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IntroductionFluctuations in water quality characteristics influence the productivity of blue crabs (Callinectes sapidus), and the risk of human exposure to pathogenic Vibrio species. Thus, this study assessed the prevalence of total and pathogenic/clinical markers of Vibrio parahaemolyticus and Vibrio vulnificus in blue crabs and seawater from the Maryland Coastal Bays (MCBs) and the correlation between Vibrio levels and physicochemical parameters.MethodsThree to five crabs and 1 L of seawater were collected monthly for 3 years (May 2018 to December 2020) from six sites within the MCBs. Hemolymph and crab tissue were extracted and pooled for each site. Extracted hemolymph, crab tissue, and seawater were analyzed for V. parahaemolyticus and V. vulnificus using the Most Probable Number (MPN) and real-time PCR methods. A one-way Analysis of Variance (ANOVA), correlations, and linear models were used to analyze the data. Akaike Information Criterion (AICc) was evaluated to determine the model that provides the best fit to the data relating to Vibrio concentrations and environmental factors.ResultsResults suggested that environmental factors could influence the growth of Vibrio spp. Both V. parahaemolyticus and V. vulnificus were more prevalent during the warmer months than colder months. Vibrio was more prevalent in crab samples compared to seawater. Vibrio vulnificus concentrations in seawater and hemolymph were positively correlated with temperature (p = 0.0143 seawater) and pH (p = 0.006 hemolymph). A negative correlation was observed between the concentration of V. vulnificus in whole crab (tissue) and dissolved oxygen level (p = 0.0256). The concentration of V. parahaemolyticus in seawater was positively correlated with temperature (p = 0.009) and negatively correlated with dissolved oxygen (p = 0.012).DiscussionThese results provide current information on the spatial and temporal distributions of Vibrio spp. in the MCBs that are useful for implementing more efficient processing and handling procedures of seafood products.

Salmonella is a foodborne pathogenic bacterium that causes salmonellosis worldwide. Also, Salmonella is considered a serious problem for food safety and public health. Several antimicrobial classes including aminoglycosides, tetracyclines, phenols, and β-Lactams are used to treat Salmonella infections. Antibiotics have been prescribed for decades to treat infections caused by bacteria in human and animal healthcare. However, intensive use of antibiotics resulted in antibiotic resistance (AR) among several foodborne bacteria including Salmonella. Furthermore, multi-drug resistance (MDR) of Salmonella has increased dramatically. In addition to MDR Salmonella, extensively drug resistant (XDR) as well as pan drug resistant (PDR) Salmonella were reported globally. Therefore, increasing AR is becoming a serious universal public health crisis. Salmonella developed many mechanisms to ensure its survival against antimicrobials. The most prominent defense mechanisms against these antibiotics include enzymatic inactivation, expelling drugs from the cell through efflux pumps, altering the structure of drugs, and changing or protecting the targets of drugs. Additionally, the formation of biofilms and plasmid-mediated AR by Salmonella, enhancing its resistance to various antibiotics, making it a challenging pathogen in both healthcare and food industry settings. This review focuses exclusively on providing a detailed overview of the mechanisms of AR in Salmonella.

ABSTRACTAlternative irrigation waters (rivers, ponds, and reclaimed water) can harbor bacterial foodborne pathogens like Salmonella enterica and Listeria monocytogenes, potentially contaminating fruit and vegetable commodities. Detecting foodborne pathogens using qPCR-based methods may accelerate testing methods and procedures compared to culture-based methods. This study compared detection of S. enterica and L. monocytogenes by qPCR (real-time PCR) and culture methods in irrigation waters to determine the influence of water type (river, pond, and reclaimed water), season (winter, spring, summer, and fall), or volume (0.1, 1, and 10 L) on sensitivity, accuracy, specificity, and positive (PPV), and negative (NPV) predictive values of these methods. Water samples were collected by filtration through modified Moore swabs (MMS) over a 2-year period at 11 sites in the Mid-Atlantic U.S. on a bi-weekly or monthly schedule. For qPCR, bacterial DNA from culture-enriched samples (n = 1,990) was analyzed by multiplex qPCR specific for S. enterica and L. monocytogenes. For culture detection, enriched samples were selectively enriched, isolated, and PCR confirmed. PPVs for qPCR detection of S. enterica and L. monocytogenes were 68% and 67%, respectively. The NPV were 87% (S. enterica) and 85% (L. monocytogenes). Higher levels of qPCR/culture agreement were observed in spring and summer compared to fall and winter for S. enterica; for L. monocytogenes, lower levels of agreement were observed in winter compared to spring, summer, and fall. Reclaimed and pond water supported higher levels of qPCR/culture agreement compared to river water for both S. enterica and L. monocytogenes, indicating that water type may influence the agreement of these results.IMPORTANCEDetecting foodborne pathogens in irrigation water can inform interventions and management strategies to reduce risk of contamination and illness associated with fresh and fresh-cut fruits and vegetables. The use of non-culture methods like qPCR has the potential to accelerate the testing process. Results indicated that pond and reclaimed water showed higher levels of agreement between culture and qPCR methods than river water, perhaps due to specific physiochemical characteristics of the water. These findings also show that season and sample volume affect the agreement of qPCR and culture results. Overall, qPCR methods could be more confidently utilized to determine the absence of Salmonella enterica and Listeria monocytogenes in irrigation water samples examined in this study.

Salmonella enterica Typhimurium DT104 (S. Typhimurium DT104) is an important foodborne pathogen that is associated with poultry and poultry products. Currently, there is very little information on the underlying molecular mechanisms that allow DT104 to survive and propagate in poultry meat and the poultry processing environment. The current study assessed the global gene expression of DT104 in ground chicken extract (GCE) compared to brain heart infusion (BHI) medium using RNA-Seq technology. DT104 was grown to the early stationary phase (ESP), inoculated into GCE or BHI, and then re-grown to the log phase before RNA was extracted and transcripts were quantified by RNA-Seq. Gene expression for DT104 grown in GCE was then compared to that of DT104 grown in BHI for samples grown to the ESP. Growth in GCE resulted in the up-regulated expression of genes related to translation, carnitine metabolism (23–283-fold change), and cobalamin (vitamin B12) biosynthesis (14-fold change). In particular, the presence of carnitine in chicken meat, and thus, in GCE, which lacks carbohydrates, may allow Salmonella to utilize this compound as a carbon and nitrogen source. This study demonstrates that RNA-Seq data can provide a comprehensive analysis of DT104 gene expression in a food model for poultry products. This study also provides additional evidence for the importance of metabolic adaptation in the ability of S. enterica to successfully adapt to and occupy niches outside of its host and provides potential targets that could be used to develop intervention strategies to control Salmonella in poultry.

IntroductionSalmonella infections have been intensely increasing and becoming a universal public health crisis. This study investigated the prevalence of Salmonella in organic and non-organic chickens and the antimicrobial resistance profiles and virulence genes (invA, pagC, and spvC) in recovered Salmonella isolates.MethodsWhole chicken carcasses [organic (n = 240) and non-organic (n = 240)] were obtained monthly for 1 year (n = 480) from a retail store on the Eastern Shore of Maryland. Salmonella isolation and identification were conducted by following the whole carcass enrichment method recommended by USDA-FSIS. Confirmed Salmonella isolates (organic n = 76; non-organic n = 137) were serotyped and tested for antibiotic susceptibility and virulence genes using standard methods.ResultsForty-nine percent (237/480) of the carcasses were positive for Salmonella. Organic and non-organic positivity rates were 37.1 and 61.8%, respectively. A significantly higher Salmonella contamination was observed in non-organic chickens (p < 0.05). The most common serovars were Salmonella Kentucky (47%), S. Infantis (35%), S. Enteritidis (6%), S. Typhimurium (5%), and S. Blockley (4%). Isolates were frequently resistant to at least one antibiotic (91.24%) or multidrug resistant (45.54%). Resistance was observed to tetracycline (82.8%), minocycline (42.3%), nitrofurantoin (40.3%), cefazolin (38.3%), ampicillin (32.1%), and ceftriaxone (26%). All isolates were susceptible to fluoroquinolone, carbapenem, and glycylcycline. The majority of isolates (99.1%) possessed at least one of three virulence genes of concern and 4.2% tested positive for all three. Ninety-five, 89, and 6.6% of isolates contained invA, pagC, and spvC genes, respectively. The spvC gene was not detected in serovars recovered from organic chickens though 92% and 82% of isolates were positive for invA and pagC. The frequency of Salmonella recovered from non-organic chickens possessing invA, pagC, and spvC genes were 97.1, 89.8, and 10.2%, respectively. Detection of invA and pagC genes showed no significant difference (p > 0.05) between organic and non-organic chickens but a significantly higher spvC gene (p < 0.05) was detected in non-organic chickens due to the majority of S. Enteritidis (92.3%) exclusively recovered from non-organic chicken carried spvC gene.DiscussionThis study reveals a high prevalence of Salmonella in both organic and non-organic chickens, which exhibit resistance to vital antibiotics and carry virulence genes, thereby creating a potential risk of salmonellosis.

Investigating the chicken microbiome is important to establish control measures for pathogens to protect consumers. This study aimed at evaluating the comparative efficiency of human pathogen detection through 16S rRNA sequencing of organic and conventional chickens processed using whole carcass enrichment (WCE) and rinse (WCR) methods. Organic and conventional whole broiler carcasses (n = 31) were vigorously shaken with 500 mL buffered peptone water (BPW). For the rinse method, a 30 mL aliquot was mixed with 30 mL of BPW. The rest of the sample, including the carcass, was used for the enrichment method. All samples were incubated at 37°C for 24 h. The samples were divided into five groups [Negative Control: only BPW without chicken (n = 5), Organic-Rinsed (n = 7), -Enriched (n = 8), Conventional-Rinsed (n = 7), and -Enriched (n = 9)]. Fifty milliliters of each sample were subjected to DNA extraction followed by 16S rRNA sequencing. Proteobacteria and Firmicutes predominated the microbiota of both conventional and organic chickens, followed by low abundances of Bacteroidetes and Fusobacterium. While the abundance of Proteobacteria and Firmicutes remained unchanged in organic chicken irrespective of the methods used, a noticeable shift in the Proteobacteria and Firmicutes ratio (59%:39% in rinsed to 38%:60% in enriched) was observed in conventional chicken. Furthermore, the choice of method did not yield any differences in Abundance-Based Coverage Estimator, and Jackknife, among conventional and organic chickens but resulted in a statistically significant difference in the Shannon, Simpson, Chao1, and phylogenetic diversity indices (p

Certified organic production prohibits chemical fertilizer use but permits the application of biological soil amendments of animal origin (BSAAOs) to croplands for fertilizing soil. For fresh produce likely to be consumed uncooked, the United States Department of Agriculture National Organic Program (USDA-NOP) stipulates a 90-day withholding period between BSAAO application and crop harvest for produce not in direct contact with soil to reduce concerns about potential pathogen contamination from BSAAOs. For fresh produce crops in direct contact with soil this withholding period is 120-days. In this study, survival and pathogenesis of three foodborne pathogens and indicator bacteria in BSAAO-amended soils were analyzed with their potential to transfer to produce harvested from three organic, integrated crop-livestock farms (ICLFs) on the Maryland Eastern Shore. In total, 428 manure/compost, soil, untreated produce, and water samples were collected before/after BSAAO incorporation and monthly for 180-days. Samples were assessed for the presence of Listeria monocytogenes (L. monocytogenes), Salmonella, and Shiga toxin-producing E. coli (STEC) with associated virulence factor (VF) genes (STEC/VF-genes), and for aerobic plate count (APC), generic E. coli (gEC), and total coliform levels using standard methods and polymerase chain reaction (PCR). Overall, 18.4% (26/141) of produce samples were positive for at least one tested pathogen, with STEC/VF-genes being detected the most. Produce was contaminated with at least one pathogen and indicators 90- and 120-days post-BSAAO incorporation. Salmonella wasn’t detected in manure/compost samples, and 6.7% (1/15) and 66.7% (10/15) of manure/compost samples were positive for L. monocytogenes and STEC/VF-genes, respectively. In BSAAO-amended soils, 29.1% (74/254) were positive for at least one of the tested pathogens in this study. STEC/VF-genes were detected in 24.0% (61/254) of soils, while L. monocytogenes and Salmonella were detected in 4.3% (11/254) and 0.8% (2/254) of soils, respectively. These three pathogens were detected in soils directly after BSAAO incorporation but were followed by a reduced prevalence. However, pathogens were detected in soils 90- and/or 120-days post-application. The results of this study indicated pathogens associated with BSAAOs (contaminated manures/composts) can persist after soil incorporation and transfer to harvestable produce grown on ICLF’s on the Maryland Eastern Shore. Therefore, further research into withholding periods and other mitigation strategies is necessary to mitigate pathogen contamination risks on ICLFs.

IntroductionFluctuations in environmental physicochemical parameters can affect the diversity and prevalence of microbial communities, including vibrios, associated with aquatic species and their surrounding environments. This study aimed to investigate the population dynamics of two Vibrio species as well as the microbial community diversity of whole crab and seawater from the Maryland Coastal Bays (MCBs), using 16S rRNA sequencing.MethodsDuring this study, three crabs and 1 L of seawater were collected monthly from two sites for 3 months. Crab tissue was extracted and pooled for each site. Extracted crab tissue and seawater were analyzed for Vibrio parahaemolyticus and V. vulnificus using Most Probable Number (MPN) real-time PCR. For 16S rRNA microbiome analysis, three different DNA extraction kits were evaluated to extract microbial DNA from individual crabs. Also, 500 mL of each seawater sample was filtered for DNA extraction.ResultsResults indicated that sample types and sampling periods had a significant effect on the alpha diversity of the microbial community of crabs and seawater (p

Blue crab (Callinectes sapidus) is a highly valuable wild fishery species of crab native to the waters of the western Atlantic Ocean and the Gulf of Mexico. The annual commercial production of live blue crabs is approximately 50,000 metric tons with a dockside value of USD 200 million. Presently the US blue crab processing industry sells crab meat in three basic forms: fresh crab meat, pasteurized crab meat, and frozen crab meat. By far “Fresh” is the most desirable form of crab meat. However, fresh crab meat has a limited shelf life. This study evaluated the effects of high-pressure processing (HPP) on enhancing the microbiological quality and shelf life of blue crab meat. Live blue crabs were pressure-cooked in a retort (≥115 °C for 4–6 min). The crab meat was handpicked, packed in plastic containers with seals, subjected to HPP treatment, and stored at 4 °C. Container integrity and water leakage issues were examined by observation in addition to weight comparison before and after HPP treatment; the shelf life of crab meat with and without HPP treatments was examined via microbiological tests and sensory evaluations. Results show that polypropylene containers sealed with 10K OTR (oxygen transmission rate) film could withstand high pressure without water leakage issues; HPP treatment at 600 MPa for 3 min could extend the shelf life of fresh, cooked, and handpicked crab meat from 6 days to 18 days based on the strictest APC (aerobic plate account) limit (APC ≤ 100,000 CFU/g). The sensory quality of the HPP-treated crab meat was well accepted throughout the 3-week storage period. The results support the use of HPP as an effective non-thermal processing technology to enhance the microbiological quality and extend the shelf life of fresh RTE blue crab meat.

Enteric bacterial pathogen levels can influence the suitability of irrigation water sources for fruits and vegetables. We hypothesize that stable spatial patterns of Salmonella enterica and Listeria monocytogenes levels may exist across surface water sources in the Mid-Atlantic U.S. Water samples were collected at four streams and two pond sites in the mid-Atlantic U.S. over 2 years, biweekly during the fruit and vegetable growing seasons, and once a month during nongrowing seasons. Two stream sites and one pond site had significantly different mean concentrations in growing and nongrowing seasons. Stable spatial patterns were determined for relative differences between the site concentrations and average concentration of both pathogens across the study area. Mean relative differences were significantly different from zero at four of the six sites for S. enterica and three of six sites for L. monocytogenes. There was a similarity between the mean relative difference distribution between sites over growing season, nongrowing season, and the entire observation period. Mean relative differences were determined for temperature, oxidation-reduction potential, specific electrical conductance, pH, dissolved oxygen, turbidity, and cumulative rainfall. A moderate-to-strong Spearman correlation (rs > 0.657) was found between spatial patterns of S. enterica and 7-day rainfall, and between relative difference patterns of L. monocytogenes and temperature (rs = 0.885) and dissolved oxygen (rs = -0.885). Persistence in ranking sampling sites by the concentrations of the two pathogens was also observed. Finding spatially stable patterns in pathogen concentrations highlights spatiotemporal dynamics of these microorganisms across the study area can facilitate the design of an effective microbial water quality monitoring program for surface irrigation water.

ABSTRACT The pathogenic marine bacterium Vibrio parahaemolyticus can cause seafood-related gastroenteritis via the consumption of raw or undercooked seafood. Infections originating from relatively cool waters in the northeast United States are typically rare, but recently, this region has shown an increase in infections attributed to the ecological introduction of pathogenic sequence type 36 (ST36) strains, which are endemic to the cool waters of the Pacific Northwest. A 2005 risk assessment performed by the Food and Drug Administration (FDA) modeled the postharvest growth of V. parahaemolyticus in oysters as a function of air temperature and the length of time the oysters remained unrefrigerated. This model, while useful, has raised questions about strain growth differences in oyster tissue and whether invasive pathogenic strains exhibit different growth rates than nonclinical strains, particularly at lower temperatures. To investigate this question, live eastern oysters were injected with ST36 clinical strains and non-ST36 nonclinical strains, and growth rates were measured using the most probable number (MPN) enumeration. The presence of V. parahaemolyticus was confirmed using PCR by targeting the thermolabile hemolysin gene (tlh), thermostable direct hemolysin (tdh), tdh-related hemolysin (trh), and a pathogenesis-related protein (prp). The growth rates of the ST36 strains were compared to the FDA model and several other data sets of V. parahaemolyticus growth in naturally inoculated oysters harvested from the Chesapeake Bay. Our data indicate that the growth rates from most studies fall within the mean of the FDA model, but with slightly higher growth at lower temperatures for ST36 strains injected into live oysters. These data suggest that further investigations of ST36 growth capability in oysters at temperatures previously thought unsuitably low for Vibrio growth are warranted. IMPORTANCE Vibrio parahaemolyticus is the leading cause of seafood-related gastroenteritis in the United States, with an estimated 45,000 cases per year. Most individuals who suffer from vibriosis consume raw or undercooked seafood, including oysters. While gastroenteritis vibriosis is usually self-limiting and treatable, V. parahaemolyticus infections are a stressor on the growing aquaculture industry. Much effort has been placed on modeling the growth of Vibrio cells in oysters in order to aid oyster growers in designing harvesting best practices and ultimately, to protect the consumer. However, ecological invasions of nonnative bacterial strains make modeling their growth complicated, as these strains are not accounted for in current models. The National Shellfish Sanitation Program (NSSP) considers 10°C (50°F) a temperature too low to enable Vibrio growth, where 15°C is considered a cutoff temperature for optimal Vibrio growth, with temperatures approaching 20°C supporting higher growth rates. However, invasive strains may be native to cooler waters. This research aimed to understand strain growth in live oysters by measuring growth rates when oysters containing ST36 strains, which may be endemic to the U.S. Pacific Northwest, were exposed to multiple temperatures postharvest. Our results will be used to aid future model development and harvesting best practices for the aquaculture industry.

Salmonella is a Gram-negative, rod-shaped, facultative anaerobic, and non-spore-forming bacterium that belongs to the family of Enterobacteriaceae and is the causative agent for typhoid/paratyphoid fever and salmonellosis. Salmonella causes the highest amount of foodborne illness among bacteria at 15.5 cases per 100,000 and causes an estimated 410,000 antibiotic-resistant infections each year in the U.S. The use of antibiotics has been a staple in poultry production for the prevention of diseases and growth promotion for the last 70 years. Due to the over-and misusage of antibiotics, there has been an emerging public health crisis. Salmonella is developing resistance and may render antibiotics inoperative in a foodborne outbreak. Poultry, when not handled properly, is a major carrier and transmitter of Salmonella, causing human illness and fatality. This review summarizes the major Salmonella outbreaks over the past three decades, the prevalence of Antimicrobial Resistant (AMR) Salmonella related to poultry, and the control measures being implemented to reduce and prevent AMR Salmonella in poultry.

The quantity of seafood imported and produced by domestic aquaculture farming has increased. Recently, it has been reported that multidrug-resistant (MDR) Salmonella Typhimurium may be associated with seafood. However, information is limited to the antimicrobial resistance, virulence properties, and genetic diversity of S. Typhimurium recovered from imported and domestic seafood. This study investigated the antimicrobial resistance, virulence properties, and genetic diversity of S. Typhimurium isolated from domestic and imported catfish, shrimp, and tilapia. A total of 127 isolates were tested for the presence of multidrug-resistance (MDR), virulence genes (invA, pagC, spvC, spvR), and genetic diversity using the Sensititre micro-broth dilution method, PCR, and pulsed-field gel electrophoresis (PFGE), respectively. All isolates were uniformly susceptible to six (amoxicillin/clavulanic acid, ceftiofur, ceftriaxone, imipenem, nitrofurantoin, and trimethoprim/sulfamethoxazole) of the 17 tested antimicrobials and genetically diverse. Fifty-three percent of the Salmonella isolates were resistant to at least one antimicrobial and 49% were multidrug resistant. Ninety-five percent of the isolates possessed the invA gene, 67% pagC, and 43% for both spvC, and spvR. The results suggest that S. Typhimurium recovered from seafood is frequently MDR, virulent, and have the ability to cause salmonellosis.

Outbreaks of human gastroenteritis have been linked to the consumption of contaminated domestic and imported seafood. This study investigated the microbiological quality of seafood obtained from retail stores on the Eastern Shore of Maryland. A total of 440 samples of domestic and imported frozen shrimp, catfish and tilapia samples were analyzed for aerobic plate count (APC), total coliforms, Escherichia coli and seafood-borne-pathogens (Vibrio parahaemolyticus, Vibrio vulnificus, Salmonella, Campylobacter jejuni). The prevalence of APC, coliforms and E. coli positive samples was 100%, 43% and 9.3%, respectively. Approximately 3.2%, 1.4%, 28.9% and 3.6% of the samples were positive for V. parahaemolyticus, V. vulnificus, Salmonella and Campylobacter jejuni, respectively. The MPN/g ranges were 150–1100 MPN/g for vibrios, 10–1100 MPN/g for Salmonella and 93–460 MPN/g for C. jejuni in seafood, respectively. Comparing bacterial prevalence by type or source of seafood, the only significant difference identified was Salmonella-positive imported tilapia (33.3%) versus domestic tilapia (19.4%). The quantitative data on pathogen levels in the present study provide additional information for quantitative risk assessment not available in previous surveys. The findings of this study suggest the association of potential food safety hazards with domestic and imported seafood and warrant further large-scale studies and risk assessment.

ABSTRACT Irrigation water sources have been shown to harbor foodborne pathogens and could contribute to the outbreak of foodborne illness related to consumption of contaminated produce. Determining the probability of and the degree to which these irrigation water sources contain these pathogens is paramount. The purpose of this study was to determine the prevalence of Salmonella enterica and Listeria monocytogenes in alternative irrigation water sources. Water samples (n = 188) were collected over 2 years (2016 to 2018) from 2 reclaimed water plants, 3 nontidal freshwater rivers, and 1 tidal brackish river on Maryland’s Eastern Shore (ESM). Samples were collected by filtration using modified Moore swabs (MMS) and analyzed by culture methods. Pathogen levels were quantified using a modified most probable number (MPN) procedure with three different volumes (10 liters, 1 liter, and 0.1 liter). Overall, 65% (122/188) and 40% (76/188) of water samples were positive for S. enterica and L. monocytogenes, respectively. For both pathogens, MPN values ranged from 0.015 to 11 MPN/liter. Pathogen levels (MPN/liter) were significantly (P

High spatiotemporal variability of pathogen concentrations in surface waters complicates the design and interpretation of microbial water quality monitoring. Empirical orthogonal function (EOF) analysis can provide spatial patterns (EOFs) of variability in deviations of concentrations in specific locations from the average concentration across the study area. These patterns can be interpreted to assess the effect of environmental factors on pathogen levels in the water. The first and the second EOFs for Listeria monocytogenes explained 84.4% and 9.7% of the total variance of deviations from average, respectively. That percentage was 50.8% and 45.0% for Salmonella enterica. The precipitation also had a strong explanatory capability (79%) of the first EOF. The first EOFs of Listeria and precipitation were similar at pond sites but were opposite to the precipitation at the stream sites. The first EOF of S. enterica and precipitation demonstrated opposite trends, whereas the second S. enterica EOF pattern had similar signs with the precipitation EOF at pond sites, indicating a relationship between rainfall and Salmonella at these sites. Overall, the rainfall data could inform on persistent spatial patterns in concentrations of the two pathogens at the pond sites in farm settings but not at stream sites located in forested areas.

Vibrio vulnificus and V. parahaemolyticus, found naturally in marine and estuarine environments, are the leading cause of seafood associated gastrointestinal illness and death. Consumption of improperly cooked crabs and handling of live crabs are potential routes of exposure to pathogenic bacteria such as V. vulnificus and V. parahaemolyticus. Little information is available on serotype genetic and antimicrobial profiles of V. vulnificus and V. parahaemolyticus recovered from Maryland estuaries. The aim of the present study was to determine the serotype of V. parahaemolyticus, evaluate antimicrobial susceptibility and genetic profiles of V. vulnificus and V. parahaemolyticus isolated from water and blue crab (Callinectes sapidus) samples collected from the Maryland Coastal Bays. One hundred and fifty (150) PCR confirmed V. parahaemolyticus including 52 tdh+ (pathogenic) and 129 V. vulnificus strains were tested for susceptibility to twenty (20) different antibiotics chosen by clinical usage for Vibrio species. The O serogroups were determined using an agglutination test with V. parahaemolyticus antisera. Pulsed-field gel electrophoresis (PFGE) was used for molecular subtyping to investigate the genetic diversity among tested strains. The most prevalent serotypes were O5 (33.3%), O3 (18.7%) and O1 (14.7%). More than 41% of all tested Vibrio isolates were resistant to three or more antibiotics. Cephalothin showed the highest resistance (42% and 61%), followed by cefoxitin (42% and 31%) and ceftazidime (36% and 29%) for V. vulnificus and V. parahaemolyticus, respectively. Most strains (99–100%) were susceptible to ampicillin/sulbactam, levofloxacin, piperacillin, piperacillin/tazobactam, and tetracycline. Fifty percent (50%) of the cephalothin resistant strains were crab isolates. Vibrio vulnificus and V. parahaemolyticus isolates demonstrated a high genetic diversity and 31% of V. vulnificus and 16% of V. parahaemolyticus strains were PFGE untypeable. No correlations were found between the V. parahaemolyticus serotype, pathogenicity, genetic and antimicrobial resistance profiles of both species of Vibrio. The observed high multiple drug resistance of V. vulnificus and V. parahaemolyticus from blue crab and its environment is of public health concern. Therefore, there is a need for frequent antibiotic sensitivity surveillance for Vibrio spp.

To address the need for simple, safe, sensitive, and scalable SARS-CoV-2 tests, we validated and implemented a PCR test that uses a saliva collection kit use at home. Individuals self-collected 300 μl saliva in vials containing Darnell Rockefeller University Laboratory (DRUL) buffer and extracted RNA was assayed by RT-PCR (the DRUL saliva assay). The limit of detection was confirmed to be 1 viral copy/μl in 20 of 20 replicate extractions. Viral RNA was stable in DRUL buffer at room temperature up to seven days after sample collection, and safety studies demonstrated that DRUL buffer immediately inactivated virus at concentrations up to 2.75x106 PFU/ml. Results from SARS-CoV-2 positive nasopharyngeal (NP) swab samples collected in viral transport media and assayed with a standard FDA Emergency Use Authorization (EUA) test were highly correlated with samples placed in DRUL buffer. Direct comparison of results from 162 individuals tested by FDA EUA oropharyngeal (OP) or NP swabs with co-collected saliva samples identified four otherwise unidentified positive cases in DRUL buffer. Over six months, we collected 3,724 samples from individuals ranging from 3 months to 92 years of age. This included collecting weekly samples over 10 weeks from teachers, children, and parents from a pre-school program, which allowed its safe reopening while at-risk pods were quarantined. In sum, we validated a simple, sensitive, stable, and safe PCR-based test using a self-collected saliva sample as a valuable tool for clinical diagnosis and screening at workplaces and schools.

This work explored the effects of salinity and temperature on the efficacy of purging V. parahaemolyticus from eastern oysters (Crassostrea virginica). Oysters were inoculated with a 5-strain cocktail of V. parahaemolyticus to levels of 104 to 105 MPN (most probable number)/g and depurated in a controlled re-circulating wet-storage system with artificial seawater (ASW). Both salinity and temperature remarkably affected the efficacy for the depuration of V. parahaemolyticus from oysters during wet-storage. The wet-storage process at salinity 20 ppt at 7.5 °C or 10 °C could achieve a larger than 3 log (MPN/g) reduction of Vibrio at Day 7, which meets the FDA’s requirement as a post-harvest process for V. parahaemolyticus control. At the conditions of 10 °C and 20 ppt, a pre-chilled system could achieve a 3.54 log (MPN/g) reduction of Vibrio in oysters on Day 7. There was no significant difference in the shelf life between inoculated and untreated oysters before the depuration, with a same survival rate (stored in a 4 °C cooler for 15 days) of 93%.

Irrigation water contaminated with Salmonella enterica and Listeria monocytogenes may provide a route of contamination of raw or minimally processed fruits and vegetables. While previous work has surveyed specific and singular types of agricultural irrigation water for bacterial pathogens, few studies have simultaneously surveyed different water sources repeatedly over an extended period of time. This study quantified S. enterica and L. monocytogenes levels (MPN/L) at 6 sites, including river waters: tidal freshwater river (MA04, n = 34), non-tidal freshwater river, (MA05, n = 32), one reclaimed water holding pond (MA06, n = 25), two pond water sites (MA10, n = 35; MA11, n = 34), and one produce wash water site (MA12, n = 10) from September 2016-October 2018. Overall, 50% (84/168) and 31% (53/170) of sampling events recovered S. enterica and L. monocytogenes, respectively. Results showed that river waters supported significantly (p < 0.05) greater levels of S. enterica than pond or reclaimed waters. The non-tidal river water sites (MA05) with the lowest water temperature supported significantly greater level of L. monocytogenes compared to all other sites; L. monocytogenes levels were also lower in winter and spring compared to summer seasons. Filtering 10 L of water through a modified Moore swab (MMS) was 43.5 (Odds ratio, p < 0.001) and 25.5 (p < 0.001) times more likely to recover S. enterica than filtering 1 L and 0.1 L, respectively; filtering 10 L was 4.8 (p < 0.05) and 3.9 (p < 0.05) times more likely to recover L. monocytogenes than 1L and 0.1 L, respectively. Work presented here shows that S. enterica and L. monocytogenes levels are higher in river waters compared to pond or reclaimed waters in the Mid-Atlantic region of the U.S., and quantitatively shows that analyzing 10 L water is more likely recover pathogens than smaller samples of environmental waters.

This study identified Vibrio parahaemolyticus in oyster and seawater samples collected from Delaware Bay from June through October of 2016. Environmental parameters including water temperature, salinity, dissolved oxygen, pH, and chlorophyll a were measured per sampling event. Oysters homogenate and seawater samples were 10-fold serially diluted and directly plated on CHROMagarᵀᴹ Vibrio medium. Presumptive V. parahaemolyticus colonies were counted and at least 20% of these colonies were selected for molecular chracterization. V. parahaemolyticus isolates (n = 165) were screened for the presence of the species-specific thermolabile hemolysin (tlh) gene, the pathogenic thermostable direct hemolysin (tdh)/ thermostable related hemolysin (trh) genes, the regulatory transmembrane DNA-binding gene (toxR), and V. parahaemolyticus metalloprotease (vpm) gene using a conventional PCR. The highest mean levels of the presumptive V. parahaemolyticus were 9.63×103 CFU/g and 1.85×103 CFU/mL in the oyster and seawater samples, respectively, during the month of July. V. parahaemolyticus levels in oyster and seawater samples were significantly positively correlated with water temperature. Of the 165 isolates, 137 (83%), 110 (66.7%), and 108 (65%) were tlh+, vpm+, and toxR+, respectively. Among the V. parahaemolyticus (tlh+) isolates, 7 (5.1%) and 15 (10.9%) were tdh+ and trh+, respectively, and 24 (17.5%), only oyster isolates, were positive for both genes. Potential pathogenic strains that possessed tdh and/or trh were notably higher in oyster (39%) than seawater (15.6%) isolates. The occurrence of total V. parahaemolyticus (tlh+) was not necessarily proportional to the potential pathogenic V. parahaemolyticus. Co-occurrence of the five genetic markers were observed only among oyster isolates. The co-occurrence of the gene markers showed a relatedness potential of tdh occurrence with vpm. We believe exploring the role of V. parahaemolyticus metalloprotease and whether it is involved in the toxic activity of the thermostable direct hemolysin (TDH) protein can be of significance. The outcomes of this study will provide some foundation for future studies regarding pathogenic Vibrio dynamics in relation to environmental quality.

As a traditional agricultural system, integrated crop-livestock farms (ICLFs) involve the production of animals and crops in a shared environment. The ICLFs in the mid-Atlantic region of the United States practice sustainable manure aging or composting processes to provide an on-farm source of soil amendment for use as natural fertilizer and soil conditioner for crop production. However, crop fertilization by soil incorporation of aged manure or compost may introduce different microbes and alter the soil microbial community. The aim of this study was to characterize the influence of aged or composted manure application on the diversity of soil bacterial community in ICLFs. Soil samples from six ICLFs in Maryland were collected before (pre-crop) and during the season (2020–2021) and used to analyze soil bacterial microbiome by 16S rDNA sequencing. Results showed that both phylum- and genus-level alterations of soil bacterial communities were associated with amendment of aged or composted manure. Particularly, Proteobacteria and Actinobacteria were enriched, while Acidobacteria, Bacteroidetes, Planctomycetes, Firmicutes, and Chloroflexi were reduced after manure product application. Meanwhile, the relative abundance of Bacillus was decreased, while two zoonotic pathogens, Salmonella and Listeria, were enriched by manure amendments. Overall, animal manure amendment of soil increased the phylogenetic diversity, but reduced the richness and evenness of the soil bacterial communities. Although manure composting management in ICLFs benefits agricultural sustainable production, the amendments altered the soil bacterial communities and were associated with the finding of two major zoonotic bacterial pathogens, which raises the possibility of their potential transfer to fresh horticultural produce crops that may be produced on the manured soils and then subsequently consumed without cooking.

Abstract The aim of this study was to determine the serogroups, antimicrobial resistance and genetic diversity of Escherichia coli isolates from samples of bivalve mollusks collected along Santa Catarina coast, Brazil, and from the Chesapeake Bay, Maryland, USA. One hundred forty‐one E. coli isolates were characterized for serogroups with 181 specific O antisera and antimicrobial susceptibility using the disk diffusion method. The genetic diversity was assessed using pulsed‐field gel electrophoresis (PFGE). The results showed that among the isolates, 19.9% were classified as multi‐drug resistant (MDR) and resistance was most frequently observed to cephalothin, nitrofurantoin, and ampicillin. The predominant serogroups were O6, O8, and O38. Some serogroups were recognized as pathogenic E. coli. PFGE dendrograms indicated extensive genetic diversity among the isolates. Although characteristics of the E. coli isolates were highly variable, it is important to note that E. coli belonging to pathogenic serogroups and MDR isolates are present in mollusks of both study areas. This is the first report on the phenotypic and genotypic characterization of E. coli from mollusks from Santa Catarina and the Chesapeake Bay that should encourage studies focusing on comparison of isolates across countries.

Growth models are predominately used in the food industry to estimate the potential growth of selected microorganisms under environmental conditions. The growth kinetics, cellular morphology, and antibiotic resistance were studied throughout the life cycle of Salmonella Typhimurium. The effect of the previous life cycle phase [late log phase (LLP), early stationary phase (ESP), late stationary phase (LSP), and early death phase (EDP)] of Salmonella after reinoculation in brain heart infusion broth (BHI), ground chicken extract (GCE), and BHI at pH 5, 7, and 9 and salt concentrations 2, 3, and 4% was investigated. The growth media and previous life cycle phase had significant effects on the lag time (λ), specific growth rate (μmax), and maximum population density (Ymax). At 2 and 4% salt concentration, the LLP had the significantly (p

Dynamic post-transcriptional control of RNA expression by RNA-binding proteins (RBPs) is critical during immune response. ZFP36 RBPs are prominent inflammatory regulators linked to autoimmunity and cancer, but functions in adaptive immunity are less clear. We used HITS-CLIP to define ZFP36 targets in mouse T cells, revealing unanticipated actions in regulating T-cell activation, proliferation, and effector functions. Transcriptome and ribosome profiling showed that ZFP36 represses mRNA target abundance and translation, notably through novel AU-rich sites in coding sequence. Functional studies revealed that ZFP36 regulates early T-cell activation kinetics cell autonomously, by attenuating activation marker expression, limiting T cell expansion, and promoting apoptosis. Strikingly, loss of ZFP36 in vivo accelerated T cell responses to acute viral infection and enhanced anti-viral immunity. These findings uncover a critical role for ZFP36 RBPs in restraining T cell expansion and effector functions, and suggest ZFP36 inhibition as a strategy to enhance immune-based therapies.

Salmonella Typhimurium is the leading cause of human non-typhoidal gastroenteritis in the US. S. Kentucky is one the most commonly recovered serovars from commercially processed poultry carcasses. This study compared the genotypic and phenotypic properties of two Salmonella enterica strains Typhimurium (ST221_31B) and Kentucky (SK222_32B) recovered from commercially processed chicken carcasses using whole genome sequencing, phenotype characterizations and an intracellular killing assay. Illumina MiSeq platform was used for sequencing of two Salmonella genomes. Phylogenetic analysis employing homologous alignment of a 1,185 non-duplicated protein-coding gene in the Salmonella core genome demonstrated fully resolved bifurcating patterns with varying levels of diversity that separated ST221_31B and SK222_32B genomes into distinct monophyletic serovar clades. Single nucleotide polymorphism (SNP) analysis identified 2,432 (ST19) SNPs within 13 Typhimurium genomes including ST221_31B representing Sequence Type ST19 and 650 (ST152) SNPs were detected within 13 Kentucky genomes including SK222_32B representing Sequence Type ST152. In addition to serovar-specific conserved coding sequences, the genomes of ST221_31B and SK222_32B harbor several genomic regions with significant genetic differences. These included phage and phage-like elements, carbon utilization or transport operons, fimbriae operons, putative membrane associated protein-encoding genes, antibiotic resistance genes, siderophore operons, and numerous hypothetical protein-encoding genes. Phenotype microarray results demonstrated that ST221_31B is capable of utilizing certain carbon compounds more efficiently as compared to SK222_3B; namely, 1,2-propanediol, M-inositol, L-threonine, α-D-lactose, D-tagatose, adonitol, formic acid, acetoacetic acid, and L-tartaric acid. ST221_31B survived for 48 h in macrophages, while SK222_32B was mostly eliminated. Further, a 3-fold growth of ST221_31B was observed at 24 hours post-infection in chicken granulosa cells while SK222_32B was unable to replicate in these cells. These results suggest that Salmonella Typhimurium can survive host defenses better and could be more invasive than Salmonella Kentucky and provide some insights into the genomic determinants responsible for these differences.

FK506 (Tacrolimus) is a potent inhibitor of calcineurin that blocks IL2 production and is widely used to prevent transplant rejection and treat autoimmunity. FK506 treatment of dendritic cells (FKDC) limits their capacity to stimulate T cell responses. FK506 does not prevent DC survival, maturation, or costimulatory molecule expression, suggesting that the limited capacity of FKDC to stimulate T cells may be due to inhibition of calcineurin signaling in the DC. Instead, we demonstrate that DC inhibit T cells by sequestering FK506 and continuously releasing the drug over several days. T cells encountering FKDC proliferate but fail to upregulate the survival factor bcl-xl and die, and IL2 restores both bcl-xl and survival. In mice, FKDC act in an antigen-specific manner to inhibit T-cell mediated autoimmune arthritis. This establishes that DCs can act as a cellular drug delivery system to target antigen specific T cells.

Studies of patients with paraneoplastic neurologic disorders (PND) have revealed that apoptotic tumor serves as a potential potent trigger for the initiation of naturally occurring tumor immunity. The purpose of this study was to assess the feasibility, safety, and immunogenicity of an apoptotic tumor-autologous dendritic cell (DC) vaccine.We have modeled PND tumor immunity in a clinical trial in which apoptotic allogeneic prostate tumor cells were used to generate an apoptotic tumor-autologous dendritic cell vaccine. Twenty-four prostate cancer patients were immunized in a Phase I, randomized, single-blind, placebo-controlled study to assess the safety and immunogenicity of this vaccine. Vaccinations were safe and well tolerated. Importantly, we also found that the vaccine was immunogenic, inducing delayed type hypersensitivity (DTH) responses and CD4+ and CD8+ T cell proliferation, with no effect on FoxP3+ regulatory T cells. A statistically significant increase in T cell proliferation responses to prostate tumor cells in vitro (p = 0.002), decrease in prostate specific antigen (PSA) slope (p = 0.016), and a two-fold increase in PSA doubling time (p = 0.003) were identified when we compared data before and after vaccination.An apoptotic cancer cell vaccine modeled on naturally occurring tumor immune responses in PND patients provides a safe and immunogenic tumor vaccine.ClinicalTrials.gov NCT00289341.