Interplay of Biologically Active Carbon Filtration and Chlorine-Based Disinfection in Mitigating the Dissemination of Antibiotic Resistance Genes in Water Reuse Distribution Systems.

Appropriate management approaches are needed to minimize the proliferation of antibiotic resistance genes (ARGs) in reclaimed water distribution systems (RWDSs). Six laboratory-scale RWDSs were operated over 3 years receiving influent with or without biologically active carbon (BAC) filtration + chlorination, chloramination, or no disinfectant residual. Shotgun metagenomic sequencing was applied toward comprehensive characterization of resistomes, focusing on total ARGs, ARG mobility, and specific ARGs of clinical concern. ARGs such as aadA, bacA, blaOXA, mphE, msrE, sul1, and sul2 were found to be particularly sensitive to varying RWDS conditions. BAC filtration with chlorination most effectively achieved and maintained the lowest levels of nearly all metagenomically derived antibiotic resistance indicators. However, BAC filtration or addition of residual disinfectants alone tended to increase these indicators. Biofilm and sediment compartments harbored ARGs in disinfected systems, presenting a concern for their release to bulk water. Relative and absolute abundances of most ARGs tended to decrease with water age (up to 5 days), with notable exceptions in BAC-filtered chloraminated and no residual systems. Superchlorination of unfiltered water especially raised concerns in terms of elevation of clinically relevant and mobile ARGs. This study revealed that BAC filtration and disinfection must be carefully coordinated in order to effectively mitigate ARG dissemination via RWDSs.

Mapping the Terrain for Pathogen Persistence and Proliferation in Non-potable Reuse Distribution Systems: Interactive Effects of Biofiltration, Disinfection, and Water Age.

Diverse pathogens can potentially persist and proliferate in reclaimed water distribution systems (RWDSs). The goal of this study was to evaluate interactive effects of reclaimed water treatments and water age on persistence and proliferation of multiple fecal (e.g., Klebsiella, Enterobacter) and non-fecal (e.g., Legionella, mycobacteria) gene markers in RWDSs. Six laboratory-scale RWDSs were operated in parallel receiving the influent with or without biologically active carbon (BAC) filtration + chlorination, chloramination, or no disinfectant residual. After 3 years of operation, the RWDSs were subject to sacrificial sampling and shotgun metagenomic sequencing. We developed an in-house metagenome-derived pathogen quantification pipeline, validated by quantitative polymerase chain reaction and mock community analysis, to estimate changes in abundance of ∼30 genera containing waterborne pathogens. Microbial community composition in the RWDS bulk water, biofilm, and sediments was clearly shaped by BAC filtration, disinfectant conditions, and water age. Key commonalities were noted in the ecological niches occupied by fecal pathogen markers in the RWDSs, while non-fecal pathogen markers were more varied in their distribution. BAC-filtration + chlorine was found to most effectively control the widest range of target genera. However, filtration alone or chlorine secondary disinfection alone resulted in proliferation of some of these genera containing waterborne pathogens.

Investigation into the misincorporation of norleucine into a recombinant protein vaccine candidate.

A high level of norleucine misincorporation was detected in a recombinant methionine-rich protein vaccine candidate expressed in E. coli K12. An investigation was conducted to evaluate a simple remediation strategy to reduce norleucine misincorporation and to determine if the phenomenon was either (a) due to the depletion of methionine during fermentation, (b) a result of the cultivation environment, or (c) a strain-specific effect. While supplementation with exogenous methionine improved product quality, the undesirable biosynthesis of non-standard amino acids such as norleucine and norvaline persisted. In contrast, non-standard amino acid biosynthesis was quickly minimized upon selection of an appropriate fed-batch process control strategy, fermentation medium, and nutrient feed. By expressing the same protein in E. coli BL21(DE3), it was determined that the biosynthesis of norleucine and norvaline, and the misincorporation of norleucine into the protein were primarily attributed to the use of E. coli K12 as the host for protein expression.

A Model for Dimerization of the SOX Group E Transcription Factor Family.

Group E members of the SOX transcription factor family include SOX8, SOX9, and SOX10. Preceding the high mobility group (HMG) domain in each of these proteins is a thirty-eight amino acid region that supports the formation of dimers on promoters containing tandemly inverted sites. The purpose of this study was to obtain new structural insights into how the dimerization region functions with the HMG domain. From a mutagenic scan of the dimerization region, the most essential amino acids of the dimerization region were clustered on the hydrophobic face of a single, predicted amphipathic helix. Consistent with our hypothesis that the dimerization region directly contacts the HMG domain, a peptide corresponding to the dimerization region bound a preassembled HMG-DNA complex. Sequence conservation among Group E members served as a basis to identify two surface exposed amino acids in the HMG domain of SOX9 that were necessary for dimerization. These data were combined to make a molecular model that places the dimerization region of one SOX9 protein onto the HMG domain of another SOX9 protein situated at the opposing site of a tandem promoter. The model provides a detailed foundation for assessing the impact of mutations on SOX Group E transcription factors.