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.

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.

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.

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.

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.

Development of a rapid and sensitive immunoassay for detection and subsequent recovery of Bacillus anthracis spores in environmental samples.

Bacillus anthracis is considered a major threat as an agent of bioterrorism. B. anthracis spores are readily dispersed as aerosols, are very persistent, and are resistant to normal disinfection treatments. Immunoassays have been developed to rapidly detect B. anthracis spores at high concentrations. However, detection of B. anthracis spores at lower concentrations is problematic due to the fact that closely related Bacillus species (e.g., B. thuringiensis) can cross-react with anti-B. anthracis antibodies, resulting in false positive detections. Subsequent polymerase chain reaction (PCR) analysis is required to differentiate virulent strains. We report here on a protocol for the rapid, sensitive detection of B. anthracis spore using the Integrating Waveguide Biosensor followed by a method for the rapid release and germination of immunocaptured spores. A detection limit of ca. 10(3) spores was achieved by incubating spores simultaneously with capture and detection antibodies ("liquid-phase" assay) prior to capture on capillary tubes/waveguides. Subsequent incubation with BHI broth directly in capillary tubes allowed for rapid germination, outgrowth, and release of spores, resulting in vegetative cells for PCR analysis.

Impact of microbial diversity on rapid detection of enterohemorrhagic Escherichia coli in surface waters.

Enterohemorrhagic Escherichia coli (EHEC) are a physiologically, immunologically and genetically diverse collection of strains that pose a serious water-borne threat to human health. Consequently, immunological and PCR assays have been developed for the rapid, sensitive detection of presumptive EHEC. However, the ability of these assays to consistently detect presumptive EHEC while excluding closely related non-EHEC strains has not been documented. We conducted a 30-month monitoring study of a major metropolitan watershed. Surface water samples were analyzed using an immunological assay for E. coli O157 (the predominant strain worldwide) and a multiplex PCR assay for the virulence genes stx(1), stx(2) and eae. The mean frequency of water samples positive for the presence of E. coli O157, stx(1) or stx(2) genes, or the eae gene was 50%, 26% and 96%, respectively. Quantitative analysis of selected enriched water samples indicated that even in samples positive for E. coli O157 cells, stx(1)/stx(2) genes, and the eae gene, the concentrations were rarely comparable. Seventeen E. coli O157 strains were isolated, however, none were EHEC. These data indicate the presence of multiple strains similar to EHEC but less pathogenic. These findings have important ramifications for the rapid detection of presumptive EHEC; namely, that current immunological or PCR assays cannot reliably identify water-borne EHEC strains.

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.

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.

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.

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.

Detection of water-borne E. coli O157 using the integrating waveguide biosensor.

Escherichia coli O157:H7, the most common serotype of enterohemorrhagic E. coli (EHEC), is responsible for numerous food-borne and water-borne infections worldwide. An integrating waveguide biosensor is described for the detection of water-borne E. coli O157, based on a fluorescent sandwich immunoassay performed inside a glass capillary waveguide. The genomic DNA of captured E. coli O157 cells was extracted and quantitative real-time PCR subsequently performed to assess biosensor-capture efficiency. In vitro microbial growth in capillary waveguide is also documented. The biosensor allows for quantitative detection of as few as 10 cells per capillary (0.075 ml volume) and can be used in conjunction with cell amplification, PCR and microarray technologies to positively identify a pathogen.

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.

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.

Titanocene(III)-promoted Reformatsky additions.

[reaction: see text] A novel method for the promotion of Reformatsky-like reactions is presented. The technique employs titanocene(III) chloride as a mild and homogeneous single-electron reductant. The reactions are rapid, operationally simple, and compatible with a wide range of functionalities. These additions are also anti diastereoselective.

A handheld real time thermal cycler for bacterial pathogen detection.

The handheld advanced nucleic acid analyzer (HANAA) is a portable real time thermal cycler unit that weighs under 1 kg and uses silicon and platinum-based thermalcycler units to conduct rapid heating and cooling of plastic reaction tubes. Two light emitting diodes (LED) provide greater than 1 mW of electrical power at wavelengths of 490 nm (blue) and 525 nm (green), allowing detection of the dyes FAM and JOE/TAMRA. Results are displayed in real time as bar graphs, and up to three, 4-sample assays can be run on the charge of the 12 V portable battery pack. The HANAA was evaluated for detection of defined Escherichia coli strains, and wild-type colonies isolated from stream water, using PCR for the lac Z and Tir genes. PCR reactions using SYBR Green dye allowed detection of E. coli ATCC 11775 and E. coli O157:H7 cells in under 30 min of assay time; however, background fluorescence associated with dye binding to nonspecific PCR products was present. DNA extracted from three isolates of Bacillus anthracis Ames, linked to a bioterrorism incident in Washington DC in October 2001, were also successfully tested on the HANAA using primers for the vrrA and capA genes. Positive results were observed at 32 and 22 min of assay time, respectively. A TaqMan probe specific to the aroQ gene of Erwinia herbicola was tested on the HANAA and when 500 cells were used as template, positive results were observed after only 7 min of assay time. Background fluorescence associated with the use of the probe was negligible. The HANAA is unique in offering real time PCR in a handheld format suitable for field use; a commercial version of the instrument, offering six reaction chambers, is available as of Fall 2002.

Comparison of immunofluorescence assay and immunomagnetic electrochemiluminescence in detection of Cryptosporidium parvum oocysts in karst water samples.

Immunofluorescence assay (IFA) and immunomagnetic electrochemiluminescence (IM-ECL) were used for comparison of the percent recovery of Cryptosporidium parvum in environmental water samples obtained from a spring draining a karst basin. The monoclonal antibodies to C. parvum, isotype IgG3 were used for optimization of the IM-ECL protocol. The combination of biotinylated and TAG-labeled anti-C. parvum antibodies with the streptavidin beads gave a linear regression slope for log ECL vs. log fresh oocysts of 0.79 (from 5 to 5,000 oocysts), which indicates a constant ECL signal per oocyst. Standard curves gave a dynamic range of 5 to 5,000 oocysts/ml (fresh) and 10 to 100,000 cells/ml (4-month-old oocysts) with the maximum limit of linear detection higher than 100,000. The linear slope of 4-month-old oocysts decreased to 0.62, which indicates that ECL signal is a function of oocyst age. The experiment associated with bead storage time shows that even after 4 months of storage of the biotinylated antibodies, the complex retains the ability for binding the oocysts and generating the ECL signal. Based on the IFA results in the experiment evaluating different protocols for oocysts recovery from karst water samples, the most efficient protocol involved dispersion, followed by flotation and immunomagnetic separation (IMS) (24% recovery). The ECL results obtained in that experiment were very similar to the results obtained in the IFA method, which indicates that the IM-ECL method is accurate. Results of the IFA in the study of the prevalence of C. parvum in the groundwater showed that oocysts were present in 78% of 1 L water samples with average number of oocysts of 6.4+/-5.5 and ranged from 0 (13 samples) to 23.3 (2 samples). The ECL signal generated from these water samples ranged from 3,771 to 622 (average 1,620+/-465). However, the background value estimated in groundwater samples with low number of oocysts detected by IFA was highly variable and elevated (from 3,702 to 272, average 1,503+/-475). The background value as a result of nonspecific binding to beads by unidentified organic components in the water can inhibit or even completely mask the signal generated by oocysts. Our investigations showed that the IM-ECL method appears to be promising for the qualitative and quantitative detection of C. parvum from the environmental water; however, the method requires further development to improve sensitivity and account for background signals.

Estimation of viable Escherichia coli O157 in surface waters using enrichment in conjunction with immunological detection.

The use of a minimal lactose enrichment broth (MLB) in conjunction with immunomagnetic electrochemiluminescence detection (IM-ECL) was evaluated for the estimation of viable Escherichia coli O157 populations in surface water samples. In principle, E. coli O157 populations (C(initial E. coli O157)) can be derived from enrichment data according to the equation: C(initial E. coli O157) = C(initial coliforms) x C(final E. coli O157)/C(final coliforms)), assuming that the growth rates and lag times of water-borne E. coli O157 and collective coliforms are sufficiently comparable, or at least consistent. We have previously described a protocol for determining C(final E. coli O157) in MLB-enriched water samples. In the present study, 80% of coliforms (red/pink colonies on MacConkey Agar) grew in MLB, indicating that this provides reasonably accurate estimates of C(initial coliforms). Estimates of C(final coliforms) were determined from turbidity data. Initial E. coli O157 populations (C(initial E. coli O157)) were calculated for 33 Baltimore watershed samples giving a positive IM-ECL response. The majority of samples contained E. coli O157 concentrations of < 1 cell per 100 ml. These data indicate that E. coli O157 are present in surface water samples but at very low levels. Growth rates for MLB-enriched coliforms were highly variable (k= 0.47 +/- 0.13 h(-1), n= 72). There was no correlation between growth rates and any measured water parameter, suggesting that coliform populations in water samples are spatially and temporally unique. Although variability in growth rates was expected to yield some low values, the fact that most E. coli O157 concentrations were < 1 suggests that other factor(s) were also responsible. Studies with E. coli O157:H7 and wild-type E. coli suggest that increased lag times due to starvation were at least partially responsible for the observed data. Based on estimates of C(initial coliforms) and k(coliforms), MLB was evaluated for sensitivity and quantitativeness. Simulated populations of E. coli O157:H7 at stationary phase varied from ca. 10(3) to 10(8) cells ml(-1) enrichment culture. Although not suitable for quantitation, MLB enrichment in conjunction with IM-ECL can detect as few as one viable water-borne E. coli O157 cell per 100 ml surface water. Experiments are in progress to evaluate alternative media for sensitivity and quantitative detection of enterohemorrhagic E. coli.