An analysis of culture-based methods used for the detection and isolation of Salmonella spp., Escherichia coli, and Enterococcus spp. from surface water: A systematic review.

Identification of methods for the standardized assessment of bacterial pathogens and antimicrobial resistance (AMR) in environmental water can improve the quality of monitoring and data collected, support global surveillance efforts, and enhance the understanding of environmental water sources. We conducted a systematic review to assemble and synthesize available literature that identified methods for assessment of prevalence and abundance of bacterial fecal indicators and pathogens in water for the purposes of monitoring bacterial pathogens and AMR. After screening for quality, 175 unique publications were identified from 15 databases, and data were extracted for analysis. This review identifies the most common and robust methods, and media used to isolate target organisms from surface water sources, summarizes methodological trends, and recognizes knowledge gaps. The information presented in this review will be useful when establishing standardized methods for monitoring bacterial pathogens and AMR in water in the United States and globally.

A comparison of methods to detect low levels of Salmonella enterica in surface waters to support antimicrobial resistance surveillance efforts performed in multiple laboratories.

Developing effective and sensitive detection methods for antimicrobial resistant Salmonella enterica from surface water is a goal of the National Antimicrobial Resistance Monitoring System (NARMS). There are no specified methods for recovery of S. enterica in surface waters in the U.S. A multi-laboratory evaluation of four methods - bulk water enrichment (BW), vertical Modified Moore Swab (VMMS), modified Standard Method 9260.B2 (SM), and dead-end ultrafiltration (DEUF) - was undertaken to recover S. enterica from surface water. In Phase 1, one-liter volumes of water were collected from the same site on five different dates. Water was shipped and analyzed at four different laboratory locations (A, B, C, and D) for recovery of 1) inoculated fluorescent S. Typhimurium strain (ca. 30 CFU/L) and 2) Salmonella present in the water sampled. At each location, BW, VMMS, or SM recovery was performed on five separate 1 L water samples. Twenty 1 L water samples were subjected to each recovery method, and overall, sixty 1 L samples were assayed for Salmonella. Inoculated, fluorescent Salmonella Typhimurium and environmental Salmonella spp. were recovered from 65 % (39/60) and 45 % (27/60) of water samples, respectively. BW, VMMS, and SM recovered fluorescent S. Typhimurium from 60 %, 60 %, and 75 % of inoculated samples, respectively. Analysis by Chi-squared test determined laboratory location had a significant (p < 0.05) effect on fluorescent S. Typhimurium recovery compared to method or date of water collection. In Phase 2, recovery of inoculated fluorescent S. Typhimurium from 1 L samples by SM and DEUF was compared at laboratory locations B and D. SM and DEUF recovered fluorescent S. Typhimurium from 100 % (20/20) and 95 % (19/20) of inoculated water samples, respectively; laboratory location (p > 0.05) did not affect Salmonella recovery. Uniform laboratory methodology and training should be prioritized in conducting Salmonella recovery from surface water in laboratories.

A newly developed Escherichia coli isolate panel from a cross section of U.S. animal production systems reveals geographic and commodity-based differences in antibiotic resistance gene carriage.

There are limited numbers of Escherichia coli isolate panels that represent United States food animal production. The majority of existing Escherichia coli isolate panels are typically designed: (i) to optimize genetic and/or phenotypic diversity; or (ii) focus on human isolates. To address this shortfall in agriculturally-related resources, we have assembled a publicly-available isolate panel (AgEc) from the four major animal production commodities in the United States, including beef, dairy, poultry, and swine, as well as isolates from agriculturally-impacted environments, and other commodity groups. Diversity analyses by phylotyping and Pulsed-field Gel Electrophoresis revealed a highly diverse composition, with the 300 isolates clustered into 71 PFGE sub-types based upon an 80% similarity cutoff. To demonstrate the panel's utility, tetracycline and sulfonamide resistance genes were assayed, which identified 131 isolates harboring genes involved in tetracycline resistance, and 41 isolates containing sulfonamide resistance genes. There was strong overlap in the two pools of isolates, 38 of the 41 isolates harboring sulfonamide resistance genes also contained tetracycline resistance genes. Analysis of antimicrobial resistance gene patterns revealed significant differences along commodity and geographical lines. This panel therefore provides the research community an E. coli isolate panel for study of issues pertinent to U.S. food animal production.

Population Analyses Reveal Preenrichment Method and Selective Enrichment Media Affect Salmonella Serovars Detected on Broiler Carcasses.

Poultry is a major Salmonella reservoir, but conventional culture-based methods typically identify the most abundant serovars while those less abundant remain undetected. Choice of enrichment procedure also introduces bias, and for broiler carcasses, a 1-min rinse before preenrichment is insufficient to release all Salmonella present. The inability to assess serovar diversity means that serovars more often associated with human illness may be masked by more abundant Salmonella. CRISPR-SeroSeq (serotyping by sequencing clustered regularly interspaced short palindromic repeats), an amplicon-based, next-generation sequencing tool, allows detection of multiple serovars and maps the relative serovar frequencies in a sample. To address the preceding limitations, CRISPR-SeroSeq was used on broiler carcasses collected prechilled at a commercial plant. Standard carcass rinse aliquot preenrichments and whole carcass preenrichments that were enriched in Rappaport-Vassiliadis (RV) and tetrathionate (TT) broths were compared. On average, five serovars were observed per carcass, including nine on one carcass. CRISPR-SeroSeq detected serovars comprising as little as 0.005% of the population. CRISPR-SeroSeq data matched (28 of 32) standard culture analysis for abundant serovars. Salmonella serovars Kentucky, Typhimurium, and Schwarzengrund were found on each carcass. Overall, serovar diversity was higher in whole carcass preenrichments that were enriched in RV (P < 0.05). Serovar Schwarzengrund was present at higher frequencies in whole carcass preenrichments compared with rinse aliquot preenrichments (t test, P < 0.05), suggesting it adheres more strongly to the carcass. Salmonella serovar Enteritidis was enriched eightfold more in TT than in RV, and serovars Schwarzengrund and Reading were preferentially enriched in RV. Comparison of preenriched and enriched samples suggests that selective enrichment in RV or TT was inhibitory to some serovars. This article addresses limitations of Salmonella surveillance protocols and provides information related to Salmonella population dynamics.

Persistence of antibiotic resistance genes in beef cattle backgrounding environment over two years after cessation of operation.

Confined animal feeding operations can facilitate the spread of genes associated with antibiotic resistance. It is not known how cattle removal from beef cattle backgrounding operation affects the persistence of antibiotic resistance genes (ARGs) in the environment. We investigated the effect of cessation of beef cattle backgrounding operation on the persistence and distribution of ARGs in the beef cattle backgrounding environment. The study was conducted at a pasture-feedlot type beef cattle backgrounding operation which consisted of feeding and grazing areas that were separated by a fence with an access gate. Backgrounding occurred for seven years before cattle were removed from the facility. Soil samples (n = 78) from 26 georeferenced locations were collected at the baseline before cattle were removed, and then one year and two years after cattle were removed. Metagenomic DNA was extracted from the soil samples and total bacterial population (16S rRNA), total Enterococcus species and class 1 integrons (intI1), and erythromycin (ermB and ermF), sulfonamide (sul1 and sul2) and tetracycline (tetO, tetW and tetQ) resistance genes were quantified. Concentrations of total bacteria, Enterococcus spp., class 1 integrons, and ARGs were higher in the feeding area and its immediate vicinity (around the fence and the gate) followed by a gradient decline along the grazing area. Although the concentrations of total bacteria, Enterococcus spp., class 1 integrons and ARGs in the feeding area significantly decreased two years after cattle removal, their concentrations were still higher than that observed in the grazing area. Higher concentrations over two years in the feeding area when compared to the grazing area suggest a lasting effect of confined beef cattle production system on the persistence of bacteria and ARGs in the soil.

One Health and Antibiotic Resistance in Agroecosystems.

Agriculture reflects One Health principals, with the job of the farmer being to sustainably balance human, animal, and soil health. It is imperative to include an agricultural perspective when addressing antibiotic resistance (AR) from a One Health perspective, as the farmers, ranchers, and agricultural professionals have an intimate working knowledge of these complex systems, and they will be on the front lines of implementing on-farm control measures. Currently, communication across the One Health triad (humans, animals, environment) regarding agricultural AR is hindered by ambiguous language, complicated by cultural and linguistic differences that can lead to the conclusion that the other participant is not aware of the facts, or has ulterior motives. This work explores and identifies the language and vocabulary of AR in the context of supporting strategic short- and long-term problem solving in a One Health context.

Using the agricultural environment to select better surrogates for foodborne pathogens associated with fresh produce.

Despite continuing efforts to reduce foodborne pathogen contamination of fresh produce, significant outbreaks continue to occur. Identification of appropriate surrogates for foodborne pathogens facilitates relevant research to identify reservoirs and amplifiers of these contaminants in production and processing environments. Therefore, the objective of this study was to identify environmental Escherichia coli isolates from manures (poultry, swine and dairy) and surface water sources with properties similar to those of the produce associated foodborne pathogens E. coli O157:H7 and Salmonella enterica serotype Typhimurium. The most similar environmental E. coli isolates were from poultry (n=3) and surface water (n=1) sources. The best environmental E. coli surrogates had cell surface characteristics (zeta potential, hydrophobicity and exopolysaccharide composition) that were similar (i.e., within 15%) to those of S. Typhimurium and/or formed biofilms more often when grown in low nutrient media prepared from lettuce lysates (24%) than when grown on high nutrient broth (7%). The rate of attachment of environmental isolates to lettuce leaves was also similar to that of S. Typhimurium. In contrast, E. coli O157:H7, a commonly used E. coli quality control strain and swine isolates behaved similarly; all were in the lowest 10% of isolates for biofilm formation and leaf attachment. These data suggest that the environment may provide a valuable resource for selection of surrogates for foodborne pathogens.

Prevalence of mcr-1 in the Cecal Contents of Food Animals in the United States.

A survey of 2,003 cecal content samples from chickens, turkeys, cattle, and swine at slaughter facilities in the United States was conducted to estimate the prevalence of the mcr-1 gene conferring resistance to colistin in Enterobacteriaceae Two cecal samples from swine had Escherichia coli with IncI2 plasmids bearing the mcr-1 gene.

Complete Genome Sequence of a Colistin Resistance Gene (mcr-1)-Bearing Isolate of Escherichia coli from the United States.

Transmissible colistin resistance conferred by the mcr-1 gene-bearing IncI2 plasmid has been recently reported in Escherichia coli in the United States. We report here the completed genome sequence of a second E. coli strain isolated from swine in the United States that carried the mcr-1 gene on an IncI2-type plasmid.

Colistin Resistance mcr-1-Gene-Bearing Escherichia coli Strain from the United States.

Transmissible colistin resistance in the form of an mcr-1-gene-bearing plasmid has been recently reported in Enterobacteriaceae in several parts of the world. We report here the completed genome sequence of an Escherichia coli strain isolated from swine in the United States that carried the mcr-1 gene on an IncI2-type plasmid.

How Should We Be Determining Background and Baseline Antibiotic Resistance Levels in Agroecosystem Research?

Although historically, antibiotic resistance has occurred naturally in environmental bacteria, many questions remain regarding the specifics of how humans and animals contribute to the development and spread of antibiotic resistance in agroecosystems. Additional research is necessary to completely understand the potential risks to human, animal, and ecological health in systems altered by antibiotic-resistance-related contamination. At present, analyzing and interpreting the effects of human and animal inputs on antibiotic resistance in agroecosystems is difficult, since standard research terminology and protocols do not exist for studying background and baseline levels of resistance in the environment. To improve the state of science in antibiotic-resistance-related research in agroecosystems, researchers are encouraged to incorporate baseline data within the study system and background data from outside the study system to normalize the study data and determine the potential impact of antibiotic-resistance-related determinants on a specific agroecosystem. Therefore, the aims of this review were to (i) present standard definitions for commonly used terms in environmental antibiotic resistance research and (ii) illustrate the need for research standards (normalization) within and between studies of antibiotic resistance in agroecosystems. To foster synergy among antibiotic resistance researchers, a new surveillance and decision-making tool is proposed to assist researchers in determining the most relevant and important antibiotic-resistance-related targets to focus on in their given agroecosystems. Incorporation of these components within antibiotic-resistance-related studies should allow for a more comprehensive and accurate picture of the current and future states of antibiotic resistance in the environment.

Impacts of antibiotic use in agriculture: what are the benefits and risks?

Antibiotic drugs provide clear benefits for food animal health and welfare, while simultaneously providing clear risks due to enrichment of resistant microorganisms. There is no consensus, however, on how to evaluate benefits and risks of antibiotic use in agriculture, or the impact on public health. Recent soil resistome work emphasizes the importance of environmental reservoirs of antibiotic resistance (AR), and provides a starting point for distinguishing AR that can be impacted by agricultural practices from AR naturally present in a system. Manure is the primary vehicle introducing antibiotic drugs, AR bacteria and AR genes from animals into the environment. Manure management, therefore, impacts the transfer of AR from agricultural to human clinical settings via soil, water, and food. Ongoing research on the ecology of naturally occurring and anthropogenically derived AR in agroecosystems is necessary to adequately quantify the benefits and risks associated with use of antibiotics in food animals.

Impact of growth conditions on transport behavior of E. coli.

The aim of this investigation is to determine the effect that growth solution has on the cell surface properties and transport behavior of eleven Escherichia coli isolates through saturated porous media. The two growth solutions used were a standard laboratory growth medium (LB) and a dairy manure extract solution. In general, cells grown in manure extract were more hydrophobic, had a more negative zeta potential, had lower amounts of surface macromolecules, and had lower attachment efficiencies than isolates grown in LB. An inverse relationship between the natural log of zeta potential and the attachment efficiency of the isolates for the cells grown in LB media was the only statistically significant correlation observed between transport behavior and cell characteristics of the isolates. This study shows the need to consider growth conditions when studying bacteria to better mimic the environmental stresses that bacteria undergo in the natural environment. This approach could lead to a better understanding of the behavior of manure-derived bacteria in aquatic and terrestrial environments.

Escherichia coli diversity in livestock manures and agriculturally impacted stream waters.

Escherichia coli (E. coli) isolate diversity enhances the likelihood of survival, spread, and/or transmission of the organism among environments. Understanding the ecology of this important organism is requisite for development of more accurate protocols for monitoring and regulatory purposes. In this study, E. coli diversity, gene profiles and transport properties of isolates from different livestock and water sources were evaluated. Strain diversity was evaluated by BOX-PCR, phylotyping, and profiling for 15 genes associated with adhesion, toxin production, iron acquisition or capsular synthesis. Attachment efficiencies were calculated for 17 isolates following transport through saturated porous media. Richness of genotype profiles for livestock isolates was relatively low (25, 12, and 11 for swine, poultry and dairy, respectively) compared to those from stream-water (115 and 126 from dry or wet weather events, respectively). Attachment efficiencies varied by an order of magnitude (0.039-0.44) and the isolate with the highest attachment efficiency possessed the largest suite of targeted genes including those for adherence (iha, agn43, and fimH), surface exclusion (traT) and the siderophore iroN ( E.coli ). Variation in E. coli isolates based on temporal and ecological source was found to translate to equally broad ranges in transport efficiency underscoring the large degree of genotypic and phenotypic variation that exists among E. coli isolates. The impact of this diversity on genetic exchange and the concomitant effect on the organisms' fate and transport under in situ environmental conditions warrant further investigation. These factors also require careful consideration for purposes of modeling, source tracking, and risk assessment.

Evaluation of nitrogen retention and microbial populations in poultry litter treated with chemical, biological or adsorbent amendments.

Poultry litter is a valuable nutrient source for crop production. Successful management to reduce ammonia and its harmful side-effects on poultry and the environment can be aided by the use of litter amendments. In this study, three acidifiers, two biological treatments, one chemical urease inhibitor and two adsorber amendments were added to poultry litter. Chemical, physical and microbiological properties of the litters were assessed at the beginning and the end of the experiment. Application of litter amendments consistently reduced organic N loss (0-15%) as compared to unamended litter (20%). Acidifiers reduced nitrogen loss through both chemical and microbiological processes. Adsorbent amendments (water treatment residuals and chitosan) reduced nitrogen loss and concentrations of ammonia-producing bacteria and fungi. The use of efficient, cost-effective litter amendments to maximum agronomic, environmental and financial benefits is essential for the future of sustainable poultry production.

Microbial mineralization of organic nitrogen forms in poultry litters.

Ammonia volatilization from the mineralization of uric acid and urea has a major impact on the poultry industry and the environment. Dry acids are commonly used to reduce ammonia emissions from poultry houses; however, little is known about how acidification affects the litter biologically. The goal of this laboratory incubation was to compare the microbiological and physiochemical effects of dry acid amendments (Al+Clear, Poultry Litter Treatment, Poultry Guard) on poultry litter to an untreated control litter and to specifically correlate uric acid and urea contents of these litters to the microbes responsible for their mineralization. Although all three acidifiers eventually produced similar effects within the litter, there was at least a 2-wk delay in the microbiological responses using Poultry Litter Treatment. Acidification of the poultry litter resulted in >3 log increases in total fungal concentrations, with both uricolytic (uric acid degrading) and ureolytic (urea degrading) fungi increasing by >2 logs within the first 2 to 4 wk of the incubation. Conversely, total, uricolytic, and ureolytic bacterial populations all significantly declined during this same time period. While uric acid and urea mineralization occurred within the first 2 wk in the untreated control litter, acidification resulted in delayed mineralization events for both uric acid and urea (2 and 4 wk delay, respectively) once fungal cell concentrations exceeded a threshold level. Therefore, fungi, and especially uricolytic fungi, appear to have a vital role in the mineralization of organic N in low-pH, high-N environments, and the activity of these fungi should be considered in best management practices to reduce ammonia volatilization from acidified poultry litter.

Correlating transport behavior with cell properties for eight porcine Escherichia coli isolates.

In this study we investigate how growth stage and depositional environment affect variability of cell properties and transport behavior of eight porcine E. coli isolates. We compared the surface properties for cells harvested during exponential and stationary growth phase and their transport behavior through columns packed with either uncoated or Fe-coated quartz sand. We then investigated correlations between measured cell properties and fitted bacterial attachment efficiencies. For both growth stages we found that bacterial attachment efficiencies in the uncoated quartz sand varied among the eight different isolates by over an order of magnitude whereas attachment efficiencies in the Fe-coated sands varied by a factor of less than two. With the exception of one isolate, growth condition had minimal impact on attachment efficiencies to the uncoated sands. A strong and statistically significant inverse relationship was observed between bacterial attachment efficiencies in the uncoated quartz sand columns and log-transformed zeta potential, whereas a mild yet statistically significant relationship between bacterial attachment efficiencies in the Fe-coated sands and cell width was observed. For the experimental conditions used in our study, we found that variability in E. coli transport was more dependent on the depositional environment than on growth conditions.

Survival of Mycobacterium avium subsp. paratuberculosis in biofilms on livestock watering trough materials.

Mycobacterium avium subsp. paratuberculosis (Map) is the causative agent of Johne's disease, a chronic enteric infection that affects ruminants. Despite the ubiquitous occurrence of Mycobacterium sp. in nature and the fact that Johne's disease has been reported worldwide, little research has been done to assess its survival in agricultural environments. The goal of this 365-day study was to evaluate the ability of Map to persist in mixed-community biofilms on materials commonly used to construct livestock watering troughs. Map was inoculated into 32l of trough water containing either concrete, plastic, galvanized or stainless steel trough materials. The concentration of Map was determined by using quantitative, real-time PCR to target the IS900 sequence in DNA extracts. High concentrations of Map were detected on all trough materials after 3 days (around 1 x 10(5)cells cm(-2)). Based on the best-fit slopes, the time required for a 99% reduction (t(99)) in biofilm-associated Map cells was 144 and 115 days for plastic and stainless steel trough materials, respectively. Map concentrations did not decrease on concrete and galvanized steel trough materials. These results suggest that Map survives well in biofilms present on livestock watering trough materials. To inhibit spread of this organism and exposure of susceptible animals to Map on infected farms, best management practices aimed at maintaining biofilm-free trough surfaces should be included in any Johne's control plan.

Spatial and temporal changes in the microbial community in an anaerobic swine waste treatment lagoon.

Microorganisms are central to both the beneficial (organic degradation, nutrient removal, biogas production) and detrimental (odor production, pathogen contamination) effects of swine waste storage systems. In this study, both quantitative (real-time polymerase chain reaction) and qualitative (denaturing gradient gel electrophoresis, cloning, sequence analysis) molecular analyses were used to track spatial and temporal changes in the microbial community of swine slurry from a 0.4 ha anaerobic lagoon. The lagoon, located in a region of western Kentucky which has a humid, subtropical environment, was sampled on a monthly basis (n=10) over a period of one year at four different depths (top, 51 cm from the top, 152 cm from the top, and bottom >198 cm). The concentration and diversity of Bacteroides sp. was seasonal (up to 90% decrease between March and June). Hespellia sp. and other clostridial species, on the other hand, were endemic in the slurry (concentrations up to 1.0x10(7) cells mL(-1) slurry) regardless of time of the year or lagoon depth. Results suggest that there were seasonal effects on the microbial community in the swine lagoon, while the effect of depth was not as pronounced. Seasonal changes in the microbial community in stored wastes may be (directly or indirectly) correlated with changes in malodor emissions from lagoons.

Comparative quantification of Campylobacter jejuni from environmental samples using traditional and molecular biological techniques.

Campylobacter jejuni is one of the most common causes of gastroenteritis in the world. Given the potential risks to human, animal, and environmental health, the development and optimization of methods to quantify this important pathogen in environmental samples is essential. Two of the most commonly used methods for quantifying C. jejuni are selective plate counting and quantitative real-time PCR (qPCR). Unfortunately, little comparative research has been performed to evaluate the accuracy of these methods for quantification of C. jejuni in aqueous and solid matricies. In this study, the limit of detection and the level of resolution obtained using these 2 methods was evaluated for C. jejuni and compared with that of the common indicator organism Escherichia coli. The use of selective plate count media for quantification of C. jejuni resulted in a 0.7-1.2 log underestimation of cell concentrations, compared with qPCR in both water and column leachate samples, whereas E. coli concentrations were found to be similar with either technique. For C. jejuni, only the qPCR assay accurately measured 2-fold changes in cell concentrations in water samples, whereas concentrations of E. coli were accurately measured regardless of method. Based on these data, qPCR assays were found to be more accurate than selective plate counts for quantification of C. jejuni from environmental samples.