Validity assessment of Michigan's proposed qPCR threshold value for rapid water-quality monitoring of E. coli contamination.

Michigan's water-quality standards specify that E. coli concentrations at bathing beaches must not exceed 300 E. coli per 100 mL, as determined by the geometric mean of culture-based concentrations in three or more representative samples from a given beach on a given day. Culture-based analysis requires 18⁠-⁠24 h to complete, so results are not available on the day of sampling. This one-day delay is problematic because results cannot be used to prevent recreation at beaches that are unsafe on the sampling day, nor do they reliably indicate whether recreation should be prevented the next day, due to high between-day variability in E. coli concentrations demonstrated by previous studies. By contrast, qPCR-based E. coli concentrations can be obtained in 3-4 h, making same-day beach notification decisions possible. Michigan has proposed a qPCR threshold value (qTV) for E. coli of 1.863 log10 gene copies per reaction as a potential equivalent value to the state standard, based on statistical analysis of a set of state-wide training data from 2016 to 2018. The main purpose of the present study is to assess the validity of the proposed qTV by determining whether the implied qPCR-based beach notification decisions agree well with culture-based decisions on two sets of test data from 2016⁠-⁠2018 (6,564 samples) and 2019-2020 (3,205 samples), and whether performance of the proposed qTV is similar on the test and training data. The results show that performance of Michigan's proposed qTV on both sets of test data was consistently good (e.g., 95% agreement with culture-based beach notification decisions during 2019⁠-⁠2020) and was as good as or better than its performance on the training data set. The false-negative rate for the proposed qTV was 25-29%, meaning that beach notification decisions based on the qTV would be expected to permit recreation on the day of sampling in 25-29% of cases where the beach exceeds the state standard for FIB contamination. This false-negative rate is higher than one would hope to see but is well below the corresponding error rate for culture-based decisions, which permit recreation at beaches that exceed the state standard on the day of sampling in 100% of cases because of the one-day delay in obtaining results. The key advantage of qPCR-based analysis is that it permits a large percentage (71-75%) of unsafe beaches to be identified in time to prevent recreation on the day of sampling.

A comparison of E. coli concentration estimates quantified by the EPA and a Michigan laboratory network using EPA Draft Method C.

We evaluated data from 10 laboratories that analyzed water samples from 82 recreational water sites across the state of Michigan between 2016 and 2018. Water sample replicates were analyzed by experienced U.S. Environmental Protection Agency (EPA) analysts and Michigan laboratories personnel, many of whom were newly trained, using EPA Draft Method C-a rapid quantitative polymerase chain reaction (qPCR) technique that provides same day Escherichia coli (E. coli) concentration results. Beach management decisions (i.e. remain open or issue an advisory or closure) based on E. coli concentration estimates obtained by Michigan labs and by the EPA were compared; the beach management decision agreed in 94% of the samples analyzed. We used the Wilcoxon one-sample signed rank test and nonparametric quantile regression to assess (1) the degree of agreement between E. coli concentrations quantified by Michigan labs versus the EPA and (2) Michigan lab E. coli measurement precision, relative to EPA results, in different years and water body types. The median quantile regression curve for Michigan labs versus EPA approximated the 1:1 line of perfect agreement more closely as years progressed. Similarly, Michigan lab E. coli estimates precision also demonstrated yearly improvements. No meaningful difference was observed in the degree of association between Michigan lab and EPA E. coli concentration estimates for inland lake and Great Lakes samples (median regression curve average slopes 0.93 and 0.95, respectively). Overall, our study shows that properly trained laboratory personnel can perform Draft Method C to a degree comparable with experienced EPA analysts. This allows health departments that oversee recreational water quality monitoring to be confident in qPCR results generated by the local laboratories responsible for analyzing the water samples.

Evaluation of multiple laboratory performance and variability in analysis of recreational freshwaters by a rapid Escherichia coli qPCR method (Draft Method C).

There is interest in the application of rapid quantitative polymerase chain reaction (qPCR) methods for recreational freshwater quality monitoring of the fecal indicator bacteria Escherichia coli (E. coli). In this study we determined the performance of 21 laboratories in meeting proposed, standardized data quality acceptance (QA) criteria and the variability of target gene copy estimates from these laboratories in analyses of 18 shared surface water samples by a draft qPCR method developed by the U.S. Environmental Protection Agency (EPA) for E. coli. The participating laboratories ranged from academic and government laboratories with more extensive qPCR experience to "new" water quality and public health laboratories with relatively little previous experience in most cases. Failures to meet QA criteria for the method were observed in 24% of the total 376 test sample analyses. Of these failures, 39% came from two of the "new" laboratories. Likely factors contributing to QA failures included deviations in recommended procedures for the storage and preparation of reference and control materials. A master standard curve calibration model was also found to give lower overall variability in log10 target gene copy estimates than the delta-delta Ct (ΔΔCt) calibration model used in previous EPA qPCR methods. However, differences between the mean estimates from the two models were not significant and variability between laboratories was the greatest contributor to overall method variability in either case. Study findings demonstrate the technical feasibility of multiple laboratories implementing this or other qPCR water quality monitoring methods with similar data quality acceptance criteria but suggest that additional practice and/or assistance may be valuable, even for some more generally experienced qPCR laboratories. Special attention should be placed on providing and following explicit guidance on the preparation, storage and handling of reference and control materials.

Standardized data quality acceptance criteria for a rapid Escherichia coli qPCR method (Draft Method C) for water quality monitoring at recreational beaches.

There is growing interest in the application of rapid quantitative polymerase chain reaction (qPCR) and other PCR-based methods for recreational water quality monitoring and management programs. This interest has strengthened given the publication of U.S. Environmental Protection Agency (EPA)-validated qPCR methods for enterococci fecal indicator bacteria (FIB) and has extended to similar methods for Escherichia coli (E. coli) FIB. Implementation of qPCR-based methods in monitoring programs can be facilitated by confidence in the quality of the data produced by these methods. Data quality can be determined through the establishment of a series of specifications that should reflect good laboratory practice. Ideally, these specifications will also account for the typical variability of data coming from multiple users of the method. This study developed proposed standardized data quality acceptance criteria that were established for important calibration model parameters and/or controls from a new qPCR method for E. coli (EPA Draft Method C) based upon data that was generated by 21 laboratories. Each laboratory followed a standardized protocol utilizing the same prescribed reagents and reference and control materials. After removal of outliers, statistical modeling based on a hierarchical Bayesian method was used to establish metrics for assay standard curve slope, intercept and lower limit of quantification that included between-laboratory, replicate testing within laboratory, and random error variability. A nested analysis of variance (ANOVA) was used to establish metrics for calibrator/positive control, negative control, and replicate sample analysis data. These data acceptance criteria should help those who may evaluate the technical quality of future findings from the method, as well as those who might use the method in the future. Furthermore, these benchmarks and the approaches described for determining them may be helpful to method users seeking to establish comparable laboratory-specific criteria if changes in the reference and/or control materials must be made.

Evidence of isoprenoid precursor toxicity in Bacillus subtilis.

The mevalonic acid (MVA) and methylerythritol phosphate (MEP) pathways for isoprenoid biosynthesis both culminate in the production of the two-five carbon prenyl diphosphates: dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP). These are the building blocks for higher isoprenoids, including many that have industrial and pharmaceutical applications. With growing interest in producing commercial isoprenoids through microbial engineering, reports have appeared of toxicity associated with the accumulation of prenyl diphosphates in Escherichia coli expressing a heterologous MVA pathway. Here we explored whether similar prenyl diphosphate toxicity, related to MEP pathway flux, could also be observed in the bacterium Bacillus subtilis. After genetic and metabolic manipulations of the endogenous MEP pathway in B. subtilis, measurements of cell growth, MEP pathway flux, and DMAPP contents suggested cytotoxicity related to prenyl diphosphate accumulation. These results have implications as to understanding the factors impacting isoprenoid biosynthesis in microbial systems.

Isoprene synthase activity parallels fluctuations of isoprene release during growth of Bacillus subtilis.

Isoprene is a volatile metabolite of uncertain function in plants, animals, and bacteria. Here, we demonstrate that the isoprene-producing bacterium, Bacillus subtilis, contains an isoprene synthase activity that catalyzes dimethylallyl diphosphate-dependent isoprene formation. Although the enzyme was very labile, it was demonstrated in both permeabilized cells and in partially purified cell extracts. Its activity was optimal at pH 6.2, required low levels of a divalent cation, and appears distinct from chloroplast isoprene synthases. When grown in a bioreactor, B. subtilis cells released isoprene in three distinct phases; using permeabilized cells, it was shown that isoprene synthase activity rose and fell in parallel with each phase. These results suggest that isoprene synthesis is highly regulated in B. subtilis and further research in this model system may shed light on the role of isoprene formation in biological systems.