Wastewater-based testing for SARS-CoV-2 is a novel tool for public health monitoring, but additional laboratory capacity is needed to provide routine monitoring at all locations where it has the potential to be useful. Few standardization practices for SARS-CoV-2 wastewater analysis currently exist, and quality assurance/quality control procedures may vary across laboratories. Alongside counterparts at many academic institutions, we built out a laboratory for routine monitoring of wastewater at the University of California, Berkeley. Here, we detail our group’s establishment of a wastewater testing laboratory including standard operating procedures, laboratory buildout and workflow, and a quality assurance plan. We present a complete data analysis pipeline and quality scoring framework and discuss the data reporting process. We hope that this information will aid others at research institutions, public health departments, and wastewater agencies in developing programs to support wastewater monitoring for public health decision-making.

The development of novel antibiotics is essential because the current arsenal of antimicrobials will soon be ineffective due to the widespread occurrence of antibiotic resistance. The development of naturally occurring cationic antimicrobial peptides (CAMPs) for therapeutics to combat antibiotic resistance has been hampered by high production costs and protease sensitivity, among other factors.

Ceragenins are a family of synthetic amphipathic molecules designed to mimic the properties of naturally-occurring cationic antimicrobial peptides (CAMPs). Although ceragenins have potent antimicrobial activity, whether their mode of action is similar to that of CAMPs has remained elusive. Here we report the results of a comparative study of the bacterial responses to two well-studied CAMPs, LL37 and colistin, and two ceragenins with related structures, CSA13 and CSA131. Using transcriptomic and proteomic analyses, we found that Escherichia coli responds similarly to both CAMPs and ceragenins by inducing a Cpx envelope stress response. However, whereas E. coli exposed to CAMPs increased expression of genes involved in colanic acid biosynthesis, bacteria exposed to ceragenins specifically modulated functions related to phosphate transport, indicating distinct mechanisms of action between these two classes of molecules. Although traditional genetic approaches failed to identify genes that confer high-level resistance to ceragenins, using a Clustered Regularly Interspaced Short Palindromic Repeats interference (CRISPRi) approach we identified E. coli essential genes that when knocked down modify sensitivity to these molecules. Comparison of the essential gene-antibiotic interactions for each of the CAMPs and ceragenins identified both overlapping and distinct dependencies for their antimicrobial activities. Overall, this study indicates that while some bacterial responses to ceragenins overlap with those induced by naturally-occurring CAMPs, these synthetic molecules target the bacterial envelope using a distinctive mode of action.

Wastewater-based testing for SARS-CoV-2 is a novel tool for public health monitoring, but additional laboratory capacity is needed to provide routine monitoring at all locations where it has the potential to be useful. Few standardization practices for SARS-CoV-2 wastewater analysis currently exist, and quality assurance/quality control procedures may vary across laboratories. Alongside counterparts at many academic institutions, we built out a laboratory for routine monitoring of wastewater at the University of California, Berkeley. Here, we detail our group's establishment of a wastewater testing laboratory including standard operating procedures, laboratory buildout and workflow, and a quality assurance plan. We present a complete data analysis pipeline and quality scoring framework and discuss the data reporting process. We hope that this information will aid others at research institutions, public health departments, and wastewater agencies in developing programs to support wastewater monitoring for public health decision-making.

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