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.

Temporal Stability of Salmonella enterica and Listeria monocytogenes in Surface Waters Used for Irrigation in the Mid-Atlantic United States.

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.

Reducing Anxiety with Nature and Gardening (RANG): Evaluating the Impacts of Gardening and Outdoor Activities on Anxiety among U.S. Adults during the COVID-19 Pandemic.

The COVID-19 pandemic impacted mental health. Growing research has identified the mental health benefits of nature contact, including gardening. We used a cross-sectional survey to investigate the association between gardening and other outdoor activities with anxiety among U.S. adults. The RANG (Reducing Anxiety with Nature and Gardening) survey was distributed online from June−September 2020 through social media (Twitter and Facebook) and a national Master Gardeners listserv. Survey questions captured demographics, COVID-19 experiences, gardening, outdoor activities, and anxiety using the Generalized Anxiety Disorder 7-item scale. Data were analyzed using chi-square, Fisher's exact, and Kruskal−Wallis tests, as well as logistic regression. Among participants, 46% reported anxiety symptoms. Participants who had gardened ≥ 15 years and those gardening > 8 h over two weeks had lower anxiety scores. Spending more time outdoors on weekdays also decreased anxiety scores. After adjusting for covariates, lower odds of anxiety were identified for 50−69 and 70−89-year-olds vs. 18−29-year-olds; males vs. females; and Texas vs. Maryland residents. These findings confirm increased anxiety during the COVID-19 pandemic and suggest that sustained gardening and other outdoor activities could help reduce anxiety.

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.

Traditional approaches for educating farmers about nontraditional water: Evaluating preferred outreach, education, and methods for alleviating concerns.

Due to increased variability in precipitation it is critical to identify alternative, nontraditional water sources for food production. Water reuse, the use of treated municipal wastewater, could be an alternative high-quality water source for agricultural systems. Tailoring education about water reuse to specific audiences is crucial for increasing public acceptance, especially for the farmers being asked to use this water source. Through a survey distributed from 2016 through 2018, farmers in the Mid-Atlantic and Southwest regions of the United States provided feedback on their preferred approaches to alleviate concerns (n = 749), and preferred outreach and education methods (n = 719) for learning about nontraditional water sources, including water reuse. Overall, farmers' top priority was water quality information to alleviate their concerns (55%). Farmers preferred to attend half-day workshops in their home county for outreach and education (57%). There were differences in preferences for alleviating concerns and receiving education by sex, geographic region, and concern about nontraditional water. Women wanted more information than men to alleviate concerns (p = 0.001) and more types of outreach and education regarding nontraditional water use (p < 0.001). Farmers who were 18-29 and 30-49 years old reported that public acceptance would alleviate concerns about nontraditional water use at a significantly higher rate (25% and 24%) than farmers in the 50-69 year old category (17%) (p = 0.04 for both). The 18-29 year olds also preferred water treatment plant tours (37%) more than all other age groups (26% or less) (p < 0.001). Information about farmer preferences for nontraditional water education and outreach can be used to develop tailored education programs and increase acceptance and use of these important water sources.

Groundwater level changes with a focus on agricultural areas in the Mid-Atlantic region of the United States, 2002-2016.

Climate change impacts all water sources, including high quality groundwater that supplies agricultural irrigation in many regions of the United States. This study assessed groundwater level changes in the U.S. Mid-Atlantic region with a focus on cultivated areas. Trends of groundwater level were estimated using linear regression, and examined for shallow, medium, and deep depths across physiographic regions of Mid-Atlantic. A hotspot analysis was conducted to identify spatial clusters of wells with rising or declining groundwater levels. In addition, differences in the percentage of cultivated area with declining groundwater between cultivated land categories was examined at the county level. From 2002-2016, the Mid-Atlantic region had an overall decline in groundwater level (0.06 m/yr, 95% CI: 0.03, 0.09) although groundwater changes varied by physiographic regions. The Coastal Plain physiographic region was dominated by declining groundwater wells (48%) and had the most significant groundwater level declines (0.23 m/yr, 95% CI: 0.19, 0.26). Significant groundwater level rises were detected in Southern Virginia adjacent to the Chesapeake Bay (0.92 m/yr on average), which could be due to the cessation of groundwater withdrawal from one of the region's largest groundwater users. In the Mid-Atlantic region, shallow groundwater was found to have slight rising trends (0.08 m, p < 0.05) while deeper groundwater showed distinctive declining trends (1.36 m, p < 0.05) between 2002 and 2016. There were significantly more cultivated areas with declining groundwater levels (88% vs. 35%, p < 0.05) in counties with high percentages of cropland (> 50%) compared to areas covered by less cropland. As climate and human pressures increase, it will be critical to identify and evaluate alternative water sources, such as reclaimed water, to sustain agricultural production and protect groundwater resources.

Prevalence of Microbiological and Chemical Contaminants in Private Drinking Water Wells in Maryland, USA.

Although many U.S. homes rely on private wells, few studies have investigated the quality of these water sources. This cross-sectional study evaluated private well water quality in Maryland, and explored possible environmental sources that could impact water quality. Well water samples (n = 118) were collected in four Maryland counties and were analyzed for microbiological and chemical contaminants. Data from the U.S. Census of Agriculture were used to evaluate associations between the presence of animal feeding operations and well water quality at the zip code level using logistic regression. Overall, 43.2% of tested wells did not meet at least one federal health-based drinking water standard. Total coliforms, fecal coliforms, enterococci, and Escherichia coli were detected in 25.4%, 15.3%, 5.1%, and 3.4% of tested wells, respectively. Approximately 26%, 3.4%, and <1% of wells did not meet standards for pH, nitrate-N, and total dissolved solids, respectively. There were no statistically significant associations between the presence of cattle, dairy, broiler, turkey, or aquaculture operations and the detection of fecal indicator bacteria in tested wells. In conclusion, nearly half of tested wells did not meet federal health-based drinking water standards, and additional research is needed to evaluate factors that impact well water quality. However, homeowner education on well water testing and well maintenance could be important for public health.

Conventional wastewater treatment and reuse site practices modify bacterial community structure but do not eliminate some opportunistic pathogens in reclaimed water.

Water recycling continues to expand across the United States, from areas that have access to advanced, potable-level treated reclaimed water, to those having access only to reclaimed water treated at conventional municipal wastewater treatment plants. This expansion makes it important to further characterize the microbial quality of these conventionally-treated water sources. Therefore, we used 16S rRNA gene sequencing to characterize total bacterial communities present in differentially-treated wastewater and reclaimed water (n = 67 samples) from four U.S. wastewater treatment plants and one associated spray irrigation site conducting on-site ultraviolet treatment and open-air storage. The number of observed operational taxonomic units was significantly lower (p < 0.01) in effluent, compared to influent, after conventional treatment. Effluent community structure was influenced more by treatment method than by influent community structure. The abundance of Legionella spp. increased as treatment progressed in one treatment plant that performed chlorination and in another that seasonally chlorinated. Overall, the alpha-diversity of bacterial communities in reclaimed water decreased (p < 0.01) during wastewater treatment and spray irrigation site ultraviolet treatment (p < 0.01), but increased (p < 0.01) after open-air storage at the spray irrigation site. The abundance of Legionella spp. was higher at the sprinkler system pumphouse at the spray irrigation site than in the influent from the treatment plant supplying the site. Legionella pneumophila was detected in conventionally treated effluent samples and in samples collected after ultraviolet treatment at the spray irrigation site, while Legionella feeleii persisted throughout on-site treatment at the spray irrigation site, and, along with Mycobacterium gordonae, was also detected at the sprinkler system pumphouse at the spray irrigation site. These data could inform the development of future treatment technologies and reuse guidelines that address a broader assemblage of the bacterial community of reclaimed water, resulting in reuse practices that may be more protective of public health.

Antibiotic Concentrations Decrease during Wastewater Treatment but Persist at Low Levels in Reclaimed Water.

Reclaimed water has emerged as a potential irrigation solution to freshwater shortages. However, limited data exist on the persistence of antibiotics in reclaimed water used for irrigation. Therefore, we examined the fate of nine commonly-used antibiotics (ampicillin, azithromycin, ciprofloxacin, linezolid, oxacillin, oxolinic acid, penicillin G, pipemidic acid, and tetracycline) in differentially treated wastewater and reclaimed water from two U.S. regions. We collected 72 samples from two Mid-Atlantic and two Midwest treatment plants, as well as one Mid-Atlantic spray irrigation site. Antibiotic concentrations were measured using liquid-chromatography- tandem mass spectrometry. Data were analyzed using Mann-Whitney-Wilcoxon tests and Kruskal Wallis tests. Overall, antibiotic concentrations in effluent samples were lower than that of influent samples. Mid-Atlantic plants had similar influent but lower effluent antibiotic concentrations compared to Midwest plants. Azithromycin was detected at the highest concentrations (of all antibiotics) in influent and effluent samples from both regions. For most antibiotics, transport from the treatment plant to the irrigation site resulted in no changes in antibiotic concentrations, and UV treatment at the irrigation site had no effect on antibiotic concentrations in reclaimed water. Our findings show that low-level antibiotic concentrations persist in reclaimed water used for irrigation; however, the public health implications are unclear at this time.

Association between community socioeconomic factors, animal feeding operations, and campylobacteriosis incidence rates: Foodborne Diseases Active Surveillance Network (FoodNet), 2004-2010.

BACKGROUND: Campylobacter is a leading cause of foodborne illness in the United States. Campylobacter infections have been associated with individual risk factors, such as the consumption of poultry and raw milk. Recently, a Maryland-based study identified community socioeconomic and environmental factors that are also associated with campylobacteriosis rates. However, no previous studies have evaluated the association between community risk factors and campylobacteriosis rates across multiple U.S. states. METHODS: We obtained Campylobacter case data (2004-2010; n = 40,768) from the Foodborne Diseases Active Surveillance Network (FoodNet) and socioeconomic and environmental data from the 2010 Census of Population and Housing, the 2011 American Community Survey, and the 2007 U.S. Census of Agriculture. We linked data by zip code and derived incidence rate ratios using negative binomial regression models. RESULTS: Community socioeconomic and environmental factors were associated with both lower and higher campylobacteriosis rates. Zip codes with higher percentages of African Americans had lower rates of campylobacteriosis (incidence rate ratio [IRR]) = 0.972; 95 % confidence interval (CI) = 0.970,0.974). In Georgia, Maryland, and Tennessee, three leading broiler chicken producing states, zip codes with broiler operations had incidence rates that were 22 % (IRR = 1.22; 95 % CI = 1.03,1.43), 16 % (IRR = 1.16; 95 % CI = 0.99,1.37), and 35 % (IRR = 1.35; 95 % CI = 1.18,1.53) higher, respectively, than those of zip codes without broiler operations. In Minnesota and New York FoodNet counties, two top dairy producing areas, zip codes with dairy operations had significantly higher campylobacteriosis incidence rates (IRR = 1.37; 95 % CI = 1.22, 1.55; IRR = 1.19; 95 % CI = 1.04,1.36). CONCLUSIONS: Community socioeconomic and environmental factors are important to consider when evaluating the relationship between possible risk factors and Campylobacter infection.

Occupational exposure to Staphylococcus aureus and Enterococcus spp. among spray irrigation workers using reclaimed water.

As reclaimed water use expands, it is important to evaluate potential occupational health risks from exposure to this alternative water source. We compared odds of colonization with methicillin-resistant Staphylococcus aureus (MRSA), methicillin-susceptible S. aureus (MSSA), vancomycin-resistant enterococci (VRE), and vancomycin-susceptible enterococci (VSE) between spray irrigation workers using reclaimed water and office worker controls. Nasal and dermal swabs from 19 spray irrigation workers and 24 office worker controls were collected and analyzed for MRSA, MSSA, VRE, and VSE. Isolates were confirmed using standard biochemical tests and polymerase chain reaction assays. Antimicrobial susceptibility testing was performed by Sensititre® microbroth dilution. Data were analyzed by two-sample proportion, chi-square, Fisher's exact tests, and logistic regression. No MRSA or VRE were detected in any samples. MSSA was detected in 26% and 29% of spray irrigators and controls, respectively. VSE was detected in 11% and 0% of spray irrigation workers and controls, respectively. The adjusted odds of MSSA, multidrug-resistant MSSA, and either MSSA or VSE colonization were greater among spray irrigation workers, however results were not statistically significant. Future studies with larger sample sizes are needed to further evaluate this relationship.

Antimicrobial susceptibility of Vibrio vulnificus and Vibrio parahaemolyticus recovered from recreational and commercial areas of Chesapeake Bay and Maryland Coastal Bays.

Vibrio vulnificus and V. parahaemolyticus in the estuarine-marine environment are of human health significance and may be increasing in pathogenicity and abundance. Vibrio illness originating from dermal contact with Vibrio laden waters or through ingestion of seafood originating from such waters can cause deleterious health effects, particularly if the strains involved are resistant to clinically important antibiotics. The purpose of this study was to evaluate antimicrobial susceptibility among these pathogens. Surface-water samples were collected from three sites of recreational and commercial importance from July to September 2009. Samples were plated onto species-specific media and resulting V. vulnificus and V. parahaemolyticus strains were confirmed using polymerase chain reaction assays and tested for antimicrobial susceptibility using the Sensititre® microbroth dilution system. Descriptive statistics, Friedman two-way Analysis of Variance (ANOVA) and Kruskal-Wallis one-way ANOVA were used to analyze the data. Vibrio vulnificus (n = 120) and V. parahaemolyticus (n = 77) were isolated from all sampling sites. Most isolates were susceptible to antibiotics recommended for treating Vibrio infections, although the majority of isolates expressed intermediate resistance to chloramphenicol (78% of V. vulnificus, 96% of V. parahaemolyticus). Vibrio parahaemolyticus also demonstrated resistance to penicillin (68%). Sampling location or month did not significantly impact V. parahaemolyticus resistance patterns, but V. vulnificus isolates from St. Martin's River had lower overall intermediate resistance than that of the other two sampling sites during the month of July (p = 0.0166). Antibiotics recommended to treat adult Vibrio infections were effective in suppressing bacterial growth, while some antibiotics recommended for pediatric treatment were not effective against some of the recovered isolates. To our knowledge, these are the first antimicrobial susceptibility data of V. vulnificus and V. parahaemolyticus recovered from the Chesapeake Bay. These data can serve as a baseline against which future studies can be compared to evaluate whether susceptibilities change over time.

Detection of vancomycin-resistant enterococci (VRE) at four U.S. wastewater treatment plants that provide effluent for reuse.

Vancomycin-resistant enterococci (VRE), a leading cause of hospital-acquired infections, can occur in wastewater. However, to date, no previous studies have evaluated the occurrence of VRE at wastewater treatment plants (WWTPs) that send their treated effluent to reuse sites. We evaluated the occurrence, concentration, and antimicrobial resistance patterns of VRE at U.S. WWTPs associated with reuse sites. We collected 44 wastewater samples, representing treatment steps from influent to effluent, from two Mid-Atlantic and two Midwest WWTPs between October 2009 and October 2010. Samples were analyzed for total enterococci and VRE using membrane filtration. Isolates were confirmed using biochemical tests and PCR. Antimicrobial susceptibility testing was performed by Sensititre microbroth dilution. Data were analyzed by two-sample proportion tests and analysis of variance. We detected VRE in 27% (12/44) of all wastewater samples collected and VRE represented 3% of total enterococci detected at all WWTPs. More samples were VRE-positive from the Mid-Atlantic compared to the Midwest WWTPs (p=0.008). VRE concentrations decreased as treatment progressed at all WWTPs, except at Mid-Atlantic WWTP1 where there was an increase in VRE concentrations in activated sludge reactor samples. VRE were not detected in chlorinated effluent, but were detected in one un-chlorinated effluent sample. All unique VRE isolates were multidrug resistant. Fifty-five percent (12/22) of the isolates displayed high-level aminoglycoside resistance. Our findings show that chlorination reduces the occurrence of VRE in wastewater. However, WWTP workers could be exposed to VRE during wastewater treatment. Our data also raise potential concerns about VRE exposure among individuals who come into contact with un-chlorinated reclaimed water.

Impact of rurality, broiler operations, and community socioeconomic factors on the risk of campylobacteriosis in Maryland.

OBJECTIVES: We evaluated the combined impact of community-level environmental and socioeconomic factors on the risk of campylobacteriosis. METHODS: We obtained Campylobacter case data (2002-2010; n = 3694) from the Maryland Foodborne Diseases Active Surveillance Network. We obtained community-level socioeconomic and environmental data from the 2000 US Census and the 2007 US Census of Agriculture. We linked data by zip code. We derived incidence rate ratios by Poisson regressions. We mapped a subset of zip code-level characteristics. RESULTS: In zip codes that were 100% rural, incidence rate ratios (IRRs) of campylobacteriosis were 6 times (IRR = 6.18; 95% confidence interval [CI] = 3.19, 11.97) greater than those in urban zip codes. In zip codes with broiler chicken operations, incidence rates were 1.45 times greater than those in zip codes without broilers (IRR = 1.45; 95% CI = 1.34, 1.58). We also observed higher rates in zip codes whose populations were predominantly White and had high median incomes. CONCLUSIONS: The community and environment in which one lives may significantly influence the risk of campylobacteriosis.

Methicillin-resistant Staphylococcus aureus (MRSA) detected at four U.S. wastewater treatment plants.

BACKGROUND: The incidence of community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) infections is increasing in the United States, and it is possible that municipal wastewater could be a reservoir of this microorganism. To date, no U.S. studies have evaluated the occurrence of MRSA in wastewater. OBJECTIVE: We examined the occurrence of MRSA and methicillin-susceptible S. aureus (MSSA) at U.S. wastewater treatment plants. METHODS: We collected wastewater samples from two Mid-Atlantic and two Midwest wastewater treatment plants between October 2009 and October 2010. Samples were analyzed for MRSA and MSSA using membrane filtration. Isolates were confirmed using biochemical tests and PCR (polymerase chain reaction). Antimicrobial susceptibility testing was performed by Sensititre® microbroth dilution. Staphylococcal cassette chromosome mec (SCCmec) typing, Panton-Valentine leucocidin (PVL) screening, and pulsed field gel electrophoresis (PFGE) were performed to further characterize the strains. Data were analyzed by two-sample proportion tests and analysis of variance. RESULTS: We detected MRSA (n = 240) and MSSA (n = 119) in 22 of 44 (50%) and 24 of 44 (55%) wastewater samples, respectively. The odds of samples being MRSA-positive decreased as treatment progressed: 10 of 12 (83%) influent samples were MRSA-positive, while only one of 12 (8%) effluent samples was MRSA-positive. Ninety-three percent and 29% of unique MRSA and MSSA isolates, respectively, were multidrug resistant. SCCmec types II and IV, the pvl gene, and USA types 100, 300, and 700 (PFGE strain types commonly found in the United States) were identified among the MRSA isolates. CONCLUSIONS: Our findings raise potential public health concerns for wastewater treatment plant workers and individuals exposed to reclaimed wastewater. Because of increasing use of reclaimed wastewater, further study is needed to evaluate the risk of exposure to antibiotic-resistant bacteria in treated wastewater.

The evaluation of a PCR-based method for identification of Salmonella enterica serotypes from environmental samples and various food matrices.

The most commonly used method for serotyping Salmonella spp. is based on the Kaufmann-White scheme, and is composed of serological reactions using antibodies to LPS agglutinins. The multiplex PCR used in this investigation was established by Kim et al. to serotype the 30 most common clinical Salmonella serotypes, as determined by CDC. The PCR assay consists of two five-plex reactions and a single two-plex PCR reaction, based on six genetic loci from Salmonella enterica serotype Typhimurium and four loci from S. enterica serotype Typhi. In this investigation, we further evaluated the method for serotyping Salmonella spp. using a reference collection, environmental samples collected from a Mid-Atlantic region tomato farm study, four food matrices spiked with different Salmonella serotypes and a proficiency test. The PCR assay was first evaluated using DNA isolated from pure cultures of isolates obtained from various clinical and environmental samples, and then DNA isolated from broth cultures of food matrices of "Red round" and Roma tomatoes, Romaine lettuce, green onions and Serrano peppers spiked with serotypes Newport, Typhimurium, Javiana and Saintpaul, respectively. The results showed that the PCR assay correctly serotyped Salmonella spp. from the clinical, environmental, spiked food matrices, and proficiency test samples. These findings are significant because the PCR assay was successful in the identification of Salmonella in the spiked samples in a broth culture containing other non-salmonella organism. This method may be a useful resource for the food safety community.

Occurrence and antibiotic resistance of multiple Salmonella serotypes recovered from water, sediment and soil on mid-Atlantic tomato farms.

Salmonella outbreaks associated with the consumption of raw tomatoes have been prevalent in recent years. However, sources of Salmonella contamination of tomatoes remain poorly understood. The objectives of this study were to identify ecological reservoirs of Salmonella on tomato farms, and to test antimicrobial susceptibilities of recovered Salmonella isolates. Fourteen Mid-Atlantic tomato farms in the U.S. were sampled in 2009 and 2010. Groundwater, irrigation pond water, pond sediment, irrigation ditch water, rhizosphere and irrigation ditch soil, leaves, tomatoes, and swabs of harvest bins and worker sanitary facilities were analyzed for Salmonella using standard culture methods and/or a flow-through immunocapture method. All presumptive Salmonella isolates (n=63) were confirmed using PCR and the Vitek(®) 2 Compact System, and serotyped using the Premi(®)Test Salmonella and a conventional serotyping method. Antimicrobial susceptibility testing was carried out using the Sensititre™ microbroth dilution system. Four of the 14 farms (29%) and 12 out of 1,091 samples (1.1%) were found to harbor Salmonella enterica subsp. enterica. Salmonella was isolated by the immunocapture method from soil, while the culture method recovered isolates from irrigation pond water and sediment, and irrigation ditch water. No Salmonella was detected on leaves or tomatoes. Multiple serotypes were identified from soil and water, four of which-S. Braenderup, S. Javiana, S. Newport and S. Typhimurium-have been previously implicated in Salmonella outbreaks associated with tomato consumption. Resistance to sulfisoxazole was prevalent and some resistance to ampicillin, cefoxitin, amoxicillin/clavulanic acid, and tetracycline was also observed. This study implicates irrigation water and soil as possible reservoirs of Salmonella on tomato farms and irrigation ditches as ephemeral habitats for Salmonella. The findings point to the potential for pre-harvest contamination of tomatoes from contaminated irrigation water or from soil or water splash from irrigation ditches onto low-lying portions of tomato plants.

Self-medication with antibiotics for the treatment of menstrual symptoms in Southwest Nigeria: a cross-sectional study.

BACKGROUND: Self-medication with antibiotics is an important factor contributing to the development of bacterial antibiotic resistance. The purpose of this study was to evaluate the prevalence of self-medication with antibiotics for the treatment of menstrual symptoms among university women in Southwest Nigeria. METHODS: A cross-sectional survey was administered to female undergraduate and graduate students (n = 706) at four universities in Southwest Nigeria in 2008. The universities were selected by convenience and the study samples within each university were randomly selected cluster samples. The survey was self-administered and included questions pertaining to menstrual symptoms, analgesic and antibiotic use patterns, and demographics. Data were analyzed using descriptive statistics and logistic regression. RESULTS: The response rate was 95.4%. Eighty-six percent (95% CI: 83-88%) of participants experienced menstrual symptoms, and 39% (95% CI: 36-43%) reported using analgesics to treat them. Overall, 24% (95% CI: 21-27%) of participants reported self-medicated use of antibiotics to treat the following menstrual symptoms: cramps, bloating, heavy bleeding, headaches, pimples/acne, moodiness, tender breasts, backache, joint and muscle pain. Factors associated with this usage were: lower levels of education (Odds Ratio (OR): 2.8, 95% CI: 1.1-7.1, p-value: 0.03); non-science major (OR: 1.58, 95% CI: 1.03-2.50, p-value: 0.04); usage of analgesics (OR: 3.17, 95% CI: 2.07-4.86, p-value: <0.001); and mild to extreme heavy bleeding (OR: 1.64, 95% CI: 1.01-2.67, p-value: 0.05) and pimples/acne (OR: 1.57, 95% CI: 0.98-2.54, p-value: 0.06). Ampicillin, tetracycline, ciprofloxacin and metronidazole were used to treat the most symptoms. Doctors or nurses (6%, 95% CI: 4-7%), friends (6%, 95% CI: 4-7%) and family members (7%, 95% CI: 5-8%) were most likely to recommend the use of antibiotics for menstrual symptoms, while these drugs were most often obtained from local chemists or pharmacists (10.2%, 95% CI: 8-12%). CONCLUSIONS: This is the first formal study to report that approximately 1 out of 4 university women surveyed in Southwest Nigeria self-medicate with antibiotics to treat menstrual symptoms. This practice could provide monthly, low-dose exposures to antibiotics among users. Further studies are necessary to evaluate the impacts of self-medication on student health.