Continuous Gradient Temperature Raman Spectroscopy of Oleic and Linoleic Acids from -100 to 50 °C.

We analyzed the unsaturated fatty acids oleic (OA, 18:1n-9) and linoleic (LA, 18:2n-3), and a 3:1 LA:OA mixture from -100 to 50 °C with continuous gradient temperature Raman spectroscopy (GTRS). The 20 Mb three-dimensional data arrays with 0.2 °C increments and first/second derivatives allowed rapid, complete assignment of solid, liquid, and transition state vibrational modes. For OA, large spectral and line width changes occurred in the solid state γ to α transition near -4 °C, and the melt (13 °C) over a range of only 1 °C. For LA, major intensity reductions from 200 to 1750 cm-1 and some peak shifts marked one solid state phase transition at -50 °C. A second solid state transition (-33 °C) had minor spectral changes. Large spectral and line width changes occurred at the melt transition (-7 °C) over a narrow temperature range. For both molecules, melting initiates at the diene structure, then progresses towards the ends. In the 3:1 LA:OA mixture, some less intense and lower frequencies present in the individual lipids are weaker or absent. For example, modes assignable to C8 rocking, C9H-C10H wagging, C10H-C11H wagging, and CH3 rocking are present in OA but absent in LA:OA. Our data quantify the concept of lipid premelting and identify the flexible structures within OA and LA, which have characteristic vibrational modes beginning at cryogenic temperatures.

Continuous gradient temperature Raman spectroscopy of N-6DPA and DHA from -100 to 20°C.

One of the great unanswered questions with respect to biological science in general is the absolute necessity of docosahexaenoic acid (DHA, 22:6n-3) in fast signal processing tissues. N-6 docosapentaenoic acid (n-6DPA, 22:5n-6), with just one less double bond, group, is fairly abundant in terrestrial food chains yet cannot substitute for DHA. Gradient temperature Raman spectroscopy (GTRS) applies the temperature gradients utilized in differential scanning calorimetry (DSC) to Raman spectroscopy, providing a straightforward technique to identify molecular rearrangements that occur near and at phase transitions. Herein we apply GTRS and DSC to n-6DPA and DHA from -100 to 20°C. 20Mb three-dimensional data arrays with 0.2°C increments and first/second derivatives allowed complete assignment of solid, liquid and transition state vibrational modes, including low intensity/frequency vibrations that cannot be readily analyzed with conventional Raman. N-6DPA and DHA show significant spectral changes with premelting (-33 and -60°C, respectively) and melting (-27 and -44°C, respectively). The CH2(HCCH)CH2 moieties are not identical in the second half of the DHA and DPA structures. DPA has bending (1450cm-1) over almost the entire temperature range. In contrast, DHA contains major CH2 twisting (1265cm-1) with no noticeable CH2 bending, consistent with a flat helical structure with a small pitch. Further modeling of neuronal membrane phospholipids must take into account torsion present in the DHA structure, which essential in determining whether the lipid chain is configured more parallel or perpendicular to the hydrophilic head group.

Modeling fate and transport of fecally-derived microorganisms at the watershed scale: State of the science and future opportunities.

Natural waters serve as habitat for a wide range of microorganisms, a proportion of which may be derived from fecal material. A number of watershed models have been developed to understand and predict the fate and transport of fecal microorganisms within complex watersheds, as well as to determine whether microbial water quality standards can be satisfied under site-specific meteorological and/or management conditions. The aim of this review is to highlight and critically evaluate developments in the modeling of microbial water quality of surface waters over the last 10 years and to discuss the future of model development and application at the watershed scale, with a particular focus on fecal indicator organisms (FIOs). In doing so, an agenda of research opportunities is identified to help deliver improvements in the modeling of microbial water quality draining through complex landscape systems. This comprehensive review therefore provides a timely steer to help strengthen future modeling capability of FIOs in surface water environments and provides a useful resource to complement the development of risk management strategies to reduce microbial impairment of freshwater sources.

Genome Sequence of Penicillium solitum RS1, Which Causes Postharvest Apple Decay.

Penicillium species cause postharvest decay, commonly known as blue mold, in pome fruits, such as apples and pears. To devise novel strategies to prevent and reduce economic losses during storage, the genome sequence of Penicillium solitum RS1 is reported here for the first time.

Aggregative adherence fimbriae I (AAF/I) mediate colonization of fresh produce and abiotic surface by Shiga toxigenic enteroaggregative Escherichia coli O104:H4.

The Shiga toxigenic Escherichia coli O104:H4 isolated during the 2011 European outbreak expresses Shiga toxin 2a and possess virulence genes associated with the enteroaggregative E. coli (EAEC) pathotype. It produces plasmid encoded aggregative adherence fimbriae I (AAF/I) which mediate cell aggregation and biofilm formation in human intestine and promote Shiga-toxin adsorption, but it is not clear whether the AAF/I fimbriae are involved in the colonization and biofilm formation on food and environmental matrices such as the surface of fresh produce. We deleted the gene encoding for the AAF/I fimbriae main subunit (AggA) from an outbreak associated E. coli O104:H4 strain, and evaluated the role of AAF/I fimbriae in the adherence and colonization of E. coli O104:H4 to spinach and abiotic surfaces. The deletion of aggA did not affect the adherence of E. coli O104:H4 to these surfaces. However, it severely diminished the colonization and biofilm formation of E. coli O104:H4 on these surfaces. Strong aggregation and biofilm formation on spinach and abiotic surfaces were observed with the wild type strain but not the isogenic aggA deletion mutant, suggesting that AAF/I fimbriae play a crucial role in persistence of O104:H4 cells outside of the intestines of host species, such as on the surface of fresh produce.

Rainfall intensity effects on removal of fecal indicator bacteria from solid dairy manure applied over grass-covered soil.

The rainfall-induced release of pathogens and microbial indicators from land-applied manure and their subsequent removal with runoff and infiltration precedes the impairment of surface and groundwater resources. It has been assumed that rainfall intensity and changes in intensity during rainfall do not affect microbial removal when expressed as a function of rainfall depth. The objective of this work was to test this assumption by measuring the removal of Escherichia coli, enterococci, total coliforms, and chloride ion from dairy manure applied in soil boxes containing fescue, under 3, 6, and 9cmh(-1) of rainfall. Runoff and leachate were collected at increasing time intervals during rainfall, and post-rainfall soil samples were taken at 0, 2, 5, and 10cm depths. Three kinetic-based models were fitted to the data on manure-constituent removal with runoff. Rainfall intensity appeared to have positive effects on rainwater partitioning to runoff, and removal with this effluent type occurred in two stages. While rainfall intensity generally did not impact the parameters of runoff-removal models, it had significant, inverse effects on the numbers of bacteria remaining in soil after rainfall. As rainfall intensity and soil profile depth increased, the numbers of indicator bacteria tended to decrease. The cumulative removal of E. coli from manure exceeded that of enterococci, especially in the form of removal with infiltration. This work may be used to improve the parameterization of models for bacteria removal with runoff and to advance estimations of depths of bacteria removal with infiltration, both of which are critical to risk assessment of microbial fate and transport in the environment.

Irrigation waters and pipe-based biofilms as sources for antibiotic-resistant bacteria.

The presence of antibiotic-resistant bacteria in environmental surface waters has gained recent attention. Wastewater and drinking water distribution systems are known to disseminate antibiotic-resistant bacteria, with the biofilms that form on the inner-surfaces of the pipeline as a hot spot for proliferation and gene exchange. Pipe-based irrigation systems that utilize surface waters may contribute to the dissemination of antibiotic-resistant bacteria in a similar manner. We conducted irrigation events at a perennial stream on a weekly basis for 1 month, and the concentrations of total heterotrophic bacteria, total coliforms, and fecal coliforms, as well as the concentrations of these bacterial groups that were resistant to ampicillin and tetracycline, were monitored at the intake water. Prior to each of the latter three events, residual pipe water was sampled and 6-in. sections of pipeline (coupons) were detached from the system, and biofilm from the inner-wall was removed and analyzed for total protein content and the above bacteria. Isolates of biofilm-associated bacteria were screened for resistance to a panel of seven antibiotics, representing five antibiotic classes. All of the monitored bacteria grew substantially in the residual water between irrigation events, and the biomass of the biofilm steadily increased from week to week. The percentages of biofilm-associated isolates that were resistant to antibiotics on the panel sometimes increased between events. Multiple-drug resistance was observed for all bacterial groups, most often for fecal coliforms, and the distributions of the numbers of antibiotics that the total coliforms and fecal coliforms were resistant to were subject to change from week to week. Results from this study highlight irrigation waters as a potential source for antibiotic-resistant bacteria, which can subsequently become incorporated into and proliferate within irrigation pipe-based biofilms.

Release and Removal of Microorganisms from Land-Deposited Animal Waste and Animal Manures: A Review of Data and Models.

Microbial pathogens present a leading cause of impairment to rivers, bays, and estuaries in the United States, and agriculture is often viewed as the major contributor to such contamination. Microbial indicators and pathogens are released from land-applied animal manure during precipitation and irrigation events and are carried in overland and subsurface flow that can reach and contaminate surface waters and ground water used for human recreation and food production. Simulating the release and removal of manure-borne pathogens and indicator microorganisms is an essential component of microbial fate and transport modeling regarding food safety and water quality. Although microbial release controls the quantities of available pathogens and indicators that move toward human exposure, a literature review on this topic is lacking. This critical review on microbial release and subsequent removal from manure and animal waste application areas includes sections on microbial release processes and release-affecting factors, such as differences in the release of microbial species or groups; bacterial attachment in turbid suspensions; animal source; animal waste composition; waste aging; manure application method; manure treatment effect; rainfall intensity, duration, and energy; rainfall recurrence; dissolved salts and temperature; vegetation and soil; and spatial and temporal scale. Differences in microbial release from liquid and solid manures are illustrated, and the influential processes are discussed. Models used for simulating release and removal and current knowledge gaps are presented, and avenues for future research are suggested.

Solid Manure As a Source of Fecal Indicator Microorganisms: Release under Simulated Rainfall.

Understanding and quantifying microbial release from manure is a precondition to estimation and management of microbial water quality. The objectives of this work were to determine the effects of rainfall intensity and surface slope on the release of Escherichia coli, enterococci, total coliforms, and dissolved chloride from solid dairy manure and to assess the performance of the one-parametric exponential model and the two-parametric Bradford-Schijven model when simulating the observed release. A controlled-intensity rainfall simulator induced 1 h of release in runoff/leachate partitioning boxes at three rainfall intensities (30, 60, and 90 mm h(-1)) and two surface slopes (5% and 20%). Bacterial concentrations in initial release were more than 1 order of magnitude lower than their starting concentrations in manure. As bacteria were released, they were partitioned into runoff and leachate at similar concentrations, but in different volumes, depending on slope. Bacterial release occurred in two stages that corresponded to mechanisms associated with release of manure liquid- and solid-phases. Parameters of the two models fitted to the bacterial release dependencies on rainfall depth were not significantly affected by rainfall intensity or slope. Based on model performance tests, the Bradford-Schijven model is recommended for simulating bacterial release from solid manure.

Continuous temperature-dependent Raman spectroscopy of melamine and structural analog detection in milk powder.

Hyperspectral Raman imaging has the potential for rapid screening of solid-phase samples for potential adulterants. We can improve mixture analysis algorithms by defining a temperature range in which the contaminant spectrum changes dramatically and uniquely compared with unadulterated material. Raman spectra were acquired for urea, biuret, cyanuric acid, and melamine (pure and at 1% in dried milk powder) from 50 to 310 °C with a gradient of 1 °C min(-1). Adulterants were clearly indentified in the milk powder. Specific frequencies that were mainly associated with ring breathing, stretching, and in-plane deformation shifted with respect to temperature up to 12 cm(-1) in all four molecules. Specific frequencies significantly increased/decreased in intensity within narrow temperature ranges independent of whether the amine was mixed in milk. Correlation of Raman and differential scanning calorimetry data identified structural components and vibrational modes, which concur with or trigger phase transitions.

Effects of environmental parameters on the dual-species biofilms formed by Escherichia coli O157:H7 and Ralstonia insidiosa, a strong biofilm producer isolated from a fresh-cut produce processing plant.

Biofilm-forming bacteria resident to food processing facilities are a food safety concern due to the potential of biofilms to harbor foodborne bacterial pathogens. When cultured together, Ralstonia insidiosa, a strong biofilm former frequently isolated from produce processing environments, has been shown to promote the incorporation of Escherichia coli O157:H7 into dual-species biofilms. In this study, interactions between E. coli O157:H7 and R. insidiosa were examined under different incubating conditions. Under static culture conditions, the incorporation of E. coli O157:H7 into biofilms with R. insidiosa was not significantly affected by either low incubating temperature (10°C) or by limited nutrient availability. Greater enhancement of E. coli O157:H7 incorporation in dual-species biofilms was observed by using a continuous culture system with limited nutrient availability. Under the continuous culture conditions used in this study, E coli O157:H7 cells showed a strong tendency of colocalizing with R. insidiosa on a glass surface at the early stage of biofilm formation. As the biofilms matured, E coli O157:H7 cells were mostly found at the bottom layer of the dual-species biofilms, suggesting an effective protection by R. insidiosa in the mature biofilms.

Dual-species biofilm formation by Escherichia coli O157:H7 and environmental bacteria isolated from fresh-cut processing facilities.

Biofilm formation is a mechanism adapted by many microorganisms that enhances the survival in stressful environments. In food processing facilities, foodborne bacterial pathogens, which many are poor biofilm formers, could potentially take advantage of this protective mechanism by interacting with other strong biofilm producers. The objective of this study was to determine the influence of bacteria native to fresh produce processing environments on the incorporation of Escherichia coli O157:H7 in biofilms. Bacteria strains representing 13 Gram-negative species isolated from two fresh produce processing facilities in a previous study were tested for forming dual-species biofilms with E. coli O157:H7. Strong biofilm producing strains of Burkholderia caryophylli and Ralstonia insidiosa exhibited 180% and 63% increase in biofilm biomass, and significant thickening of the biofilms (B. caryophylli not tested), when co-cultured with E. coli O157:H7. E. coli O157:H7 populations increased by approximately 1 log in dual-species biofilms formed with B. caryophylli or R. insidiosa. While only a subset of environmental isolates with strong biofilm formation abilities increased the presence of E. coli O157:H7 in biofilms, all tested E. coli O157:H7 exhibited higher incorporation in dual-species biofilms with R. insidiosa. These observations support the notion that E. coli O157:H7 and specific strong biofilm producing bacteria interact synergistically in biofilm formation, and suggest a route for increased survival potential of E. coli O157:H7 in fresh produce processing environments.

Native microflora in fresh-cut produce processing plants and their potentials for biofilm formation.

Representative food contact and nonfood contact surfaces in two mid-sized, fresh-cut processing facilities were sampled for microbiological analyses after routine daily sanitization. Mesophilic and psychrotrophic bacteria on the sampled surfaces were isolated by plating on nonselective bacterial media. Alternatively, bacteria were isolated after an incubation period that allowed the formation of heterogeneous biofilms on stainless steel beads. Of over 1,000 tested isolates, most were capable of forming biofilms, with approximately 30 % being strong or moderate biofilm formers. Selected isolates (117) were subjected to species identification by using the Biolog Gen III microbial identification system. They distributed among 23 genera, which included soil bacteria, plant-related bacteria, coliforms, and opportunistic plant- or human-pathogenic bacteria. The most commonly identified bacteria species were Pseudomonas fluorescens, Rahnella aquatilis, and Ralstonia insidiosa. The high prevalence of R. insidiosa, a strong biofilm former, and P. fluorescens, a moderate biofilm former, suggests that they were established residents in the sampled plants. These results suggest that native microflora capable of forming biofilms are widely distributed in fresh-produce processing environments.

Using the Q10 model to simulate E. coli survival in cowpats on grazing lands.

Microbiological quality of surface waters can be affected by microbial load in runoff from grazing lands. This effect, with other factors, depends on the survival of microorganisms in animal waste deposited on pastures. Since temperature is a leading environmental parameter affecting survival, it indirectly impacts water microbial quality. The Q10 model is widely used to predict the effect of temperature on rates of biological processes, including survival. Objectives of this work were to (i) evaluate the applicability of the Q10 model to Escherichia coli inactivation in bovine manure deposited on grazing land (i.e., cowpats) and (ii) identify explanatory variables for the previously reported E. coli survival dynamics in cowpats. Data utilized in this study include published results on E. coli concentrations in natural and repacked cowpats from research conducted the U.S. (Virginia and Maryland), New Zealand, and the United Kingdom. Inspection of the datasets led to conceptualizing E. coli survival (in cowpats) as a two-stage process, in which the initial stage was due to growth, inactivation or stationary state of the population and the second stage was the approximately first-order inactivation. Applying the Q10 model to these datasets showed a remarkable similarity in inactivation rates, using the thermal time. The reference inactivation rate constant of 0.042 (thermal days)(-1) at 20 °C gave a good approximation (R(2)=0.88) of all inactivation stage data with Q10=1.48. The reference inactivation rate constants in individual studies were no different from the one obtained by pooling all data (P<0.05). The rate of logarithm of the E. coli concentration change during the first stage depended on temperature. Duration of the first stage, prior to the first-order inactivation stage and the initial concentration of E. coli in cowpats, could not be predicted from available data. Diet and age are probable factors affecting these two parameters however, until their environmental and management predictors are known, microbial water quality modeling must treat them as a stochastic source of uncertainty in simulation results.

Potential pollutant sources in a Choptank River (USA) subwatershed and the influence of land use and watershed characteristics.

Row-crop and poultry production have been implicated as sources of water pollution along the Choptank River, an estuary and tributary of the Chesapeake Bay. This study examined the effects of land use, subwatershed characteristics, and climatic conditions on the water quality parameters of a subwatershed in the Choptank River watershed. The catchments within the subwatershed were defined using advanced remotely-sensed data and current geographic information system processing techniques. Water and sediment samples were collected in May-October 2009 and April-June 2010 under mostly baseflow conditions and analyzed for select bacteria, nitrate-N, ammonium-N, total arsenic, total phosphorus (TP), orthophosphate (ortho-P), and particle-phase phosphorus (PP); n=96 for all analytes except for arsenic, n=136, and for bacteria, n=89 (aqueous) and 62 (sediment). Detections of Enterococci and Escherichia coli concentrations were ubiquitous in this subwatershed and showed no correlation to location or land use, however larger bacterial counts were observed shortly after precipitation. Nitrate-N concentrations were not correlated with agricultural lands, which may reflect the small change in percent agriculture and/or the similarity of agronomic practices and crops produced between catchments. Concentration data suggested that ammonia emission and possible deposition to surface waters occurred and that these processes may be influenced by local agronomic practices and climatic conditions. The negative correlation of PP and arsenic concentrations with percent forest was explained by the stronger signal of the head waters and overland flow of particulate phase analytes versus dissolved phase inputs from groundwater. Service roadways at some poultry production facilities were found to redirect runoff from the facilities to neighboring catchment areas, which affected water quality parameters. Results suggest that in this subwatershed, catchments with poultry production facilities are possible sources for arsenic and PP as compared to catchment areas where these facilities were not present.

Detection of E. coli O157:H7 by immunomagnetic separation coupled with fluorescence immunoassay.

Conventional culture-based methods for detection of E. coli O157:H7 in foods and water sources are time-consuming, and results can be ambiguous, requiring further confirmation by biochemical testing and PCR. A rapid immunoassay prior to cultivation to identify presumptive positive sample would save considerable time and resources. Immunomagnetic separation (IMS) techniques are routinely used for isolation of E. coli O157:H7 from enriched food and water samples, typically in conjunction with cultural detection followed by biochemical and serological confirmation. In this study, we developed a new method that combines IMS with fluorescence immunoassay, termed immunomagnetic fluorescence assay (IMFA), for the detection of E. coli O157:H7. E. coli O157:H7 cells were first captured by anti-O157 antibody-coated magnetic beads and then recognized by a fluorescent detector antibody, forming an immunosandwich complex. This complex was subsequently dissociated for measurement of fluorescence intensity with Signalyte™-II spectrofluorometer. Experiments were conducted to evaluate both linearity and sensitivity of the assay. Capture efficiencies were greater than 98%, as determined by cultural plating and quantitative real-time PCR, when cell concentrations were <10(5) cells/mL. Capture efficiency decreased at higher cell concentrations, due to the limitation of bead binding capacity. At lower cell concentrations (10-10(4) cells/mL), the fluorescence intensity of dissociated Cy5 solution was highly correlated with E. coli 157:H7 cell concentrations. The detection limit was 10 CFU per mL of water. The assay can be completed in less than 3 h since enrichment is not required, as compared to existing techniques that typically require a 24 h incubation for pre-enrichment, followed by confirmatory tests.

Relationship between eae and stx virulence genes and Escherichia coli in an agricultural watershed: implications for irrigation water standards and leafy green commodities.

The California Leafy Greens Marketing Agreement (LGMA) was adopted in an effort to minimize the risk of contamination of leafy greens with enteric pathogens from a variety of sources, including ground and surface irrigation waters. The LGMA contains standards similar to those established for recreational waters, based on Escherichia coli concentrations. However, no correlation between E. coli and any specific waterborne pathogen(s) has been reported. We conducted this monitoring study in an agricultural watershed to (i) evaluate spatial and temporal fluctuations in E. coli populations and virulence genes associated with pathogenic E. coli and (ii) investigate whether a relationship could be established between E. coli and virulence genes. The virulence genes targeted for analysis were the eae and stx genes, encoding for intimin and Shiga-like toxins, respectively; they were detected with PCR methods. E. coli concentrations and eae and stx prevalence varied both spatially and temporally. In general, both were higher in agricultural than in forested areas and were higher in the summer and fall seasons than in winter. The eae and stx genes were prevalent throughout the watershed. However, in the absence of actual isolates, no conclusions could be drawn regarding the prevalence of specific pathogenic E. coli. No correlation was observed between E. coli concentrations and virulence genes; lower E. coli concentrations were not necessarily associated with decreased prevalence of eae and stx genes. These results suggest that the LGMA standards might not adequately address the issue of waterborne contamination, and that alternative criteria might be required.

Integrating waveguide biosensor.

The Integrating Waveguide Biosensor was developed for rapid and sensitive detection of bacterial cells, spores, and toxins. A sandwich format of immunoassay was employed using Salmonella as model. The analyte was immunocaptured on the inner surface of the waveguide and then detected by the antibody conjugated with fluorescent dye. The waveguide was illuminated by an excitation light at a 90 degrees angle. The emitted light from fluorescent labels on the surface of the waveguide was efficiently collected and channeled to a detector at the end of the waveguide, while minimizing interference from the excitation light. Utilizing fluorescent dye Cy5, a 635-nm diode laser for excitation, and a photomultiplier tube detector, the Integrating Waveguide Sensor System was able to detect approximately ten captured cells of Salmonella.

Size distributions of manure particles released under simulated rainfall.

Manure and animal waste deposited on cropland and grazing lands serve as a source of microorganisms, some of which may be pathogenic. These microorganisms are released along with particles of dissolved manure during rainfall events. Relatively little if anything is known about the amounts and sizes of manure particles released during rainfall, that subsequently may serve as carriers, abode, and nutritional source for microorganisms. The objective of this work was to obtain and present the first experimental data on sizes of bovine manure particles released to runoff during simulated rainfall and leached through soil during subsequent infiltration. Experiments were conducted using 200 cm long boxes containing turfgrass soil sod; the boxes were designed so that rates of manure dissolution and subsequent infiltration and runoff could be monitored independently. Dairy manure was applied on the upper portion of boxes. Simulated rainfall (ca. 32.4 mm h(-1)) was applied for 90 min on boxes with stands of either live or dead grass. Electrical conductivity, turbidity, and particle size distributions obtained from laser diffractometry were determined in manure runoff and soil leachate samples. Turbidity of leachates and manure runoff samples decreased exponentially. Turbidity of manure runoff samples was on average 20% less than turbidity of soil leachate samples. Turbidity of leachate samples from boxes with dead grass was on average 30% less than from boxes with live grass. Particle size distributions in manure runoff and leachate suspensions remained remarkably stable after 15 min of runoff initiation, although the turbidity continued to decrease. Particles had the median diameter of 3.8 microm, and 90% of particles were between 0.6 and 17.8 microm. The particle size distributions were not affected by the grass status. Because manure particles are known to affect transport and retention of microbial pathogens in soil, more information needs to be collected about the concurrent release of pathogens and manure particles during rainfall events.

A multiple protocol to improve diagnosis and isolation of Shiga toxin-producing Escherichia coli from human stool specimens.

Many infections caused by Shiga toxin-producing Escherichia coli (STEC) are undiagnosed, particularly non-O157 STEC. We evaluated the use of a multiple protocol approach to improve diagnosis, isolation, and characterization of STEC strains. Among 18 presumptive STEC-positive stool samples received by the INOVA Fairfax Hospital, Falls Church, VA, in 2006, 16 were Shiga toxin positive. From these 16 stool samples, 8 O157:H7 and 5 non-O157 STEC were isolated by plating onto sorbitol MacConkey (SMAC) agar. The remaining 5 stool samples that did not yield colonies on SMAC agar plates were enriched. All enriched samples were Shiga toxin positive, and 2 O157:H7 and 1 non-O157 STEC were subsequently isolated. The 2 remaining enriched samples did not yield isolates; however, based on polymerase chain reaction (PCR) analysis, both samples contained STEC genes. Based on PCR analysis of non-O157 strains, 3 strain types were identified. Samples from 3 patients, received within 2 days of one another, had a similar gene profile-eae and stx(1) negative and stx(2) positive-suggesting that these patients were likely infected with the same strain. Our results indicate that a multiple protocol approach is necessary to reliably diagnose and isolate STEC strains, and that PCR profiling of strains could allow for more rapid identification of outbreaks.