Multicenter Evaluation of the Novel ETEST Fosfomycin for Antimicrobial Susceptibility Testing of Enterobacterales, Enterococcus faecalis, and Staphylococcus Species.

Fosfomycin is a phosphonic acid derivative active against a wide spectrum of Gram-positive and Gram-negative pathogens. It is used for the treatment of uncomplicated urinary tract infections (uUTI) or severe infections by oral or intravenous (i.v.) administration. In order to improve its performance and robustness, the fosfomycin strip, an antibiotic gradient diffusion strip, was redeveloped and evaluated in the multicenter study summarized in this paper. ETEST fosfomycin (ETEST FO) clinical performance was evaluated by three study sites on 152 Enterococcus faecalis, 100 Staphylococcus spp. and 330 Enterobacterales in comparison with the CLSI and EUCAST agar dilution reference method. Referring to FDA performance criteria, the ETEST FO achieved 91.0% of essential (EA) and 99.0% of categorical agreement (CA) for Escherichia coli. In addition, 98.0% EA and 93.4% CA were achieved for E. faecalis, with no very major errors (VME) or major errors (ME). According to EUCAST breakpoints for intravenous fosfomycin use, Enterobacterales and Staphylococcus spp. also met ISO acceptance criteria for EA and CA (EA 91.5%, 94.0%, respectively, and CA 98.0% for both). A VME rate of 8.8% was observed for Enterobacterales but the MICs were within EA. A trend to predict lower MICs for Citrobacter spp., E. coli and Salmonella enterica and to predict higher MICs for Klebsiella pneumoniae MICs was observed, while ETEST FO should not be used for Enterobacter cloacae, because of low EA and a high VME rate. The study results support the efficiency of the novel ETEST FO, making it an easy-to-handle tool as a substitute to the classical agar dilution method.

Survey of Toxoplasma gondii Exposure in Muskrats in a Relatively Pristine Ecosystem.

Waterborne transmission of Toxoplasma gondii is assumed to be enhanced in areas with human-altered landscapes (e.g., urbanization, agriculture) and increased populations of non-native domestic and feral cats (Felis catus). However, little is known concerning T. gondii exposure risks in more natural watersheds (e.g., reduced human footprint, no domestic or feral cats) to establish a baseline for comparisons. In this study, muskrats (Ondatra zibethicus) were used as sentinels to assess baseline T. gondii exposure in a relatively pristine watershed in the Greater Voyageurs Ecosystem, northern Minnesota, during the summers of 2018-2019. Toxoplasma gondii antibodies were assayed in sera of live-trapped muskrats (n = 70) using a modified agglutination test. None of our samples were positive for T. gondii antibodies (P = 0.00, 95% Wald Score Confidence Interval = 0.00-0.05). This study establishes a baseline to compare T. gondii waterborne transmission risks in other human-modified watersheds.

Inactivation of airborne porcine reproductive and respiratory syndrome virus (PRRSv) by a packed bed dielectric barrier discharge non-thermal plasma.

Porcine reproductive and respiratory syndrome virus (PRRSv) is one of the most significant airborne viruses impacting the pork industry in the US. Non-thermal plasmas (NTPs) are electrical discharges comprised of reactive radicals and excited species that inactivate viruses and bacteria. Our previous experiments using a packed bed NTP reactor demonstrated effective inactivation of bacteriophage MS2 as a function of applied voltage and power. The present study examined the effectiveness of the same reactor in inactivating aerosolized PRRSv. A PRRSv solution containing ∼105 TCID50/ml of PRRSv VR2332 strain was aerosolized at 3 ml/min by an air-jet nebulizer and introduced into 5 or 12 cfm air flow followed by NTP exposure in the reactor. Twin impingers upstream and downstream of the reactor collected samples of the virus-laden air flow for subsequent TCID50 assay and qPCR analyses. An optical particle sizer measured upstream and downstream aerosol size distributions, giving estimates of aerosol filtration by the reactor. The results showed that PRRSv was inactivated to a similar degree as MS2 at the same conditions, with the maximum 1.3-log inactivation of PRRSv achieved at 20 kV and 12 cfm air flow rate. The results demonstrate the potential of properly optimized NTPs in controlling PRRSv transmission.

Screening for Diabetes Risk Using Integrated Dental and Medical Electronic Health Record Data.

Undiagnosed diabetes and prediabetes present a serious public health challenge. We previously reported that data available in the dental setting can serve as a tool for early dysglycemia identification in a primarily Hispanic, urban population. In the present study, we sought to determine how the identification approach can be recalibrated to detect diabetes or prediabetes in a White, rural cohort and whether an integrated dental-medical electronic health record (iEHR) offers further value to the process. We analyzed iEHR data from the Marshfield Clinic, a health system providing care in rural Wisconsin, for dental patients who were ≥21 y of age, reported that they had never been told they had diabetes, had an initial periodontal examination of at least 2 quadrants, and had a glycemic assessment within 3 mo of that examination. We then assessed the performance of multiple predictive models for prediabetes/diabetes. The study outcome, glycemic status, was gleaned from the medical module of the iEHR based on American Diabetes Association blood test cutoffs. The sample size was 4,560 individuals. Multivariate logistic regression revealed that the best performance was achieved by a model that took advantage of the iEHR. Predictors included age, sex, race, ethnicity, number of missing teeth, percentage of teeth with at least 1 pocket ≥5 mm from the dental EHR, and overweight/obesity, hypertension, hyperlipidemia, and smoking status from the medical EHR. The model achieved an area under the receiver operating characteristic curve of 0.71 (95% confidence interval, 0.69-0.72), yielding a sensitivity of 0.70 and a specificity of 0.62. Across a range of populations, informed by certain patient characteristics, dental care team members can play a role in helping to identify dental patients with undiagnosed diabetes or prediabetes. The accuracy of the prediction increases when dental findings are combined with information from the medical EHR. Knowledge Transfer Statement: Prediabetes and diabetes often go undiagnosed for many years. Early identification and care can lead to improved glycemic outcomes and prevent wide-ranging morbidity, including adverse oral health consequences, in affected individuals. Information available in the dental office can be used by clinicians to identify those who remain undiagnosed or are at risk; the accuracy of this prediction increases when combined with information from the medical electronic health record.

Genetic basis for soma is present in undifferentiated volvocine green algae.

Somatic cellular differentiation plays a critical role in the transition from unicellular to multicellular life, but the evolution of its genetic basis remains poorly understood. By definition, somatic cells do not reproduce to pass on genes and so constitute an extreme form of altruistic behaviour. The volvocine green algae provide an excellent model system to study the evolution of multicellularity and somatic differentiation. In Volvox carteri, somatic cell differentiation is controlled by the regA gene, which is part of a tandem duplication of genes known as the reg cluster. Although previous work found the reg cluster in divergent Volvox species, its origin and distribution in the broader group of volvocine algae has not been known. Here, we show that the reg cluster is present in many species without somatic cells and determine that the genetic basis for soma arose before the phenotype at the origin of the family Volvocaceae approximately 200 million years ago. We hypothesize that the ancestral function was involved in regulating reproduction in response to stress and that this function was later co-opted to produce soma. Determining that the reg cluster was co-opted to control somatic cell development provides insight into how cellular differentiation, and with it greater levels of complexity and individuality, evolves.

Improving qPCR methodology for detection of foaming bacteria by analysis of broad-spectrum primers and a highly specific probe for quantification of Nocardia spp. in activated sludge.

AIMS: To develop qPCR broad-spectrum primers combined with a Nocardia genus-specific probe for the identification of a broad spectrum of Nocardia spp. and to analyse the effects of using this developed primer and probe set on the ability to quantify Nocardia spp. in mixed DNA. METHODS AND RESULTS: The consequences of using a degenerative primer set and species-specific probe for the genus Nocardia on qPCR assays were examined using DNA extracts of pure cultures and activated sludge. The mixed DNA extracts where the target organism Nocardia flavorosea concentration ranged from 5 × 102 to 5 × 106 copies per reaction, while the background organism's DNA (Mycobacterium bovis) concentration was held at 5 × 106 copies per reaction, only produced comparable cycle threshold florescence levels when N. flavorosea concentration was greater than or equal to the background organism concentration. When concentrations of N. flavorosea were lowered in increments of 1 log, while holding M. bovis concentrations constant at 5 × 106 copies per reaction, all assays demonstrated delayed cycle threshold values with a maximum 34·6-fold decrease in cycle threshold at a ratio of 106 M. bovis: 102 N. flavorosea copies per reaction. CONCLUSIONS: The data presented in this study indicated that increasing the ability of a primer set to capture a broad group of organisms can affect the accuracy of quantification even when a highly specific probe is used. This study examined several applications of molecular tools in complex communities such as evaluating the effect of mispriming vs interference. It also elucidates the importance of understanding the community genetic make-up on primer design. SIGNIFICANCE AND IMPACT OF THE STUDY: Degenerative primers are very useful in amplifying bacterial DNA across genera, but reduce the efficiency of qPCR reactions. Therefore, standards that address closely related background species must be used to obtain accurate qPCR results.

Hypothermia in Uremic Dogs and Cats.

BACKGROUND: The prevalence of uremic hypothermia (UH) and the effects of improving uremia on body temperature have not been determined in veterinary patients. OBJECTIVES: To determine the prevalence of UH and correlations between uremia and body temperature in patients undergoing intermittent hemodialysis (IHD). ANIMALS: Uremic dogs (n = 122) and cats (n = 79) treated by IHD at the Bobst Hospital of the Animal Medical Center from 1997 to 2013. METHODS: Retrospective review of medical records. RESULTS: The prevalence of hypothermia was 38% in azotemic cats and 20.5% in azotemic dogs. Statistically significant temperature differences were observed between uremic and nonuremic dogs (nonuremic: mean, 100.8°F; range, 91.2-109.5°F; uremic: mean, 99.9°F; range, 95.6-103.8°F; P < .0001) and cats (nonuremic: mean, 100.6°F; range, 94.0-103.8°F; uremic: mean, 99.3°F; range, 92.3-103.4°F; P < .0001). In dog dialysis patients, significant models included (1) timing (pre-dialysis versus post-dialysis) with weight class (small [P < .0001], medium [P = .016], and large breed [P = .033] dogs), (2) timing with serum creatinine concentration (P = .021), and (3) timing with BUN concentration (P < .0001). In cat dialysis patients, there was a significant interaction between timing and weight as a categorical variable (<5 kg and ≥5 kg). CONCLUSIONS AND CLINICAL IMPORTANCE: Uremic hypothermia appears to be a clinical phenomenon that occurs in cats and dogs. Uremic patients are hypothermic compared to ill nonuremic patients and body temperatures increase when uremia is corrected with IHD in dogs and in cats >5 kg. In cats, UH seems to be a more prevalent phenomenon driven by uremia. Uremic hypothermia does occur in dogs, but body weight is a more important predictor of body temperature.

Communication and technology in genetic counseling for familial cancer.

When a cancer predisposing germline mutation is detected in an index case, the presence of the underlying syndrome is confirmed and the potential for predictive testing of at-risk relatives is established. However, the reporting of a positive family history does not routinely lead to communication of information about risk to close, much less distant relatives. This review summarizes information technology utilized to address penetration or 'reach' of knowledge of risk within extended families, including the use of telephone and video counseling to reach distant patients, and anticipate novel internet-based processes for communication between investigators and relatives.

Thermal characterization of polyamide 11/nanographene platelet nanocomposites.

The objective of this study is to evaluate thermal properties of polyamide 11 (PA11)/nanographene platelet (NGP) nanocomposites. Samples were prepared using 1 wt%, 3 wt%, and 5 wt% of NGPs. Isopropyl alcohol (IPA) was used as a solvent to assist in dispersion of the NGPs within the PA11 powder. The NGPs were hand mixed evenly into the PA11 powder using a wooden dowel. Morphological characterization of the PA11/NGP nanocomposite was conducted using scanning electron microscopy. Thermal characterization of nanocomposites includes thermogravimetric analysis, differential scanning calorimetry and thermomechanical analysis. Results indicate that the addition of NGPs shows an initial increase in thermal stability and crystallization temperature (T(c)) along with a decrease in glass transition temperature (T(g)) and no improvement in coefficient of thermal expansion (alpha). These results are attributed to improved interfacial adhesion between NGPs and PA11, restricting polymer chain mobility.

Streptococcus mutans strains recovered from caries-active or caries-free individuals differ in sensitivity to host antimicrobial peptides.

Antimicrobial peptides (AMPs) are among the repertoire of host innate immune defenses. In the oral cavity, several AMPs are present in saliva and have antimicrobial activities against oral bacteria, including Streptococcus mutans, a primary etiological agent of dental caries. In this study, we hypothesized that unique S. mutans strains, as determined by DNA fingerprinting from sixty 13-year-old subjects with or without experience of caries, would have different susceptibilities to α-defensins-1-3 (HNP-1-3), ß-defensins-2-3 (HBD-2-3) and LL-37. The salivary levels of these peptides in subjects were also measured by enzyme-linked immunosorbent assays. We found that S. mutans strains from children with active caries showed greater resistance to salivary HNP-1-2, HBD-2-3 and LL-37 at varying concentrations than those from caries-free subjects. In addition, combinations of these peptides increased their antimicrobial activity against S. mutans either additively or synergistically. The salivary levels of these peptides were highly variable among subjects with no correlation to host caries experience. However, the levels of a number of these peptides in saliva appeared to be positively correlated within an individual. Our findings suggest that the relative ability of S. mutans to resist host salivary AMPs may be considered a potential virulence factor for this species such that S. mutans strains that are more resistant to these peptides may have an ecological advantage to preferentially colonize within dental plaque and increase the risk of dental caries.

First Report of Fusarium Wilt of Canary Island Date Palm Caused by Fusarium oxysporum f. sp. canariensis in Louisiana.

Canary Island date palm (Phoenix canariensis Hort. Ex Chabaud) is a signature palm planted in New Orleans, LA. Currently, the city has approximately 1,000 mature Canary Island date palms. During the fall of 2009, 153 palms were inspected with 27 palms exhibiting typical symptoms of Fusarium wilt. Symptoms included one-sided death and a reddish brown streak on the rachis of affected fronds and death of the leaflets. Longitudinal sections of affected fronds showed vascular discoloration. Severely infected palms were completely dead. Small pieces of diseased tissue from five palms were surface sterilized with sodium hypochlorite (0.6%) for 2 to 3 min, then rinsed in sterile distilled water, blotted dry, and plated on potato dextrose agar (PDA). Fungal colonies on PDA produced a purple pigment, and both macro- and microconidia that are typical of Fusarium oxysporum were observed under a light microscope. A single-spore culture of isolate PDC-4701 was obtained. DNA from this isolate was extracted with a DNeasy Plant Mini kit (Qiagen Inc., Valencia, CA) and primers ef1 and ef2 were used to amplify and sequence the translation elongation factor 1-α gene (2). NCBI BLAST analysis of the 616-bp sequence resulted in 100% identity with F. oxysporum f. sp. canariensis isolates PLM-385B from Texas and PLM-511A from South Carolina (GenBank Accession Nos. HM 591538 and HM 591537, respectively). Isolate PDC-4701, grown on PDA for 2 weeks, was used to inoculate 10 9-month-old P. canariensis seedlings. An 18-gauge needle was used to inject 15 ml of a 107 conidia/ml suspension into the stem near the soil line. Each seedling was inoculated at two locations and covered with Parafilm at the inoculation sites. Ten control seedlings were injected with sterile distilled water in the same manner. Inoculated and control seedlings were maintained in a greenhouse at 28 ± 2°C. Leaves of all 10 inoculated seedlings started to wilt 3 months after inoculation. Internal vascular discoloration was observed and the pathogen was reisolated from the symptomatic seedlings. No symptoms developed on any of the 10 control seedlings. On the basis of morphology and DNA sequence data, this pathogen is identified as F. oxysporum f. sp. canariensis. Fusarium wilt of Canary Island date palm has been previously reported from California, Florida, Nevada, Texas, and South Carolina (1). To our knowledge, this is the first report of Fusarium wilt of Canary Island date palm caused by F. oxysporum f. sp. canariensis in Louisiana, extending its geographic range. The disease may adversely affect the tradition of planting Canary Island date palms in New Orleans. The sequence of isolate PDC-4701 has been submitted to the NCBI database (GenBank Accession No. JF826442) and a culture specimen has been deposited in the Fusarium Research Center culture collection (Accession No. O-2602) at the Pennsylvania State University, University Park, PA. References: (1) M. L. Elliott et al. Plant Dis. 95:356, 2011. (2) D. M. Geiser et al. Eur. J. Plant Pathol. 110:473, 2004.

TEMPO EC for the enumeration of Escherichia coli in foods: collaborative study.

The automated method for enumeration of Escherichia coli, TEMPO EC, in foods uses a dehydrated culture medium and enumeration card containing 48 wells across three different dilutions for the automatic determination of the most probable number (MPN). The alternative method was compared in a multilaboratory collaborative study to AOAC Official Method 966.24. Six food types were artificially contaminated with E. coli: raw ground beef, bagged lettuce, cooked chicken, pasteurized crabmeat, frozen green beans, and pasteurized whole milk. All foods were analyzed for E. coli counts by 11 collaborating laboratories throughout the United States. Test portions from the six food types each contaminated at four different contamination levels were evaluated. The study demonstrated that the TEMPO EC method is a reliable, automated assay for the enumeration of E. coli in foods.

Comparative whole-genome analysis of Streptococcus mutans isolates within and among individuals of different caries status.

INTRODUCTION: Genotypic analyses of Streptococcus mutans using fingerprinting methods depend on a few genetic loci being different but do not reveal the underlying genome-wide differences between strains. METHODS: We used comparative genomic hybridization (CGH) with 70-mer oligonucleotide microarrays containing open reading frames (ORFs) from S. mutans strain UA159 to examine the genetic diversity of 44 isolates from nine children selected from a local study population in Eastern Iowa. RESULTS: Unique strains (clones) within each child initially identified by arbitrary-priming polymerase chain reaction were confirmed by CGH. There was a wide range of variation in the hybridization patterns of the 1948 ORFs among the test isolates examined. Between 87 and 237 ORFs failed to give a positive signal among individual isolates. A total of 323 of the UA159 ORFs were absent from one or more of the test strains. These 323 variable genes seemed to be distributed across the entire UA159 genome and across all the predicted functional categories. CONCLUSION: This set of very close geographically and temporally collected S. mutans isolates had a degree of gene content variation as high as a previously examined global set of strains. Comparing the frequency of these variable genes, the majority of which have unknown function, among strains of different origins (i.e. different caries status) could help to determine their relevance in S. mutans cariogenicity.

Nicotine increases the collagen-degrading ability of human gingival fibroblasts.

BACKGROUND AND OBJECTIVE: The objective of this study was to determine the effects that nicotine and the combination of nicotine and Porphyromonas gingivalis supernatant have on human gingival fibroblast-mediated collagen degradation. MATERIAL AND METHODS: Human gingival fibroblasts were cultured with 25-500 microg/ml of nicotine in collagen-coated six-well plates. On days 1-5, the conditioned media was collected for zymography and western blot analyses of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). The cells were then removed and the collagen cleavage visualized by Coomassie blue staining. To examine the combined effect, 250 microg/ml of nicotine and 10% v/v culture supernatant of P. gingivalis ATCC 33277 were added to the human gingival fibroblasts. The mRNA levels of multiple MMPs and TIMPs were monitored. RESULTS: Nicotine increased the human gingival fibroblast-mediated collagen cleavage. The MMP-14 and MMP-2 produced by the nicotine-treated human gingival fibroblasts more readily underwent zymogen activation. Nicotine treatment resulted in TIMP-2 redistribution to the cell surface. The mRNAs of multiple MMPs and TIMPs were unaltered by nicotine. An additive collagen cleavage effect was observed when the human gingival fibroblasts were treated with both nicotine and P. gingivalis. CONCLUSION: Nicotine increased human gingival fibroblast-mediated collagen degradation, in part through the activation of membrane-associated MMPs. Nicotine and P. gingivalis had an additive effect on human gingival fibroblast-mediated collagen degradation.

Thermal balance of cattle grazing winter range: model application.

Beef cattle grazing semiarid foothill rangeland of the Northern Rockies during winter may be exposed to cold temperatures and high winds while grazing pastures with low nutritional value. Cattle can physiologically and behaviorally respond to the changing environment to lower their metabolic requirements and reduce the effects of cold exposure. Requirements of grazing cattle may be overpredicted with models developed in controlled settings that do not account for energy-conserving behaviors. We refined a simple thermal balance equation to model heat exchange of free-ranging cattle. We accounted for the complex interactions between animal behavior and the changing natural environment by applying the insulation characteristics of the cattle's tissue and coat to a simple geometric shape of an asymmetric ellipsoid at different orientations to the sun and wind. We compared the model predictions with heat production measured in 3 studies, and in all cases the model predictions were similar to those reported. Model simulations indicate behaviors, such as lying and orientation to the sun, mitigated the effects of extreme weather. For many combinations of winter weather variables, metabolic requirements increased only slightly due to cold exposure of mature beef cattle in a near-maintenance state. The results indicate that solar radiation contributes strongly to the thermal balance of a cow. Thus, previous models that do not account for the irradiative environment may overestimate metabolic requirements of cattle acclimated to grazing winter range.

First Report of Xylella fastidiosa in Oklahoma.

In Oklahoma, during the late summer of 2004, an elm tree (Ulmus americanus L.) located in the Oklahoma Botanical Gardens near Stillwater showed symptoms of marginal leaf scorch bordered by a yellow band between necrotic and green tissues, indicating possible Xylella fastidiosa infection. Three leaves from the symptomatic tree and one leaf from an asymptomatic nearby elm were sampled. DNA was extracted with the Extract-N-Amp kit (Sigma, St. Louis, MO). Samples were tested for X. fastidiosa using real-time polymerase chain reaction (PCR) with Xylella genus specific primers XfF1/XfR2 and dual-labeled TaqMan probe XfP2 (2). Infected oleander from California was used as a positive control. All three samples from symptomatic leaves and the positive control were PCR positive, and the sample from the asymptomatic tree was PCR negative. Attempts to culture an isolate of the bacteria from petioles and branch tissues on PD3 and PW, media selective for X. fastidiosa, failed. For more detailed molecular characterization of the putative pathogen, DNA from additional symptomatic petioles from the same tree was isolated using the cetyltrimethylammoniumbromide (CTAB) extraction. X. fastidiosa specific primers BBXFOUTF1 (5'-AAGCGCCTCCGTGAGTTATC-3') and BBXFOUTR1 (5'-CCTTCACGCATATCATCACC-3') were used to PCR amplify the gyrB gene. The amplification product was recovered after gel electrophoresis with QIAquick gel extraction kit (Qiagen, Valencia, CA) and was subjected to automated sequencing (Oklahoma State University Recombinant DNA/Protein Resource Facility). BLASTN alignment (1) of the obtained 381 bp sequence revealed 100% identity with the gyrB gene from elm (GenBank Accession No. AF534966) and mulberry (GenBank Accession No. AF534965) isolates of X. fastidiosa. During 2005, petiole samples from the tree were collected and serological diagnosis was confirmed using enzyme-linked immunosorbent assay (Agdia, Inc., Elkhart, IN). Some strains of X. fastidiosa have very wide host ranges and many of the hosts may be asymptomatic. Therefore, the economic importance and implications of the detection of X. fastidiosa in the state of Oklahoma remain to be determined. To our knowledge, this is the first report of X. fastidiosa in Oklahoma. References: (1) S. F. Altschul et al. J. Mol. Biol. 215:403, 1990. (2) N. W. Schaad et al. Phytopathology 92:721, 2002.

Principle component feature detector for motor cortical control.

Principle component analysis (PCA) was performed on recorded neuronal action potentials from neural ensembles in rat's motor cortex when the rat was involved in a closed-loop real-time brain machine interface (BCI). The implanted rat was placed in a conditioning chamber, but freely moving, to decide which one of the two paddles should be activated to shift the light to the center. It is found that the principle component feature vectors revealed the importance of individual neurons and their temporal dynamics in relation to the intention of activating either left or right paddle. In addition, the first principle component feature has much higher discriminative capability than others although it represents only a few percentage of the total variance. Using the first principle component with the Bayes classifier achieved 90% classification accuracy, which is comparable with the accuracy obtained by a more sophisticated high performance support vector classifiers.

A biomarker for the identification of swine fecal pollution in water, using the STII toxin gene from enterotoxigenic Escherichia coli.

This research developed a PCR method to identify swine fecal pollution in water, using a portion of the STII toxin gene from enterotoxigenic Escherichia coli as the target sequence. This method showed the gene to have a wide-spread geographical distribution and temporal stability; and the primers demonstrated high specificity, sensitivity, and reliability. A total of 110 DNA extracts from different animal fecal and human sewage samples were screened using the primers and no positives resulted. Centrifugation and filtration methods for concentrating E. coli seeded into stream, ocean, secondary effluent, and dairy lagoon waters resulted in detection limits at the femtogram and attogram levels. E. coli with the biomarker seeded into stream, ocean, and secondary effluent waters remained stable for approximately 2 weeks for all water types. Of the farm lagoon and waste samples tested, 94% were positive for the STII trait, regardless of the number of E. coli screened and 100% were positive when > or =35 E. coli isolates were screened. As the PCR product of the target sequence yielded a single band, the method is applicable to dot blot detection methodology, yielding great accuracy in determining the presence of swine fecal sources.

A biomarker for the identification of cattle fecal pollution in water using the LTIIa toxin gene from enterotoxigenic Escherichia coli.

This research describes a method based on PCR to identify cattle fecal pollution in water using a portion of the heat labile toxin IIA (LTIIa) gene from enterotoxigenic Escherichia coli (ETEC). We describe the development of the primers and target. DNA extracts (221) from different animal fecal and human sewage samples were screened and showed no cross-reactivity. Minimum detection limits using centrifugation and filtration methods to concentrate E. coli seeded into stream, ocean, and secondary effluent waters were found to be at femtogram and attogram levels, respectively. Stability of the biomarker in stream, ocean, and secondary effluent waters was 2-4 weeks for all water types. Finally, 33 farm lagoon and waste samples were collected and 31 tested to validate the method; 93% were positive for the LTIIa trait when >1,000 E. coli were screened and 100% positive when >10(5) E. coli were screened. Prevalence of the toxin gene in the E. coli population affected the outcome of the analyses. The cow biomarker can be used in watershed studies to identify cattle waste with great accuracy if the appropriate numbers of E. coli are screened.

Inhibition of transforming growth factor (TGF)-beta1-induced extracellular matrix with a novel inhibitor of the TGF-beta type I receptor kinase activity: SB-431542.

Transforming growth factor beta1 (TGF-beta1) is a potent fibrotic factor responsible for the synthesis of extracellular matrix. TGF-beta1 acts through the TGF-beta type I and type II receptors to activate intracellular mediators, such as Smad proteins, the p38 mitogen-activated protein kinase (MAPK), and the extracellular signal-regulated kinase pathway. We expressed the kinase domain of the TGF-beta type I receptor [activin receptor-like kinase (ALK)5] and the substrate, Smad3, and determined that SB-431542 is a selective inhibitor of Smad3 phosphorylation with an IC50 of 94 nM. It inhibited TGF-beta1-induced nuclear Smad3 localization. The p38 mitogen-activated protein kinase inhibitors SB-203580 and SB-202190 also inhibit phosphorylation of Smad3 by ALK5 with IC50 values of 6 and 3 microM, respectively. This suggests that these p38 MAPK inhibitors must be used at concentrations of less than 10 microM to selectively address p38 MAPK mechanisms. However, the p38 MAPK inhibitor SB-242235 did not inhibit ALK5. To evaluate the relative contribution of Smad signaling and p38 MAPK signaling in TGF-beta1-induced matrix production, the effect of SB-431542 was compared with that of SB-242235 in renal epithelial carcinoma A498 cells. All compounds inhibited TGF-beta1-induced fibronectin (FN) mRNA, indicating that FN synthesis is mediated in part via the p38 MAPK pathway. In contrast, SB-431542, but not the selective p38 MAPK inhibitor SB-242235, inhibited TGF-beta1-induced collagen Ialpha1 (col Ialpha1). These data indicate that some matrix markers that are stimulated by TGF-beta1 are mediated via the p38 MAPK pathway (i.e., FN), whereas others seem to be activated via ALK5 signaling independent of the p38 MAPK pathway (i.e., col Ialpha1).