Extended Spectrum ß-Lactamase Activity and Cephalosporin Resistance in Escherichia coli from U.S. Mid-Atlantic Surface and Reclaimed Water.

Phylogenetic distribution and extended spectrum ß-lactamase (ESBL) activity of Escherichia coli recovered from surface and reclaimed water in the mid-Atlantic U.S. were evaluated. Among 488 isolates, phylogroups B1 and A were the most and least prevalent, respectively. Water type, but not season, affected phylogroup distribution. The likelihood of detecting group A isolates was higher in reclaimed than pond (P < 0.01), freshwater river (P < 0.01) or brackish river (P < 0.05) water. Homogeneity in group distribution was lowest in pond water, where group B1 comprised 50% of isolates. Only 16 (3.3%) isolates exhibited phenotypic resistance to one or more cephalosporins tested and only four had ESBL activity, representing groups B1, B2 isolates, and D. Phylogroup was a factor in antimicrobial resistance (P < 0.05), with group A (8.7%) and D (1.6%) exhibiting the highest and lowest rates. Resistance to cefoxitin was the most prevalent. Multi- versus single drug resistance was affected by phylogroup (P < 0.05) and more likely in groups D and B1 than A which carried resistance to cefoxitin only. The most detected ß-lactam resistance genes were blaCMY-2 and blaTEM. Water type was a factor for blaCTX-M gene detection (P < 0.05). Phenotypic resistance to cefotaxime, ceftriaxone, cefuroxime and ceftazidime, and genetic determinants for ESBL-mediated resistance were found predominantly in B2 and D isolates from rivers and reclaimed water. Overall, ESBL activity and cephalosporin resistance in reclaimed and surface water isolates were low. Integrating data on ESBL activity and ß-lactam resistance among E. coli populations can inform decisions on safety of irrigation water sources and One Health. IMPORTANCE Extended spectrum ß-lactamase (ESBL) producing bacteria, that are resistant to a broad range of antimicrobial agents, are spreading in the environment but data remain scarce. ESBL-producing Escherichia coli infections in the community are on the rise. This work was conducted to assess presence of ESBL-producing E. coli in water that could be used for irrigation of fresh produce. The study provides the most extensive evaluation of ESBL-producing E. coli in surface and reclaimed water in the mid-Atlantic United States. The prevalence of ESBL producers was low and phenotypic resistance to cephalosporins (types of ß-lactam antibiotics) was affected by season but not water type. Data on antimicrobial resistance among E. coli populations in water can inform decisions on safety of irrigation water sources and One Health.

Effects of season and water type on the distribution and antimicrobial resistance of Enterococcus faecalis and Ent. faecium from surface and reclaimed water.

AIMS: To evaluate the safety of irrigation water sources based on phenotypic antimicrobial resistance (AMR) in Enterococcus spp., a potential environmental reservoir for AMR determinants. METHODS AND RESULTS: Eleven sites representing fresh and brackish water rivers, ponds and reclaimed water, were sampled over 2 years. Samples (n = 333) yielded 198 unique isolates of Ent. faecalis and Ent. faecium which were tested for antimicrobial susceptibility by microbroth dilution. Species distribution was influenced by water type and season. Enterococcus faecalis was more likely found in freshwater rivers and in summer, and Ent. faecium in reclaimed water and in spring. Only 11% of isolates were pansusceptible, while 48.5% and 26.3% were single (SDR) and multidrug resistant (MDR), respectively. MDR was more likely detected in Ent. faecium than Ent. faecalis. Winter isolates were more likely than summer isolates to exhibit MDR than SDR. CONCLUSIONS: Enterococcus faecalis and Ent. faecium in surface and reclaimed water exhibited diverse phenotypic AMR and a low-level resistance to clinically important antimicrobials such as ampicillin, vancomycin and linezolid. SIGNIFICANCE AND IMPACT OF THE STUDY: Single and multidrug resistance in E. faecalis and E. faecium varied by season but not water type. Antimicrobial resistance prevalence can assist decisions on the safety of irrigation water sources for fresh produce crops.

Impact of irrigation water type and sampling frequency on Microbial Water Quality Profiles required for compliance with U.S. Food Safety Modernization Act Produce Safety Rule standards.

The U.S. Food Safety Modernization Act (FSMA) Produce Safety Rule (PSR) requires that farmers generate a Microbial Water Quality Profile (MWQP) from 20 samples per agricultural water source, taken over 2-4 years and five annual samples thereafter. Farmers must use the MWQP to ascertain a geometric mean (GM) of ≤126 CFU/100 mL and statistical threshold value (STV) of ≤410 CFU/100 mL of generic Escherichia coli. Farmers are responsible for collecting samples and paying for testing, incurring a financial and time burden. To determine if testing frequency can be reduced without compromising accuracy, water samples (n = 279) were collected from twelve sites in the U.S. Mid-Atlantic region from 2016 to 2018 comprising tidal brackish river, non-tidal fresh river, pond, vegetable processing, and reclaimed water. The GM and STV were calculated for all sites and water types using all samples, and for multiple sub-samples of <20 from each site and water type. A Monte Carlo simulation was used to determine the proportion of sub-sample sizes that yielded the same determination as the entire sample size of PSR standard compliance. Four sites, two pond and two reclaimed water sites, complied with PSR GM and STV requirements when using the entire sample set. When a water source's calculated GM and STV using the entire sample set hovered close to the PSR thresholds, sub-sample sizes approached the recommended 20 samples to reach a congruent compliance determination. However, 99% agreement was obtained with a sub-sample of five when the absolute difference between the GM and STV from total samples and the PSR thresholds was ≥2.6 and 4.5 log CFU/100 mL E. coli, respectively. These findings suggest that under certain conditions the MWQP may be generated with well below 20 samples, reducing the economic burden on farmers while still maintaining a representative MWQP.

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.

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.

Quenching by sodium thiosulfate does not influence 16S rRNA gene sequencing profiles of reclaimed water from three sites in the Mid-Atlantic, United States.

A quenching agent is commonly added to chlorinated, reclaimed water during sample collection to prevent chlorine-mediated die-off of viable microbiota. However, the effect of quenching on downstream 16S rRNA-based bacterial community analyses is unclear. We conducted a side-by-side comparison of 16S rRNA sequencing data from reclaimed water samples quenched with sodium thiosulfate and non-quenched samples. Our data showed that 16 S rRNA processing and sequencing methods, and resulting bacterial profiles, were not negatively impacted by quenching.

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.

Persistence comparison of two Shiga-toxin producing Escherichia coli (STEC) serovars during long-term storage and thermal inactivation in various wheat flours.

Foodborne outbreaks associated with Shiga toxin-producing Escherichia coli (STEC) contaminated wheat flour have been an increasing food safety concern in recent decades. However, there is little literature aimed at investigating the impact of different flour types on the persistence of STEC during storage and thermal inactivation. Therefore, two serovars of STEC, O121 and O157, were selected to inoculate each of five different types of common wheat flours: whole wheat, bleached, unbleached, bread, and self-rising. Inoculated flours were examined for the stability of STEC during storage for up to 42 days at room temperature (RT) and aw ~0.56. Additionally, the thermal resistance of O121 and O157 under isothermal conditions at 60, 70, 80, and 90°C was analyzed for the inoculated flours. STEC storage persistence at RT was generally not affected by flour type, however, decreases of 1.2 and 2.4 log CFU/day within whole wheat flour for O121 and O157, respectively, were significantly lower than other flours. Though few differences were identified in relation to flour type, O121 exhibited significantly better survival rates than O157 during both equilibrium and storage periods. Compared to an approximate 6 log reduction in the population of O157, O121 population levels were reduced by a significantly lower amount (~3 log) during the entire storage period at RT. At each isothermal temperature, the impact of flour type on the thermal resistance capabilities of O121 or O157 was not a significant factor and resulted in similar survival curves regardless of serovar. Instead of exhibiting linear survival curves, both O121 and O157 displayed nonlinear curves with some shoulder/tail effect. Similar for both O121 and O157, the predicted decimal reduction time (D-value) decreased from approximately 25 min to around 8 min as the isothermal temperature increased from 60°C to 90°C. Results reported here can contribute to risk assessment models concerning contamination of STEC in wheat flour and add to our understanding of the impacts of flour type and STEC serovar on desiccation stability during storage and isothermal inactivation during thermal treatment.

Differential methylation analysis in neuropathologically confirmed dementia with Lewy bodies.

Dementia with Lewy bodies (DLB) is a common form of dementia in the elderly population. We performed genome-wide DNA methylation mapping of cerebellar tissue from pathologically confirmed DLB cases and controls to study the epigenetic profile of this understudied disease. After quality control filtering, 728,197 CpG-sites in 278 cases and 172 controls were available for the analysis. We undertook an epigenome-wide association study, which found a differential methylation signature in DLB cases. Our analysis identified seven differentially methylated probes and three regions associated with DLB. The most significant CpGs were located in ARSB (cg16086807), LINC00173 (cg18800161), and MGRN1 (cg16250093). Functional enrichment evaluations found widespread epigenetic dysregulation in genes associated with neuron-to-neuron synapse, postsynaptic specialization, postsynaptic density, and CTCF-mediated synaptic plasticity. In conclusion, our study highlights the potential importance of epigenetic alterations in the pathogenesis of DLB and provides insights into the modified genes, regions and pathways that may guide therapeutic developments.

Genome-wide structural variant analysis identifies risk loci for non-Alzheimer's dementias.

We characterized the role of structural variants, a largely unexplored type of genetic variation, in two non-Alzheimer's dementias, namely Lewy body dementia (LBD) and frontotemporal dementia (FTD)/amyotrophic lateral sclerosis (ALS). To do this, we applied an advanced structural variant calling pipeline (GATK-SV) to short-read whole-genome sequence data from 5,213 European-ancestry cases and 4,132 controls. We discovered, replicated, and validated a deletion in TPCN1 as a novel risk locus for LBD and detected the known structural variants at the C9orf72 and MAPT loci as associated with FTD/ALS. We also identified rare pathogenic structural variants in both LBD and FTD/ALS. Finally, we assembled a catalog of structural variants that can be mined for new insights into the pathogenesis of these understudied forms of dementia.

Aeromonas spp. diversity in U.S. mid-Atlantic surface and reclaimed water, seasonal dynamics, virulence gene patterns and attachment to lettuce.

Aeromonas, a ubiquitous taxon in water environments, is emerging as a foodborne pathogen of concern that remains understudied and under-reported. We evaluated the distribution of 331 Aeromonas spp. isolates collected from irrigation water over one year and characterised their virulence profile, attachment and ability to persist on lettuce. Water sources included non-tidal and tidal river, farm pond and reclaimed water. Twenty Aeromonas species were identified; A. veronii, A. hydrophila and A. jandaei predominated in all water types and seasons, comprising ~63% of isolates. Species distribution was most affected by water type. The highest and lowest diversity were detected in river and pond water, respectively. A. hydrophila and A. veronii ranked highest in frequency in fresh river and reclaimed water, while A. jandaei ranked first in pond water. Only two isolates carried all five virulence genes tested, while 46% of A. hydrophila (n = 50), 54% of A. veronii (n = 61) and 50% of A. jandaei (n = 32) isolates harboured multiple enterotoxin genes. Detection of alt and ast genes was more likely in summer collections, while ast detection was less likely in tidal brackish river and pond water isolates. Season was a factor in attachment to polystyrene, being strongest in spring isolates. The gene flaA was associated with strong attachment and was more likely to be detected in non-tidal fresh river isolates. A. hydrophila and A. jandaei isolates persisted on lettuce leaves for 24 h, but populations dwindled over 120 h, while loosely and strongly attached cells of A. veronii isolates persisted for 120 h. This study provides comprehensive data on Aeromonas species distribution and environmental traits. The associations revealed among diversity, water type, season, virulence factors and phyllosphere attachment capacity can inform agricultural water standards in novel ways. Moreover, understanding Aeromonas-plant interactions is an important step in advancing food safety of fruit and vegetables.

Levels of Salmonella enterica and Listeria monocytogenes in Alternative Irrigation Water Vary Based on Water Source on the Eastern Shore of Maryland.

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 < 0.05) greater for the nontidal freshwater river sites and the tidal brackish river site than the reclaimed water sites. L. monocytogenes levels in water varied based on season. Detection of S. enterica was more likely with 10-liter filtration compared to 0.1-liter filtration. The physicochemical factors measured attributed only 6.4% of the constrained variance to the levels of both pathogens. This study shows clear variations in S. enterica and L. monocytogenes levels in irrigation water sources on ESM. IMPORTANCE In the last several decades, Maryland's Eastern Shore has seen significant declines in groundwater levels. While this area is not currently experiencing drought conditions or water scarcity, this research represents a proactive approach. Efforts, to investigate the levels of pathogenic bacteria and the microbial quality of alternative irrigation water are important for sustainable irrigation practices into the future. This research will be used to determine the suitability of alternative irrigation water sources for use in fresh produce irrigation to conserve groundwater.

Microbiological quality assessment of milk at different stages of the dairy value chain in a developing country setting.

The main objective of the study was to assess the microbiological quality of milk at different stages of the dairy value chain from farm to the factory in Bangladesh. A total of 438 raw milk samples (387 from primary producers, 32 from collectors, 15 from chilling plants, 4 from local restaurants) and 95 commercially processed milk samples were collected from northern part of Bangladesh. Almost 72% (n = 280) of samples at producer level and 100% from both collectors (n = 32) and chilling plants (n = 15) were contaminated with coliforms while 57% (n = 220) of samples from producers, 91% (n = 29) of samples from collectors and 100% (n = 15) from chilling plants were contaminated with fecal coliforms. Around 31% (n = 119) of samples from producers were positive for E. coli whereas >60% (n = 20) and 100% (n = 15) samples from collectors and chilling plants, respectively were positive for E. coli. One quarter of samples from collectors were positive for B. cereus and coagulase positive staphylococci and 33% (n = 5) of samples from chilling plants were positive for both of these microorganisms. In case of commercially processed milk, 77% (n = 46) and 37% (n = 22) of pasteurized milk samples had a high aerobic plate count (APC) (104 CFU/ml) and coliform count (>10 CFU/ml), respectively. None of the samples was positive for Shigella spp., Salmonella spp., and Campylobacter spp. Among 158 E. coli positive raw milk samples, 9% (n = 14) contained pathogenic E. coli, and enteroaggregative E. coli (EAEC) and Shiga-toxin producing E. coli (STEC) were found to be the predominant pathotypes. Of the 23 pathogenic E. coli identified from 14 samples based on their gene contents, >95% (n = 22) were resistant to at least one antibiotic and 13% (n = 3) of isolates were resistant to ≥3 classes of antibiotics. Several factors including the time of milking, hygiene practices of the producers, cow breed and amount of milk produced by the cow were found to be significantly associated with high APC of milk samples. In conclusion, both raw and commercially pasteurized milk are highly contaminated with fecal organisms. For intervention, more emphasis should be given at producer's level as microorganisms introduced to milk at this stage get the longest time for survival and multiplication.