Biomedical subjects

Daniel R Shelton

Publications and source records attributed to Daniel R Shelton.

9 recordsLinked to original sources

PubMed2006

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.

Adhesins, Bacterial↗

PubMed2005

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.

Biosensing Techniques↗

PubMed2005

Effect of bovine manure on Cryptosporidium parvum oocyst attachment to soil.

The objective of this work was to assess the effect of dilute bovine manure (1.0% and 0.1%) versus that of no manure on attachment and subsequent detachment of Cryptosporidium parvum oocysts to soil. Manure enhanced the attachment of oocysts to soil particles; the maximum attachment was observed with 0.1% manure. Oocyst attachment was partially reversible; maximum detachment was observed with dilute manure. These results indicate that oocyst attachment to soil is substantially affected by bovine manure in a complex manner and should have implications for how oocysts may be transported through or over soils.

Animals↗

PubMed2005

tir- and stx-positive Escherichia coli in stream waters in a metropolitan area.

Diarrheagenic Escherichia coli, which may include the enteropathogenic E. coli and the enterohemorrhagic E. coli, are a significant cause of diarrheal disease among infants and children in both developing and developed areas. Disease outbreaks related to freshwater exposure have been documented, but the presence of these organisms in the urban aquatic environment is not well characterized. From April 2002 through April 2004 we conducted weekly surveys of streams in the metropolitan Baltimore, Md., area for the prevalence of potentially pathogenic E. coli by using PCR assays targeting the tir and stx(1) and stx(2) genes. Coliforms testing positive for the presence of the tir gene were cultured from 653 of 1,218 samples (53%), with a greater prevalence associated with urban, polluted streams than in suburban and forested watershed streams. Polluted urban streams were also more likely to test positive for the presence of one of the stx genes. Sequence analysis of the tir amplicon, as well as the entire tir gene from three isolates, indicated that the pathogenic E. coli present in the stream waters has a high degree of sequence homology with the E. coli O157:H7 serotype. Our data indicate that pathogenic E. coli are continually deposited into a variety of stream habitats and suggest that this organism may be a permanent member of the gastrointestinal microflora of humans and animals in the metropolitan Baltimore area.

Amino Acid Sequence↗

PubMed2004

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.

Bacteriological Techniques↗

PubMed2003

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.

Journal Article↗

PubMed2003

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.

Bacillus anthracis↗

PubMed2003

Evaluation of parameters affecting quantitative detection of Escherichia coli O157 in enriched water samples using immunomagnetic electrochemiluminescence.

We report here the use of immunomagnetic (IM) electrochemiluminescence (ECL) for quantitative detection of Esherichia coli O157:H7 in water samples following enrichment in minimal lactose broth (MLB). IM beads prepared in-house with four commercial anti-O157 monoclonal antibodies were compared for efficiency of cell capture. IM-ECL responses for E. coli O157:H7 (strain SEA13B88) were similar for all four commercial anti-O157 LPS monoclonal antibodies. The ECL signal was linearly correlated with E. coli O157:H7 cell concentration, indicating a constant ECL response per cell. Twenty-two strains of E. coli O157:H7 or O157:NM gave comparable ECL signals using IM beads prepared in-house. To assess the potential for interference from background bacteria in MLB-enriched water samples, 10(4) cells of E. coli O157:H7 (strain SEA13B88) were added to enriched samples prior to analysis. There was considerable variability in recovery of E. coli O157:H7 cells; net ECL signals ranged from 1% to 100% of expected values (i.e., percent inhibition from 0% to 99%). Cultures of Klebsiella pneumoniae, Klebsiella oxytoca, and Enterobacter cloacae, subsequently isolated from MLB-enriched water samples via IM separation (IMS), were observed to interfere with the binding of E. coli O157:H7 cells to IM beads. Recoveries of 10(4) E. coli O157:H7 cells were </=10% in the presence of ca. 10(8) K. pneumoniae, K. oxytoca, or E. cloacae cells. None of these strains gave a positive IM-ECL signal. Although competitive binding decreased sensitivity, there still was a linear correlation between ECL signal and higher E. coli O157:H7 cell concentrations. These studies indicate that IM-ECL in conjunction with MLB enrichment is capable of quantitatively detecting as few as 10(3) to 10(5) E. coli O157:H7 cells ml(-1), depending on percent recoveries, in enriched samples that contain ca. 10(9) total lactose-fermenting bacteria ml(-1). Assuming comparable growth rates for E. coli O157:H7 and other lactose-fermenting bacteria in MLB, it may be possible to detect as few as one E. coli O157:H7 in 100 ml of raw water containing as many as 10(4) to 10(6) lactose-fermenting bacteria (i.e., total coliforms).

Escherichia coli O157↗

PubMed2003

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.

Animals↗