RNA Identification of PRIME Cells Predicting Rheumatoid Arthritis Flares.

BACKGROUND: Rheumatoid arthritis, like many inflammatory diseases, is characterized by episodes of quiescence and exacerbation (flares). The molecular events leading to flares are unknown. METHODS: We established a clinical and technical protocol for repeated home collection of blood in patients with rheumatoid arthritis to allow for longitudinal RNA sequencing (RNA-seq). Specimens were obtained from 364 time points during eight flares over a period of 4 years in our index patient, as well as from 235 time points during flares in three additional patients. We identified transcripts that were differentially expressed before flares and compared these with data from synovial single-cell RNA-seq. Flow cytometry and sorted-blood-cell RNA-seq in additional patients were used to validate the findings. RESULTS: Consistent changes were observed in blood transcriptional profiles 1 to 2 weeks before a rheumatoid arthritis flare. B-cell activation was followed by expansion of circulating CD45-CD31-PDPN+ preinflammatory mesenchymal, or PRIME, cells in the blood from patients with rheumatoid arthritis; these cells shared features of inflammatory synovial fibroblasts. Levels of circulating PRIME cells decreased during flares in all 4 patients, and flow cytometry and sorted-cell RNA-seq confirmed the presence of PRIME cells in 19 additional patients with rheumatoid arthritis. CONCLUSIONS: Longitudinal genomic analysis of rheumatoid arthritis flares revealed PRIME cells in the blood during the period before a flare and suggested a model in which these cells become activated by B cells in the weeks before a flare and subsequently migrate out of the blood into the synovium. (Funded by the National Institutes of Health and others.).

Investigating the Relationship between Nitrate, Total Dissolved Nitrogen, and Phosphate with Abundance of Pathogenic Vibrios and Harmful Algal Blooms in Rehoboth Bay, Delaware.

Vibrio spp. and phytoplankton are naturally abundant in marine environments. Recent studies have suggested that the co-occurrence of phytoplankton and the pathogenic bacterium Vibrio parahaemolyticus is due to shared ecological factors, such as nutrient requirements. We compared these communities at two locations in the Delaware Inland Bays, representing a site with high anthropogenic inputs (Torquay Canal) and a less developed area (Sloan Cove). In 2017 to 2018, using light microscopy, we were able to identify the presence of many bloom-forming algal species, such as Karlodinium veneficum, Dinophysis acuminata, Heterosigma akashiwo, and Chattonella subsalsa. Dinoflagellate biomass was higher at Torquay Canal than that at Sloan Cove. D. acuminata and Chloromorum toxicum were found only at Torquay Canal and were not observed in Sloan Cove. Most probable number real-time PCR revealed V. parahaemolyticus and Vibrio vulnificus in environmental samples. The abundance of vibrios and their virulence genes varied between sites, with a significant association between total dissolved nitrogen (TDN), PO4-, total dissolved phosphorus (TDP), and pathogenic markers. A generalized linear model revealed that principal component 1 of environmental factors (temperature, dissolved oxygen, salinity, TDN, PO4-, TDP, NO3:NO2, NO2-, and NH4+) was the best at detecting total (tlh+) V. parahaemolyticus, suggesting that they are the prime drivers for the growth and distribution of pathogenic Vibrio spp. IMPORTANCE Vibrio-associated illnesses have been expanding globally over the past several decades (A. Newton, M. Kendall, D. J. Vugia, O. L. Henao, and B. E. Mahon, Clin Infect Dis 54:S391-S395, 2012, https://doi.org/10.1093/cid/cis243). Many studies have linked this expansion with an increase in global temperature (J. Martinez-Urtaza, B. C. John, J. Trinanes, and A. DePaola, Food Res Int 43:10, 2010, https://doi.org/10.1016/j.foodres.2010.04.001; L. Vezzulli, R. R. Colwell, and C. Pruzzo, Microb Ecol 65:817-825, 2013, https://doi.org/10.1007/s00248-012-0163-2; R. N. Paranjpye, W. B. Nilsson, M. Liermann, and E. D. Hilborn, FEMS Microbiol Ecol 91:fiv121, 2015, https://doi.org/10.1093/femsec/fiv121). Temperature and salinity are the two major factors affecting the distribution of Vibrio spp. (D. Ceccarelli and R. R. Colwell, Front Microbiol 5:256, 2014, https://doi.org/10.3389/fmicb.2014.00256). However, Vibrio sp. abundance can also be affected by nutrient load and marine plankton blooms (V. J. McKenzie and A. R. Townsend, EcoHealth 4:384-396, 2007; L. Vezzulli, C. Pruzzo, A. Huq, and R. R. Colwell, Environ Microbiol Rep 2:27-33, 2010, https://doi.org/10.1111/j.1758-2229.2009.00128.x; S. Liu, Z. Jiang, Y. Deng, Y. Wu, J. Zhang, et al. Microbiologyopen 7:e00600, 2018, https://doi.org/10.1002/mbo3.600). The expansion of Vibrio spp. in marine environments calls for a deeper understanding of the biotic and abiotic factors that play a role in their abundance. We observed that pathogenic Vibrio spp. were most abundant in areas that favor the proliferation of harmful algal bloom (HAB) species. These results can inform managers, researchers, and oyster growers on factors that can influence the growth and distribution of pathogenic Vibrio spp. in the Delaware Inland Bays.

Variations in Bacterial Communities and Antibiotic Resistance Genes Across Diverse Recycled and Surface Water Irrigation Sources in the Mid-Atlantic and Southwest United States: A CONSERVE Two-Year Field Study.

Reduced availability of agricultural water has spurred increased interest in using recycled irrigation water for U.S. food crop production. However, there are significant knowledge gaps concerning the microbiological quality of these water sources. To address these gaps, we used 16S rRNA gene and metagenomic sequencing to characterize taxonomic and functional variations (e.g., antimicrobial resistance) in bacterial communities across diverse recycled and surface water irrigation sources. We collected 1 L water samples (n = 410) between 2016 and 2018 from the Mid-Atlantic (12 sites) and Southwest (10 sites) U.S. Samples were filtered, and DNA was extracted. The V3-V4 regions of the 16S rRNA gene were then PCR amplified and sequenced. Metagenomic sequencing was also performed to characterize antibiotic, metal, and biocide resistance genes. Bacterial alpha and beta diversities were significantly different (p < 0.001) across water types and seasons. Pathogenic bacteria, such as Salmonella enterica, Staphylococcus aureus, and Aeromonas hydrophilia were observed across sample types. The most common antibiotic resistance genes identified coded against macrolides/lincosamides/streptogramins, aminoglycosides, rifampin and elfamycins, and their read counts fluctuated across seasons. We also observed multi-metal and multi-biocide resistance across all water types. To our knowledge, this is the most comprehensive longitudinal study to date of U.S. recycled water and surface water used for irrigation. Our findings improve understanding of the potential differences in the risk of exposure to bacterial pathogens and antibiotic resistance genes originating from diverse irrigation water sources across seasons and U.S. regions.

Enteric Viruses and Pepper Mild Mottle Virus Show Significant Correlation in Select Mid-Atlantic Agricultural Waters.

Enteric viruses (EVs) are the largest contributors to foodborne illnesses and outbreaks globally. Their ability to persist in the environment, coupled with the challenges experienced in environmental monitoring, creates a critical aperture through which agricultural crops may become contaminated. This study involved a 17-month investigation of select human EVs and viral indicators in nontraditional irrigation water sources (surface and reclaimed waters) in the Mid-Atlantic region of the United States. Real-time quantitative PCR was used for detection of Aichi virus, hepatitis A virus, and norovirus genotypes I and II (GI and GII, respectively). Pepper mild mottle virus (PMMoV), a common viral indicator of human fecal contamination, was also evaluated, along with atmospheric (air and water temperature, cloud cover, and precipitation 24 h, 7 days, and 14 days prior to sample collection) and physicochemical (dissolved oxygen, pH, salinity, and turbidity) data, to determine whether there were any associations between EVs and measured parameters. EVs were detected more frequently in reclaimed waters (32% [n = 22]) than in surface waters (4% [n = 49]), similar to PMMoV detection frequency in surface (33% [n = 42]) and reclaimed (67% [n = 21]) waters. Our data show a significant correlation between EV and PMMoV (R2 = 0.628, P < 0.05) detection levels in reclaimed water samples but not in surface water samples (R2 = 0.476, P = 0.78). Water salinity significantly affected the detection of both EVs and PMMoV (P < 0.05), as demonstrated by logistic regression analyses. These results provide relevant insights into the extent and degree of association between human (pathogenic) EVs and water quality data in Mid-Atlantic surface and reclaimed waters, as potential sources for agricultural irrigation. IMPORTANCE Microbiological analysis of agricultural waters is fundamental to ensure microbial food safety. The highly variable nature of nontraditional sources of irrigation water makes them particularly difficult to test for the presence of viruses. Multiple characteristics influence viral persistence in a water source, as well as affecting the recovery and detection methods that are employed. Testing for a suite of viruses in water samples is often too costly and labor-intensive, making identification of suitable indicators for viral pathogen contamination necessary. The results from this study address two critical data gaps, namely, EV prevalence in surface and reclaimed waters of the Mid-Atlantic region of the United States and subsequent evaluation of physicochemical and atmospheric parameters used to inform the potential for the use of indicators of viral contamination.

Seasonal and Geographical Differences in Total and Pathogenic Vibrio parahaemolyticus and Vibrio vulnificus Levels in Seawater and Oysters from the Delaware and Chesapeake Bays Determined Using Several Methods.

Oyster and seawater samples were collected from five sites in the Chesapeake Bay, MD, and three sites in the Delaware Bay, DE, from May to October 2016 and 2017. Abundances and detection frequencies for total and pathogenic Vibrio parahaemolyticus and Vibrio vulnificus were compared using the standard most-probable-number-PCR (MPN-PCR) assay and a direct-plating (DP) method on CHROMagar Vibrio for total (tlh+ ) and pathogenic (tdh+ and trh+ ) V. parahaemolyticus genes and total (vvhA) and pathogenic (vcgC) V. vulnificus genes. The colony overlay procedure for peptidases (COPP) assay was evaluated for total Vibrionaceae DP had high false-negative rates (14 to 77%) for most PCR targets and was deemed unsatisfactory. Logistic regression models of the COPP assay showed high concordances with MPN-PCR for tdh+ and trh+V. parahaemolyticus and vvhA+V. vulnificus in oysters (85.7 to 90.9%) and seawater (81.1 to 92.7%) when seawater temperature and salinity were factored into the model, suggesting that the COPP assay could potentially serve as a more rapid method to detect vibrios in oysters and seawater. Differences in total Vibrionaceae and pathogenic Vibrio abundances between state sampling sites over different collection years were contrasted for oysters and seawater by MPN-PCR. Abundances of tdh+ and trh+V. parahaemolyticus were ∼8-fold higher in Delaware oysters than in Maryland oysters, whereas abundances of vcgC+V. vulnificus were nearly identical. For Delaware oysters, 93.5% were both tdh+ and trh+, compared to only 19.2% in Maryland. These results indicate that pathogenic V. parahaemolyticus was more prevalent in the Delaware Bay than in the Chesapeake Bay.IMPORTANCE While V. parahaemolyticus and V. vulnificus cause shellfish-associated morbidity and mortality among shellfish consumers, current regulatory assays for vibrios are complex, time-consuming, labor-intensive, and relatively expensive. In this study, the rapid, simple, and inexpensive COPP assay was identified as a possible alternative to MPN-PCR for shellfish monitoring. This paper shows differences in total Vibrionaceae and pathogenic vibrios found in seawater and oysters from the commercially important Delaware and Chesapeake Bays. Vibrio parahaemolyticus isolates from the Delaware Bay were more likely to contain commonly recognized pathogenicity genes than those from the Chesapeake Bay.

Longitudinal Assessment of the Dynamics of Escherichia coli, Total Coliforms, Enterococcus spp., and Aeromonas spp. in Alternative Irrigation Water Sources: a CONSERVE Study.

As climate change continues to stress freshwater resources, we have a pressing need to identify alternative (nontraditional) sources of microbially safe water for irrigation of fresh produce. This study is part of the center CONSERVE, which aims to facilitate the adoption of adequate agricultural water sources. A 26-month longitudinal study was conducted at 11 sites to assess the prevalence of bacteria indicating water quality, fecal contamination, and crop contamination risk (Escherichia coli, total coliforms [TC], Enterococcus, and Aeromonas). Sites included nontidal freshwater rivers/creeks (NF), a tidal brackish river (TB), irrigation ponds (PW), and reclaimed water sites (RW). Water samples were filtered for bacterial quantification. E. coli, TC, enterococci (∼86%, 98%, and 90% positive, respectively; n = 333), and Aeromonas (∼98% positive; n = 133) were widespread in water samples tested. Highest E. coli counts were in rivers, TC counts in TB, and enterococci in rivers and ponds (P < 0.001 in all cases) compared to other water types. Aeromonas counts were consistent across sites. Seasonal dynamics were detected in NF and PW samples only. E. coli counts were higher in the vegetable crop-growing (May-October) than nongrowing (November-April) season in all water types (P < 0.05). Only one RW and both PW sites met the U.S. Food Safety Modernization Act water standards. However, implementation of recommended mitigation measures of allowing time for microbial die-off between irrigation and harvest would bring all other sites into compliance within 2 days. This study provides comprehensive microbial data on alternative irrigation water and serves as an important resource for food safety planning and policy setting.IMPORTANCE Increasing demands for fresh fruit and vegetables, a variable climate affecting agricultural water availability, and microbial food safety goals are pressing the need to identify new, safe, alternative sources of irrigation water. Our study generated microbial data collected over a 2-year period from potential sources of irrigation (rivers, ponds, and reclaimed water sites). Pond water was found to comply with Food Safety Modernization Act (FSMA) microbial standards for irrigation of fruit and vegetables. Bacterial counts in reclaimed water, a resource that is not universally allowed on fresh produce in the United States, generally met microbial standards or needed minimal mitigation. We detected the most seasonality and the highest microbial loads in river water, which emerged as the water type that would require the most mitigation to be compliant with established FSMA standards. This data set represents one of the most comprehensive, longitudinal analyses of alternative irrigation water sources in the United States.

Incidence of fecal indicator and pathogenic bacteria in reclaimed and return flow waters in Arizona, USA.

The quality of irrigation water used to cultivate produce that is consumed raw is an important issue with regard to food safety. In this study, the microbiological quality of potential irrigation water sources in Arizona was evaluated by testing for the presence of indicator and pathogenic bacteria. Reclaimed water samples were collected from two wastewater treatment plants and return flow samples were collected from two drainage canals and one return flow pond. Standard membrane filtration methods were used for detection of indicator bacteria. Water samples (n = 28) were filtered through cellulose ester membrane filters and bacterial populations were enumerated by placing the filters on selective agar. For detection of pathogens (Salmonella enterica, Listeria monocytogenes and Shiga toxin-producing E. coli (STEC)), water samples were filtered through Modified Moore swabs and enriched in Universal Pre-enrichment Broth, followed by selective enrichment broth for each pathogen. The enriched broth was streaked onto agar media selective for each pathogen. Presumptive colonies were confirmed by PCR/real-time PCR. Among the 14 reclaimed water samples from two sites, the ranges of recovered populations of E. coli, total coliforms, and enterococci were 0-1.3, 0.5-8.3 × 103, and 0-5.5 CFU/100 mL, respectively. No L. monocytogenes, Salmonella or STEC were found. In the 13 return flow water samples from 3 sites, the ranges of recovered populations of E. coli, total coliforms and enterococci were 1.9-5.3 × 102, 6.5 × 102-9.1 × 104, and 2.9-3.7× 103 CFU/100 mL, respectively. All samples were negative for L. monocytogenes. One (7.1%) of the return flow samples was positive for E. coli O145. Nine (64.3%) of the samples were positive for Salmonella. Both real-time PCR and culture-based methods were used for the detection of Salmonella and L. monocytogenes, and the results from the two methods were comparable. The findings of this study provide evidence that irrigation waters in Arizona, including reclaimed water and return flows, could be potential sources of bacterial contamination of produce. Additional work is needed to evaluate whether bacteria present in irrigation water sources transfer to the edible portion of irrigated plants and are capable of persisting through post-harvest activities.

Prevalence of Shiga-toxigenic and atypical enteropathogenic Escherichia coli in untreated surface water and reclaimed water in the Mid-Atlantic U.S.

The microbial quality of irrigation water has increasingly become a concern as a source of contamination for fruits and vegetables. Non-traditional sources of water are being used by more and more growers in smaller, highly diversified farms in the Mid-Atlantic region of the U.S. Shiga-toxigenic E. coli (STEC) have been responsible for several outbreaks of infections associated with the consumption of leafy greens. Our study evaluated the prevalence of the "big seven" STEC serogroups and the associated enterohemorrhagic E. coli (EHEC) virulence factors (VF) genes in conventional and nontraditional irrigation waters in the Mid-Atlantic region of the U.S. Water samples (n = 510) from 170 sampling events were collected from eight untreated surface water sites, two wastewater reclamation facilities, and one vegetable processing plant, over a 12-month period. Ten liters of water were filtered through Modified Moore swabs (MMS); swabs were then enriched into Universal Pre-enrichment Broth (UPB), followed by enrichment into non-O157 STEC R&F broth and isolation on R & F non-O157 STEC chromogenic plating medium. Isolates (n = 2489) from enriched MMS from water samples were screened for frequently reported STEC serogroups that cause foodborne illness: O26, O45, O103, O111, O121, O145, and O157, along with VF genes stx1, stx2, eae, and ehxA. Through this screening process, STEC isolates were found in 2.35% (12/510) of water samples, while 9.0% (46/510) contained an atypical enteropathogenic E. coli (aEPEC) isolate. The eae gene (n = 88 isolates) was the most frequently detected EHEC VF of the isolates screened. The majority of STEC isolates (stx1 or stx2) genes mainly came from either a pond or reclamation pond water site on two specific dates, potentially indicating that these isolates were not spatially or temporally distributed among the sampling sites. STEC isolates at reclaimed water sites may have been introduced after wastewater treatment. None of the isolates containing eae were determined to be Escherichia albertii. Our work showed that STEC prevalence in Mid-Atlantic untreated surface waters over a 12-month period was lower than the prevalence of atypical EPEC.

Pharmaceuticals, herbicides, and disinfectants in agricultural water sources.

Agricultural water withdrawals account for the largest proportion of global freshwater use. Increasing municipal water demands and droughts are straining agricultural water supplies. Therefore, alternative solutions to agricultural water crises are urgently needed, including the use of nontraditional water sources such as advanced treated wastewater or reclaimed water, brackish water, return flows, and effluent from produce processing facilities. However, it is critical to ensure that such usage does not compromise soil, crop, and public health. Here, we characterized five different nontraditional water types (n = 357 samples) for the presence of pharmaceuticals, herbicides, and disinfectants using ultra-high-pressure liquid chromatography tandem mass spectrometry based method (UPLC-MS/MS). We then evaluated whether the levels of these contaminants were influenced by season. The highest level of herbicides (atrazine) was detected in untreated pond water (median concentration 135.9 ng/L). Reclaimed water had the highest levels of antibiotics and stimulants including azithromycin (215 ng/L), sulfamethoxazole (232.1 ng/L), and caffeine (89.4 ng/L). Produce processing plant water also tended to have high levels of atrazine (102.7 ng/L) and ciprofloxacin (80.1 ng/L). In addition, we observed seasonal variability across water types, with the highest atrazine concentrations observed during summer months, while the highest median azithromycin concentrations were observed in reclaimed water during the winter season. Further studies are needed to evaluate if economically feasible on-farm water treatment technologies can effectively remove such contaminants from nontraditional irrigation water sources.

Development and application of a real-time polymerase chain reaction method for quantification of Escherichia coli in oysters (Crassostrea gigas).

Oysters are important mariculture species worldwide. Because of their filter-feeding behaviors, oysters can accumulate microorganisms, including pathogens, from surrounding water and concentrate bacteria in high numbers. Rapid and suitable methods for quantification of Escherichia coli in oysters are necessary considering that oysters are perishable foods often consumed raw and some countries use E. coli as the regulatory limit. The objective of this study was to develop a qPCR method for quantification of E. coli in oysters. Additionally, different methods were evaluated for DNA extraction from oyster samples and the more reliable method was chosen. Primers and probe were designed targeting uidA gene of E. coli and shown to specifically amplify DNA from E. coli. Standard curves with bacterial DNA extracted from oysters samples artificially inoculated with E. coli were conducted. A good correlation was noticed when the qPCR method was compared to a culture method in oyster samples. This is the first report of a method exclusively developed for direct quantification of E. coli in oyster, the method showed to be suitable for quantification of E. coli in oysters and could be useful in routine analyses, as it requires less time than the culture method.

Vibrio parahaemolyticus and Vibrio vulnificus Recovered from Oysters during an Oyster Relay Study.

Vibrio parahaemolyticus and Vibrio vulnificus are naturally occurring estuarine bacteria and are the leading causes of seafood-associated infections and mortality in the United States. Though multiple-antibiotic-resistant V. parahaemolyticus and V. vulnificus strains have been reported, resistance patterns in vibrios are not as well documented as those of other foodborne bacterial pathogens. Salinity relaying (SR) is a postharvest processing (PHP) treatment to reduce the abundances of these pathogens in shellfish harvested during the warmer months. The purpose of this study was to evaluate the antimicrobial susceptibility (AMS), pathogenicity, and genetic profiles of V. parahaemolyticus and V. vulnificus recovered from oysters during an oyster relay study. Isolates (V. parahaemolyticus [n = 296] and V. vulnificus [n = 94]) were recovered from oysters before and during the 21-day relaying study to detect virulence genes (tdh and trh) and genes correlated with virulence (vcgC) using multiplex quantitative PCR (qPCR). AMS to 20 different antibiotics was investigated using microbroth dilution, and pulsed-field gel electrophoresis (PFGE) was used to study the genetic profiles of the isolates. Twenty percent of V. vulnificus isolates were vcgC+, while 1 and 2% of V. parahaemolyticus were tdh+ and trh+, respectively. More than 77% of the V. vulnificus isolates and 30% of the V. parahaemolyticus isolates were resistant to at least one antimicrobial. Forty-eight percent of V. vulnificus and 8% of V. parahaemolyticus isolates were resistant to two or more antimicrobials. All isolates demonstrated a high genetic diversity, even among those isolated from the same site and having a similar AMS profile. No significant effects of the relaying process on AMS, virulence genes, or PFGE profiles of V. vulnificus and V. parahaemolyticus were observed.IMPORTANCE Analysis of the antibiotic resistance profiles of V. vulnificus and V. parahaemolyticus isolated from oysters during this study indicated that more than 48% of V. vulnificus isolates were resistant to two or more antimicrobials, including those recommended by the CDC for treating Vibrio infections. Also, the V. parahaemolyticus isolates showed high MICs for some of the Vibrio infection treatment antibiotics. Monitoring of AMS profiles of this bacterium is important to ensure optimal treatment of infections and improve food safety. Our study showed no significant differences in the AMS profiles of V. vulnificus (P = 0.26) and V. parahaemolyticus (P = 0.23) isolated from the oysters collected before versus after relaying. This suggests that the salinity of the relaying sites did not affect the AMS profiles of the Vibrio isolates, although it did reduce the numbers of these bacteria in oysters (S. Parveen et al., J Food Sci 82:484-491, 2017, https://doi.org/10.1111/1750-3841.13584).

Antibiotic resistance of Vibrio parahaemolyticus and Vibrio vulnificus in various countries: A review.

Vibrio parahaemolyticus and Vibrio vulnificus are the leading causes of seafood associated infections and mortality in the United States. The main syndromes caused by these pathogens are gastroenteritis, wound infections, and septicemia. This article reviewed the antibiotic resistance profile of V. parahaemolyticus and V. vulnificus in the United States and other countries including Italy, Brazil, Philippines, Malaysia, Thailand, China, India, Iran, South Africa and Australia. The awareness of antimicrobial resistance of these two pathogens is not as well documented as other foodborne bacterial pathogens. Vibrio spp. are usually susceptible to most antimicrobials of veterinary and human significance. However, many studies reported that V. vulnificus and V. parahaemolyticus showed multiple-antibiotic resistance due to misuse of antibiotics to control infections in aquaculture production. In addition, both environmental and clinical isolates showed similar antibiotic resistance profiles. Most frequently observed antibiotic resistance profiles involved ampicillin, penicillin and tetracycline regardless of the countries. The presence of multiple-antibiotic resistant bacteria in seafood and aquatic environments is a major concern in fish and shellfish farming and human health.

Microbiological quality of fresh produce obtained from retail stores on the Eastern Shore of Maryland, United States of America.

The aim of this study was to investigate the microbiological quality of six types of fresh produce obtained from three retail stores located on the Eastern Shore of Maryland, USA. A total of 414 samples representing basil, cilantro, lettuce, scallion, spinach, and parsley were analyzed for total aerobic bacteria (APC), total coliforms, Escherichia coli, and three pathogenic bacteria (E. coli O157:H7, Listeria monocytogenes, and Salmonella), using standard methods. Presumptive pathogenic isolates were confirmed using BAX Polymerase Chain Reaction. Total aerobic populations varied widely between samples, while 38.41% were positive for total coliforms and only 10.15% for E. coli. Median abundance (log CFU/g) of total coliforms and E. coli were less than the limit of detection and that of APC ranged from 5.78 to 6.61 over the six produce types. There was a statistically significant difference in prevalence of total coliforms among the retail stores, but not for abundance of APC or prevalence of E. coli. E. coli O157:H7 and L. monocytogenes were detected in one spinach sample each, while one parsley and one cilantro sample were positive for Salmonella. There were no statistically significant differences in microbiological quality among produce types. Although the results of this study provided some indices of sanitary and/or spoilage level, no relationship was observed among the total aerobic bacteria, total coliforms, E. coli, and the presence of pathogenic bacteria in the samples tested.

Salmonella and Campylobacter: Antimicrobial resistance and bacteriophage control in poultry.

Salmonella and Campylobacter are major causes of foodborne related illness and are traditionally associated with consuming undercooked poultry and/or consuming products that have been cross contaminated with raw poultry. Many of the isolated Salmonella and Campylobacter that can cause disease have displayed antimicrobial resistance phenotypes. Although poultry producers have reduced on-the-farm overuse of antimicrobials, antimicrobial resistant Salmonella and Campylobacter strains still persist. One method of bio-control, that is producing promising results, is the use of lytic bacteriophages. This review will highlight the current emergence and persistence of antimicrobial resistant Salmonella and Campylobacter recovered from poultry as well as bacteriophage research interventions and limitations.

Dendritic cell vaccines containing lymphocytes produce improved immunogenicity in patients with cancer.

BACKGROUND: Dendritic cells are currently under investigation for their ability to generate anti-cancer immune responses. No consensus has been reached as to the optimal method of dendritic cell vaccine preparation and is a barrier to success in the field. METHODS: Over a course of three separate dendritic cell vaccine studies to treat cancer, we tested two different methods for preparing dendritic cells from peripheral blood mononuclear cells: adherence and antibody-selected CD14+ cells. RESULTS: Surprisingly, we found that patients who received dendritic cell vaccines generated by the adherence method mounted increased T cell proliferation in response to vaccination. This difference could not be accounted for by dendritic cell vaccine dose, cell surface phenotype or dendritic cell function in vitro. One notable difference between the two vaccine preparation methods was that the dendritic cell vaccine cultures generated by the adherence method contained up to 10% lymphocytes, and these lymphocytes were proliferating and producing IFNγ in response to antigen in vitro at the time of administration. CONCLUSIONS: Enhanced immunogenicity of adherence dendritic cell vaccinations may be due to the presence of lymphocytes during dendritic cell culture. TRIAL REGISTRATION: Clinicaltrials.gov identifiers: NCT00289341, NCT00345293, and NCT00893945.

Molecular characterization of antibiotic resistant Salmonella Typhimurium and Salmonella Kentucky isolated from pre- and post-chill whole broilers carcasses.

There is conflicting data regarding whether commercial chilling has any effect on persistence of Salmonella serovars, including antibiotic resistant variants, on chicken carcasses. A total of 309 Salmonella Typhimurium and Salmonella Kentucky isolates recovered from pre- and post-chill whole broiler carcasses were characterized for genetic relatedness using Pulsed Field Gel Electrophoresis (PFGE) and for the presence of virulence factors (invA, pagC, spvC) by PCR and for aerobactin and colicin production by bioassays. A subset of these isolates (n = 218) displaying resistance to either sulfisoxazole and/or ceftiofur [S. Typhimurium (n = 66) and S. Kentucky (n = 152)] were further tested for the presence of associated antibiotic resistance elements (class-I integrons and blaCMY genes) by PCR. All 145 ceftiofur resistant S. Kentucky and S. Typhimurium isolates possessed blaCMY genes. Class-I integrons were only detected in 6.1% (n = 4/66) of sulfisoxazole resistant S. Typhimurium isolates. The PFGE analysis revealed the presence of genetically diverse populations within the recovered isolates but clusters were generally concordant with serotypes and antimicrobial resistance profiles. At a 100% pattern similarity index, thirty-six percent of the undistinguishable S. Typhimurium and 22% of the undistinguishable S. Kentucky isolates were recovered from the same chilling step. All isolates possessed the invA and pagC genes, but only 1.4%possessed spvC. Irrespective of the chilling step, there was a significant difference (P < 0.05) in the production of aerobactin and colicin between S. Typhimurium and S. Kentucky isolates. Taken together, these results indicate that chilling impacted the recovery of particular Salmonella clonal groups but had no effect on the presence of class-I integrons, blaCMY genes, and tested virulence factors.

Machine learning to predict the relationship between Vibrio spp. concentrations in seawater and oysters and prevalent environmental conditions.

Vibrio parahaemolyticus and Vibrio vulnificus are bacteria with a significant public health impact. Identifying factors impacting their presence and concentrations in food sources could enable the identification of significant risk factors and prevent incidences of foodborne illness. In recent years, machine learning has shown promise in modeling microbial presence based on prevalent external and internal variables, such as environmental variables and gene presence/absence, respectively, particularly with the generation and availability of large amounts and diverse sources of data. Such analyses can prove useful in predicting microbial behavior in food systems, particularly under the influence of the constant changes in environmental variables. In this study, we tested the efficacy of six machine learning regression models (random forest, support vector machine, elastic net, neural network, k-nearest neighbors, and extreme gradient boosting) in predicting the relationship between environmental variables and total and pathogenic V. parahaemolyticus and V. vulnificus concentrations in seawater and oysters. In general, environmental variables were found to be reliable predictors of total and pathogenic V. parahaemolyticus and V. vulnificus concentrations in seawater, and pathogenic V. parahaemolyticus in oysters (Acceptable Prediction Zone >70 %) when analyzed using our machine learning models. SHapley Additive exPlanations, which was used to identify variables influencing Vibrio concentrations, identified chlorophyll a content, seawater salinity, seawater temperature, and turbidity as influential variables. It is important to note that different strains were differentially impacted by the same environmental variable, indicating the need for further research to study the causes and potential mechanisms of these variations. In conclusion, environmental variables could be important predictors of Vibrio growth and behavior in seafood. Moreover, the models developed in this study could prove invaluable in assessing and managing the risks associated with V. parahaemolyticus and V. vulnificus, particularly in the face of a changing environment.

Prevalence and antimicrobial resistance of major foodborne pathogens isolated from pangas and tilapia fish sold in retail markets of Dhaka city, Bangladesh.

Fish sold at retail markets are often contaminated with harmful bacterial pathogens, posing significant health risks. Despite the growing aquaculture industry in Bangladesh to meet high demand, little attention has been paid to ensuring the safety of fish. The objective of this study was to evaluate the microbiological quality of tilapia and pangas fish sold in retail markets across Dhaka city, Bangladesh. Specifically, the study aimed to compare the quality of fish from traditional wet markets and modern supermarkets, as well as fish samples collected during morning and evening hours. A total of 500 raw cut-fish samples (250 tilapia and 250 pangas) were collected at the point of sale from 32 wet markets and 25 supermarkets. All samples were tested for Escherichia coli, extended-spectrum ß-lactamase-producing E. coli (ESBL-Ec), along with the foodborne pathogens Salmonella, Shigella, Vibrio, and Cryptosporidium spp. Bacterial isolates were characterized using antibiotic susceptibility tests (AST) and the presence of common virulence and antibiotic-resistant genes. Fish samples from retail markets had higher prevalence of tested bacteria including E. coli (92 %), V. cholerae (62 %), ESBL-Ec (48 %), and Salmonella spp. (24 %). There was a significant difference in the prevalence of E. coli (97 % vs. 71 %), ESBL-Ec (58 % vs. 8 %) and Salmonella spp. (28 % vs. 8 %) on the wet market samples compared to supermarket samples (p < 0.005). The mean concentration of E. coli on fish from the wet market was 3.0 ± 0.9 log10 CFU/g, while that from supermarkets was 1.6 ± 0.9 log10 CFU/g. The mean concentration of ESBL-Ec in fish from wet markets and supermarkets were 2.3 ± 0.8 log10 CFU/g and 1.6 ± 0.5 log10 CFU/g, respectively. AST revealed that 46 % of E. coli isolates were multi-drug resistant (MDR), while 4 %, 2 % and 5 % of E. coli, Salmonella spp. and Vibrio spp. isolates, respectively, were resistant to carbapenems. At least 3 % of total E. coli isolates were found to be diarrheagenic, while 40 % of Salmonella isolates harbored pathogenic genes (stn, bcfC, ssaQ, avrA and sodC1), and none of the V. cholerae isolates harbored ctxA and tcpA. Our research shows that raw-cut fish samples from retail markets are contaminated with pathogenic and antibiotic-resistant bacteria, which could be a significant food safety concern. Public health interventions should be implemented to improve food safety and hygiene practices in the retail fish markets.

Detection of Salmonella enterica and Listeria monocytogenes in alternative irrigation water by culture and qPCR-based methods in the Mid-Atlantic U.S.

Alternative 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. IMPORTANCE: Detecting 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.

Synovial fibroblast gene expression is associated with sensory nerve growth and pain in rheumatoid arthritis.

It has been presumed that rheumatoid arthritis (RA) joint pain is related to inflammation in the synovium; however, recent studies reveal that pain scores in patients do not correlate with synovial inflammation. We developed a machine-learning approach (graph-based gene expression module identification or GbGMI) to identify an 815-gene expression module associated with pain in synovial biopsy samples from patients with established RA who had limited synovial inflammation at arthroplasty. We then validated this finding in an independent cohort of synovial biopsy samples from patients who had early untreated RA with little inflammation. Single-cell RNA sequencing analyses indicated that most of these 815 genes were most robustly expressed by lining layer synovial fibroblasts. Receptor-ligand interaction analysis predicted cross-talk between human lining layer fibroblasts and human dorsal root ganglion neurons expressing calcitonin gene-related peptide (CGRP+). Both RA synovial fibroblast culture supernatant and netrin-4, which is abundantly expressed by lining fibroblasts and was within the GbGMI-identified pain-associated gene module, increased the branching of pain-sensitive murine CGRP+ dorsal root ganglion neurons in vitro. Imaging of solvent-cleared synovial tissue with little inflammation from humans with RA revealed CGRP+ pain-sensing neurons encasing blood vessels growing into synovial hypertrophic papilla. Together, these findings support a model whereby synovial lining fibroblasts express genes associated with pain that enhance the growth of pain-sensing neurons into regions of synovial hypertrophy in RA.