Draft Genome Sequence of the Multiple Antibiotic Resistant Pseudomonas aeruginosa PAO1-UB Subline.

We report the draft genome sequence and antibiotic susceptibility of Pseudomonas aeruginosa strain PAO1-UB, a subline of the common reference strain PAO1. This strain was sequenced in order to provide information on the genome dynamics of PAO1 sublines and their genes conferring resistance to multiple antibiotics.

Draft Genome Sequence of a Multiple Antibiotic Resistant Staphylococcus aureus NCTC 6571-UB Laboratory Strain.

We report the draft genome sequence of the laboratory strain Staphylococcus aureus NCTC 6571-UB, a strain that was derived from S. aureus NCTC 6571. This strain was selected for sequencing in order to provide information on the genome dynamics and the acquired resistance genes for penicillin G, trimethoprim, and sulfamethoxazole resistance.

Organizational influences on healthcare system adoption and use of advanced health information technology capabilities.

OBJECTIVES: The adoption of advanced health information technology (HIT) capabilities, such as predictive analytic functions and patient access to records, remains variable among healthcare systems across the United States. This study is the first to identify characteristics that may drive this variability among health systems. STUDY DESIGN: Responses from the 2017/2018 National Survey of Healthcare Organizations and Systems were used to assess the extent to which healthcare system organizational structure, electronic health record (EHR) standardization, and resource allocation practices were associated with use of 5 advanced HIT capabilities. Of 732 systems surveyed, 446 responded (60.9%), 425 (58.1%) met sample inclusion criteria, and 389 (53.1%) reported consistent EHR use. METHODS: Measures of adoption, resource allocation, and organizational structure were developed based on survey responses. Multivariate linear regression with control variables estimated the relationships. RESULTS: Adoption of advanced HIT capabilities is low and variable, with a mean of 2.4 capabilities adopted and only 8.4% of systems reporting widespread adoption of all 5 capabilities. In adjusted analyses, EHR standardization (ß = 0.76; P = .001) was the strongest predictor of the number of advanced capabilities adopted, and ownership and management of medical groups (ß = 0.32; P = .04) was also a significant predictor. CONCLUSIONS: Health systems that standardize their EHRs and that own and manage hospitals and medical groups have higher rates of advanced HIT adoption and use. System leaders looking to increase the use of advanced HIT capabilities should consider ways to better standardize their EHRs across organizations.

Validation of Modifications to the ANSR(®) Listeria Method for Improved Ease of Use and Performance.

A study was conducted to validate minor reagent formulation, enrichment, and procedural changes to the ANSR(®) Listeria method, Performance-Tested Method(SM) 101202. In order to improve ease of use and diminish risk of amplicon contamination, the lyophilized reagent components were reformulated for increased solubility, thus eliminating the need to mix by pipetting. In the alternative procedure, an aliquot of the lysate is added to lyophilized ANSR reagents, immediately capped, and briefly mixed by vortexing. When three foods (hot dogs, Mexican-style cheese, and cantaloupe) and sponge samples taken from a stainless steel surface were tested, significant differences in performance between the ANSR and U.S. Food and Drug Administration Bacteriological Analytical Manual or U.S. Department of Agriculture, Food Safety and Inspection Service Microbiology Laboratory Guidebook reference culture procedures were seen with hot dogs and Mexican-style cheese after 16 h enrichment, with the reference methods producing more positive results. After 24 h enrichment, however, there were no significant differences in method performance for any of the four matrixes tested. Robustness testing was also conducted, with variations to lysis buffer volume, lysis time, and sample volume having no demonstrable effect on assay results. Accelerated stability testing was carried out over a 10-week period and showed no diminishment in assay performance. A second phase of the study examined performance of the ANSR assay following enrichment in a new medium, LESS Plus broth, designed for use with all food and environmental sample types. With the alternative LESS Plus broth, there were no significant differences in performance between the ANSR method and the reference culture procedures for any of the matrixes tested after either 16 or 24 h enrichment, although 24 h enrichment is recommended for hot dogs due to higher sensitivity. Results of inclusivity and exclusivity testing using LESS Plus broth showed that the ANSR assay is highly specific, with 100% expected results for target and nontarget bacteria.

Validation of the ANSR(®) Listeria monocytogenes Method for Detection of Listeria monocytogenes in Selected Food and Environmental Samples.

Work was conducted to validate performance of the ANSR(®) for Listeria monocytogenes method in selected food and environmental matrixes. This DNA-based assay involves amplification of nucleic acid via an isothermal reaction based on nicking enzyme amplification technology. Following single-step sample enrichment for 16-24 h for most matrixes, the assay is completed in 40 min using only simple instrumentation. When 50 distinct strains of L. monocytogenes were tested for inclusivity, 48 produced positive results, the exceptions being two strains confirmed by PCR to lack the assay target gene. Forty-seven nontarget strains (30 species), including multiple non-monocytogenes Listeria species as well as non-Listeria, Gram-positive bacteria, were tested, and all generated negative ANSR assay results. Performance of the ANSR method was compared with that of the U.S. Department of Agriculture, Food Safety and Inspection Service Microbiology Laboratory Guidebook reference culture procedure for detection of L. monocytogenes in hot dogs, pasteurized liquid egg, and sponge samples taken from an inoculated stainless steel surface. In addition, ANSR performance was measured against the U.S. Food and Drug Administration Bacteriological Analytical Manual reference method for detection of L. monocytogenes in Mexican-style cheese, cantaloupe, sprout irrigation water, and guacamole. With the single exception of pasteurized liquid egg at 16 h, ANSR method performance as quantified by the number of positives obtained was not statistically different from that of the reference methods. Robustness trials demonstrated that deliberate introduction of small deviations to the normal assay parameters did not affect ANSR method performance. Results of accelerated stability testing conducted using two manufactured lots of reagents predicts stability at the specified storage temperature of 4°C of more than 1 year.

Evaluation of the ANSR for Salmonella assay for identification of Salmonella spp. from colony picks from selective/differential agar media: first action 2013.14.

A collaborative study was conducted to evaluate performance of the ANSR for Salmonella assay for identification of Salmonella spp. from colony picks taken from selective/differential agar media. The ANSR Salmonella assay is an isothermal nucleic acid amplification test based on the nicking enzyme amplification reaction chemistry. The test can be completed in less than 40 min including sample preparation. A total of 18 laboratories representing industry, government, academic, and commercial testing laboratories participated in the study. Each collaborator tested up to 84 samples, comprised of colony picks of six Salmonella spp. and six non-salmonellae taken from six selective/differential agar media as well as tryptic soy agar. A total of 1441 analyses were performed, 1416 of which gave the correct identification, for overall accuracy of 98.3%. For identification of Salmonella spp., 755 of 756 tests (99.9%) produced the correct result. For identification of non-salmonellae as such, 661 of 685 assays (96.5%) produced the correct result. Of the 18 laboratories, 15 produced data sets with 99-100% accuracy. The majority of false-positive results were clustered in three laboratories; analysis of raw data suggests procedural difficulties in at least two cases, which may explain the atypical data from these collaborators. The ANSR Salmonella assay can be used as a rapid, accurate adjunct or alternative to biochemical testing for identification of presumptive Salmonella spp. isolates.

Validation of the ANSR Salmonella method for detection of Salmonella spp. in selected foods and environmental samples.

ANSR Salmonella is a new molecular diagnostic assay for detection of Salmonella spp. in foods and environmental samples. The test is based on the nicking enzyme amplification reaction (NEAR) isothermal nucleic acid amplification technology. The assay platform features simple instrumentation, minimal labor, and, following a single-step 10-24 h enrichment (depending on sample type), an extremely short assay time of 30 min, including sample preparation. Detection is real-time using fluorescent molecular beacon probes. Inclusivity testing was performed using a panel of 113 strains of S. enterica and S. bongori, representing 109 serovars and all genetic subgroups. With the single exception of the rare serovar S. Weslaco, all serovars and genetic subgroups were detected. Exclusivity testing of 38 non-salmonellae, mostly Enterobacteriaceae, yielded no evidence of cross-reactivity. In comparative testing of chicken carcass rinse, raw ground turkey, raw ground beef, hot dogs, and oat cereal, there were no statistically significant differences in the number of positive results obtained with the ANSR and the U.S. Department of Agriculture-Food Safety and Inspection Service or U.S. Food and Drug Administration/Bacteriological Analytical Manual reference culture methods. In testing of swab or sponge samples from five different environmental surfaces, four trials showed no statistically significant differences in the number of positive results by the ANSR and the U.S. Food and Drug Administration/ Bacteriological Analytical Manual reference methods; in the trial with stainless steel surface, there were significantly more positive results by the ANSR method. Ruggedness experiments showed a high degree of assay robustness when deviations in reagent volumes and incubation times were introduced.

Validation of the ANSR Listeria method for detection of Listeria spp. in environmental samples.

ANSR Listeria is a new diagnostic assay for detection of Listeria spp. in sponge or swab samples taken from a variety of environmental surfaces. The method is an isothermal nucleic acid amplification assay based on the nicking enzyme amplification reaction technology. Following single-step sample enrichment for 16-24 h, the assay is completed in 40 min, requiring only simple instrumentation. In inclusivity testing, 48 of 51 Listeria strains tested positive, with only the three strains of L. grayi producing negative results. Further investigation showed that L. grayi is reactive in the ANSR assay, but its ability to grow under the selective enrichment conditions used in the method is variable. In exclusivity testing, 32 species of non-Listeria, Gram-positive bacteria all produced negative ANSR assay results. Performance of the ANSR method was compared to that of the U.S. Department of Agriculture-Food Safety and Inspection Service reference culture procedure for detection of Listeria spp. in sponge or swab samples taken from inoculated stainless steel, plastic, ceramic tile, sealed concrete, and rubber surfaces. Data were analyzed using Chi-square and probability of detection models. Only one surface, stainless steel, showed a significant difference in performance between the methods, with the ANSR method producing more positive results. Results of internal trials were supported by findings from independent laboratory testing. The ANSR Listeria method can be used as an accurate, rapid, and simple alternative to standard culture methods for detection of Listeria spp. in environmental samples.

Reveal E. coli 2.0 method for detection of Escherichia coli O157:H7 in raw beef.

Reveal E. coli 2.0 is a new lateral-flow immunodiagnostic test for detection of E. coli O157:H7 and O157:NM in raw beef trim and ground beef. Compared with the original Reveal E. coli O157:H7 assay, the new test utilizes a unique antibody combination resulting in improved test specificity. The device architecture and test procedure have also been modified, and a single enrichment protocol was developed which allows the test to be performed at any point during an enrichment period of 12 to 20 h. Results of inclusivity and exclusivity testing showed that the test is specific for E. coli serotypes O157:H7 and O157:NM, with the exception of two strains of O157:H38 and one strain of O157:H43 which produced positive reactions. In internal and independent laboratory trials comparing the Reveal 2.0 method to the U.S. Department of Agriculture-Food Safety and Inspection Service reference culture procedure for detection of E. coli O157:H7 in 65 and 375 g raw beef trim and ground beef samples, there were no statistically significant differences in method performance with the exception of a single internal trial with 375 g ground beef samples in which the Reveal method produced significantly more positive results. There were no unconfirmed positive results by the Reveal assay, for specificity of 100%. Results of ruggedness testing showed that the Reveal test produces accurate results even with substantial deviation in sample volume or device incubation time or temperature. However, addition of the promoter reagent to the test sample prior to introducing the test device is essential to proper test performance.

A hierarchical approach employing metabolic and gene expression profiles to identify the pathways that confer cytotoxicity in HepG2 cells.

BACKGROUND: Free fatty acids (FFA) and tumor necrosis factor alpha (TNF-alpha) have been implicated in the pathogenesis of many obesity-related metabolic disorders. When human hepatoblastoma cells (HepG2) were exposed to different types of FFA and TNF-alpha, saturated fatty acid was found to be cytotoxic and its toxicity was exacerbated by TNF-alpha. In order to identify the processes associated with the toxicity of saturated FFA and TNF-alpha, the metabolic and gene expression profiles were measured to characterize the cellular states. A computational model was developed to integrate these disparate data to reveal the underlying pathways and mechanisms involved in saturated fatty acid toxicity. RESULTS: A hierarchical framework consisting of three stages was developed to identify the processes and genes that regulate the toxicity. First, discriminant analysis identified that fatty acid oxidation and intracellular triglyceride accumulation were the most relevant in differentiating the cytotoxic phenotype. Second, gene set enrichment analysis (GSEA) was applied to the cDNA microarray data to identify the transcriptionally altered pathways and processes. Finally, the genes and gene sets that regulate the metabolic responses identified in step 1 were identified by integrating the expression of the enriched gene sets and the metabolic profiles with a multi-block partial least squares (MBPLS) regression model. CONCLUSION: The hierarchical approach suggested potential mechanisms involved in mediating the cytotoxic and cytoprotective pathways, as well as identified novel targets, such as NADH dehydrogenases, aldehyde dehydrogenases 1A1 (ALDH1A1) and endothelial membrane protein 3 (EMP3) as modulator of the toxic phenotypes. These predictions, as well as, some specific targets that were suggested by the analysis were experimentally validated.