s, and S. Newport C4.2 at approximate 10 8 CFU/g and stored at 25 • C. Transposon sequencing analysis (Tn-seq) was then employed to determine the relative abundance of each Tn5 insertion site immediately after inoculation (T 0), after drying (T 1), and at 120 days (T 120). In S. Enteritidis, S. Typhimurium, and S. Newport mutant libraries, the relative abundance of 51, 80, and 101 Tn5 insertion sites, respectively, was significantly lower at T 1 compared to T 0 , while in libraries of S. Enteritidis and S. Typhimurium the relative abundance of 42 and 68 Tn5 insertion sites, respectively, was significantly lower at T 120 compared to T 1. Tn5 insertion sites with reduced relative abundance in this competition assay were localized in DNA repair, lipopolysaccharide biosynthesis and stringent response genes. Twelve genes among those under strong negative selection in the competition assay were selected for further study. Whole gene deletion mutants in ten of these genes, sspA, barA, uvrB, damX, rfbD, uvrY, lrhA, yifE, rbsR, and ompR, were impaired for individual survival on pistachios. The findings highlight the value of combined mutagenesis and sequencing to identify novel genes important for the survival of Salmonella in low-moisture foods.

The 2011 listeriosis outbreak attributed to whole cantaloupe involved several genetically distinct strains of serotypes 1/2a and 1/2b that had not been previously reported in invasive listeriosis outbreaks. Here we investigated the potential of strains from the 2011 cantaloupe outbreak to adhere, survive, and grow on cantaloupe rind and flesh and in juice extracted from cantaloupe at different temperatures (4, 8, and 25°C). All strains were able to adhere and grow, with ∼10-fold increases after 7 days at 4 or 8°C and after 24 h at 25°C, with a propensity for more growth on rind than on flesh or in extract. No significant differences in growth potential were noted among the different strains or between them and unrelated strains from other listeriosis outbreaks involving celery, deli meats, or hot dogs. Similarly to the cantaloupe outbreak strains, these other strains exhibited greater propensity for growth on rind than on flesh or in extract. Rinsing of cantaloupe fragments in sterile water resulted in temporary reductions of the populations by 50- to 100-fold, suggesting the potential of such washing to reduce risk if the produce is promptly consumed. The absence of marked differences in adherence or growth between the cantaloupe outbreak strains and strains from other outbreaks highlights the need to further characterize the 2011 cantaloupe outbreak strains and elucidate potential biological attributes that contributed to their implication in the outbreak.

Listeria monocytogenes has been extensively studied as a model facultative intracellular pathogen. While the roles of major virulence factors in host-pathogen interactions have been extensively characterized, recent work suggests that some of these factors can also contribute to environmental proliferation of this pathogen. In this study, we characterized two non-hemolytic transposon mutants of strain 2011L-2858 (serotype 1/2b), implicated in the 2011 listeriosis outbreak via whole cantaloupe, for their capacity to form biofilms on polystyrene, aggregate, and colonize cantaloupe rind. One mutant harbored a single mariner-based transposon insertion in hly, encoding the hemolysin Listeriolysin O, while the other harbored a single insertion in prfA, encoding PrfA, a master regulator for hly and numerous other virulence genes. Biofilm formation was significantly reduced in the prfA mutant, and to a lesser extent, in the hly mutant. Inactivation of either hly or prfA significantly reduced L. monocytogenes aggregation. However, both mutants adhered similarly to the wildtype parental strain on cantaloupe rind at either 25 or 37°C. Furthermore, growth and competitive fitness of the mutants on cantaloupe rind was not significantly impacted at either temperature. The findings suggest that, in spite of their involvement in biofilm formation and aggregation, these key virulence determinants may not be required for the ability of L. monocytogenes to colonize fresh produce.

Temperature is arguably the most important factor affecting microbial proliferation in fresh-cut produce. In this study, growth of Listeria monocytogenes in diced onions and celery and Salmonella Typhimurium in diced tomatoes was determined in modified atmosphere packages and snap-fit containers using three fluctuating temperature scenarios for transport, retail storage, and display. As expected, L. monocytogenes growth in diced onions and celery varied depending on the extent of temperature abuse, with exposure to high and intermediate temperature-abuse scenarios generally being growth supportive. A Baranyi primary model with a square-root secondary model for maximum growth rate, and a linear model for maximum population density, were used to estimate Listeria growth under fluctuating temperature. Accuracy and acceptability of the model prediction were evaluated in terms of root mean square error (RMSE) and acceptable prediction zone (APZ), respectively. Overall, growth predictions for L. monocytogenes were more accurate for celery (RMSE, 0.28 to 0.47) than onions (RMSE, 0.42 to 1.53) under the fluctuating temperature scenarios tested. However, both predictions yielded APZ values that ranged from 82 to 100% for celery and 36 to 78% for onions. In contrast, Salmonella Typhimurium populations increased more than 1 log CFU/g in diced tomatoes under the three fluctuating temperature scenarios studied. Overall, these diced products packaged under a high-oxygen atmosphere showed decreased pathogen growth compared with product stored in a passive modified atmosphere. Findings from this study will be particularly useful in assessing the risk associated with consumption of diced celery, tomatoes, and onions and in designing effective packaging strategies to minimize pathogen growth in fresh-cut produce.

Salmonella can become viable but non-culturable (VBNC) in response to environmental stressors but the induction of the VBNC state in Salmonella contaminating ready-to-eat dried fruit is poorly characterized. Dried apples, strawberries and raisins were mixed with a five-strain cocktail of Salmonella at 4% volume per weight of dried fruit at 10 9 CFU/g. The inoculated dried fruit were then dried in desiccators at 25°C until the water activity (a w ) approximated that of the uninoculated dried fruit. However, Salmonella could not be recovered after drying, not even after enrichment, suggesting a population reduction of approx. 8 log CFU/g. To assess the potential impact of storage temperature on survival, dried apples were spot-inoculated with the Salmonella cocktail, dried under ambient atmosphere at 25°C and stored at 4 and 25°C. Spot-inoculation permitted recovery of Salmonella on dried apple after drying, with the population of Salmonella decreasing progressively on dried apples stored at 25°C until it was undetectable after about 46 days, even following enrichment. The population decline was noticeably slower at 4°C, with Salmonella being detected until 82 days. However, fluorescence microscopy and laser scanning confocal microscopy with the LIVE-DEAD Bac Light Bacterial Viability system at timepoints at which no Salmonella could be recovered on growth media even following enrichment, showed that a large proportion (56-85%) of the Salmonella cells on the dried fruit were viable. The data suggest that the unique combination of stressors in dried fruit can induce large numbers of VBNC cells of Salmonella . IMPORTANCE Salmonella is a leading foodborne pathogen globally causing numerous outbreaks of foodborne illnesses and remains the leading contributor to deaths attributed to foodborne disease in the United States and other industrialized nations. Therefore, efficient detection methods for Salmonella contaminating food are critical for public health and food safety. Culture-based microbiological methods are considered the gold standard for the detection and enumeration of Salmonella in food. Findings from this study suggest that unique stressors on dried fruit can induce the VBNC state in Salmonella , thus rendering it undetectable with culture-based methods even though the bacteria remain viable. Therefore, strong consideration should be given to using, in addition to culture-based methods, microscopic and molecular methods for the accurate detection of all viable and/or culturable cells of Salmonella contaminating dried fruit, as all these cells have the potential to cause human illness.

Methicillin-resistant Staphylococcus aureus (MRSA) infections cause substantive morbidity and mortality in neonates. Using publicly available resources from the National Center of Biotechnology Information (NCBI) and Food and Drug Administration’s (FDA) GalaxyTrakr pipeline, we illustrate the dynamics of MRSA colonization and infection in neonates.

MRSA infections cause significant morbidity and mortality in neonates. Clinical testing and routine surveillance screening identified an increase in neonates with MRSA colonization and infection which triggered prospective genomic surveillance. Here we show the complex transmission dynamics of MRSA in a NICU setting. Analyses revealed concurrent transmission chains affecting 16 of 22 MRSA-colonized patients (68%), and 3.1% of all NICU patients (n=517). Prematurity and longer lengths of stay increased risks for colonization. Intervals of up to 7 months occurred among some cluster-related isolates. 3 of 22 MRSA-colonized patients developed invasive infections with the colonizing strain. Comparisons with 21,521 isolates in the NCBI Pathogen Detection Resource revealed NICU strains to be distinct from MRSA seen locally and internationally. Integration of international strain datasets in analyses increased the resolution of strain clusters and helped rule-out suspected transmission events. Analyses also identified sequence type 1535 isolates, emergent in the Middle East, carrying a unique SCCmec with fusC and aac(6)-Ie/aph(2)-1a that provided a multi-drug resistant phenotype. NICU genomic surveillance identified cryptic MRSA colonization events, including NICU-endemic strains not linked with local hospital or international clusters, and has rich potential to guide improvements in infection prevention for this vulnerable patient population.