Assessing cross-laboratory performance for quantifying coliphage using EPA Method 1642.

AIMS: Widespread adoption of the new U.S. Environmental Protection Agency (USEPA) Method 1642 for enumeration of coliphage in recreational water requires demonstration that laboratories consistently meet internal method performance goals and yield results that are consistent across laboratories. METHODS AND RESULTS: Here we assess the performance of six laboratories processing a series of blind wastewater- and coliphage-spiked samples along with laboratory blanks. All laboratories met the method-defined recovery requirements when performance was averaged across samples, with the few failures on individual samples mostly occurring for less-experienced laboratories on the initial samples processed. Failures that occurred on later samples were generally attributed to easily correctable activities. Failure rates were higher for somatic vs. F+ coliphage, attributable to the more stringent performance criteria associated with somatic coliphage. There was no difference in failure rate between samples prepared in a marine water matrix compared to that in phosphate-buffered saline. CONCLUSIONS: Variation among laboratories was similar to that previously reported for enterococci, the current bacterial indicator used for evaluating beach water quality for public health protection. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings suggest that laboratory performance is not an inhibitor to the adoption of coliphage as a new indicator for assessing recreational health risk.

Examining Racial/Ethnic Differences in the Association of Victimization and Suicidal Thoughts and Behaviors with Alcohol Use Among Sexual Minority Youth.

Purpose: Integrating Minority Stress Theory and Bagge and Sher's Theoretical Framework of the Alcohol-Suicide Attempt Relation, this study aimed to test whether experiencing both minority stress and suicidal thoughts and behaviors (STBs) had a greater strength of association with Latinx and Black sexual minority youth (SMY)'s alcohol use compared with that of White SMY. Methods: Using data on 2341 non-Latinx Black, Latinx, and non-Latinx White SMY from the 2015 and 2017 Youth Risk Behavior Surveys, we tested the prevalence of STBs, victimization, and alcohol use for Black and Latinx participants compared with White participants. Multivariate logistic regression analyses tested the main effects of STBs, victimization, and race/ethnicity on alcohol use. Finally, interaction terms assessed the interaction among STBs, victimization, and race/ethnicity on alcohol use. Results: The results supported our hypothesis, based on Minority Stress Theory, that experiencing victimization would be associated with greater alcohol use. Results also supported Bagge and Sher's theoretical framework showing that suicide plan and attempts were associated with greater alcohol use. When taking all of these stressors into account, results showed that Latinx SMY who experienced victimization had greater current prevalence of alcohol use than their White counterparts. However, Latinx SMY who were victimized and experienced suicidal ideation reported lower alcohol use than White SMY. Conclusion: These findings support the double jeopardy and resiliency hypotheses, which suggest that minority stressors have differing associations for each racial/ethnic SMY group's alcohol use. More research is needed that helps to disentangle the protective and risk factors for alcohol use among Black and Latinx SMY.

Relationship between coliphage and Enterococcus at southern California beaches and implications for beach water quality management.

Coliphage have been suggested as an alternative to fecal indicator bacteria for assessing recreational beach water quality, but it is unclear how frequently and at what types of beaches coliphage produces a different management outcome. Here we conducted side-by-side sampling of male-specific and somatic coliphage by the new EPA dead-end hollow fiber ultrafiltration (D-HFUF-SAL) method and Enterococcus at southern California beaches over two years. When samples were combined for all beach sites, somatic and male-specific coliphage both correlated with Enterococcus. When examined categorically, Enterococcus would have resulted in approximately two times the number of health advisories as somatic coliphage and four times that of male-specific coliphage,using recently proposed thresholds of 60 PFU/100 mL for somatic and 30 PFU/100 mL for male-specific coliphage. Overall, only 12% of total exceedances would have been for coliphage alone. Somatic coliphage exceedances that occurred in the absence of an Enterococcus exceedance were limited to a single site during south swell events, when this beach is known to be affected by nearby minimally treated sewage. Thus, somatic coliphage provided additional valuable health protection information, but may be more appropriate as a supplement to FIB measurements rather than as replacement because: (a) EPA-approved PCR methods for Enterococcus allow a more rapid response, (b) coliphage is more challenging owing to its greater sampling volume and laboratory time requirements, and (c) Enterococcus' long data history has yielded predictive management models that would need to be recreated for coliphage.

Expression and Purification of Recombinant Proteins in Escherichia coli Tagged with the Metal-Binding Proteins SmbP and CusF3H.

The bacterium Escherichia coli is still considered the first option as a microbial cell factory for recombinant protein production, and affinity chromatography is by far the preferred technique for initial purification after protein expression and cell lysis. In this chapter, we describe the methodology to express and purify recombinant proteins in E. coli tagged with the first two metal-binding proteins proposed as fusion partners. They are the small metal-binding protein SmbP and a mutant of the copper resistance protein CusF3H+. There are several advantages of using them as protein tags: they prevent the formation of inclusion bodies by increasing solubility of the target proteins, they enable purification by immobilized metal-affinity chromatography using Ni(II) ions with high purity, and because of their low molecular weights, excellent final yields are obtained for the target proteins after cleavage and removal of the protein tag. Here we also describe the protocol for the production of proteins in the periplasm of E. coli tagged with two SmbP variants that include the PelB or the TorA signal sequences for transport via the Sec or the Tat pathway, respectively. Based on these methods, we consider CusF3H+ and SmbP excellent alternatives as fusion proteins for the production of recombinant proteins in E. coli.

Optimizing Periplasmic Expression in Escherichia coli for the Production of Recombinant Proteins Tagged with the Small Metal-Binding Protein SmbP.

We have previously shown that the small metal-binding protein (SmbP) extracted from the gram-negative bacterium Nitrosomonas europaea can be employed as a fusion protein for the expression and purification of recombinant proteins in Escherichia coli. With the goal of increasing the amounts of SmbP-tagged proteins produced in the E. coli periplasm, we replaced the native SmbP signal peptide with three different signal sequences: two were from the proteins CusF and PelB, for transport via the Sec pathway, and one was the signal peptide from TorA, for transport via the Tat pathway. Expression of SmbP-tagged Red Fluorescent Protein (RFP) using these three alternative signal peptides individually showed a considerable increase in protein levels in the periplasm of E. coli as compared to its level using the SmbP signal sequence. Therefore, for routine periplasmic expression and purification of recombinant proteins in E. coli, we highly recommend the use of the fusion proteins PelB-SmbP or CusF-SmbP, since these signal sequences increase periplasmic production considerably as compared to the wild-type. Our work, finally, demonstrates that periplasmic expression for SmbP-tagged proteins is not limited to the Sec pathway, in that the TorA-SmbP construct can export reasonable quantities of folded proteins to the periplasm. Although the Sec route has been the most widely used, sometimes, depending on the nature of the protein of interest, for example, if it contains cofactors, it is more appropriate to consider using the Tat route over the Sec. SmbP therefore can be recommended in terms of its particular versatility when combined with signal peptides for the two different routes.