Accounting for the microbial assembly of each process in wastewater treatment plants (WWTPs): study of four WWTPs receiving similar influent streams.

We evaluated a unique model in which four full-scale wastewater treatment plants (WWTPs) with the same treatment schematic and fed with similar influent wastewater were tracked over an 8-month period to determine whether the community assembly would differ in the activated sludge (AS) and sand filtration (SF) stages. For each WWTP, AS and SF achieved an average of 1-log10 (90%) and <0.02-log10 (5%) reduction of total cells, respectively. Despite the removal of cells, both AS and SF had a higher alpha and beta diversity compared to the influent microbial community. Using the Sloan neutral model, it was observed that AS and SF were individually dominated by different assembly processes. Specifically, microorganisms from influent to AS were predominantly determined by the selective niche process for all WWTPs, while the microbial community in the SF was relatively favored by a stochastic, random migration process, except two WWTPs. AS also contributed more to the final effluent microbial community compared with the SF. Given that each WWTP operates the AS independently and that there is a niche selection process driven mainly by the chemical oxygen demand concentration, operational taxonomic units unique to each of the WWTPs were also identified. The findings from this study indicate that each WWTP has its distinct microbial signature and could be used for source-tracking purposes.IMPORTANCEThis study provided a novel concept that microorganisms follow a niche assembly in the activated sludge (AS) tank and that the AS contributed more than the sand filtration process toward the final microbial signature that is unique to each treatment plant. This observation highlights the importance of understanding the microbial community selected by the AS stage, which could contribute toward source-tracking the effluent from different wastewater treatment plants.

SARS-CoV-2 wastewater-based epidemiology in an enclosed compound: A 2.5-year survey to identify factors contributing to local community dissemination.

Long-term (>2.5 years) surveillance of SARS-CoV-2 RNA concentrations in wastewater was conducted within an enclosed university compound. This study aims to demonstrate how coupling wastewater-based epidemiology (WBE) with meta-data can identify which factors contribute toward the dissemination of SARS-CoV-2 within a local community. Throughout the pandemic, the temporal dynamics of SARS-CoV-2 RNA concentrations were tracked by quantitative polymerase chain reaction and analyzed in the context of the number of positive swab cases, the extent of human movement, and intervention measures. Our findings suggest that during the early phase of the pandemic, when strict lockdown was imposed, the viral titer load in the wastewater remained below detection limits, with <4 positive swab cases reported over a 14-day period in the compound. After the lockdown was lifted and global travel gradually resumed, SARS-CoV-2 RNA was first detected in the wastewater on 12 August 2020 and increased in frequency thereafter, despite high vaccination rates and mandatory face-covering requirements in the community. Accompanied by a combination of the Omicron surge and significant global travel by community members, SARS-CoV-2 RNA was detected in most of the weekly wastewater samples collected in late December 2021 and January 2022. With the cease of mandatory face covering, SARS-CoV-2 was detected in at least two of the four weekly wastewater samples collected from May through August 2022. Retrospective Nanopore sequencing revealed the presence of the Omicron variant in the wastewater with a multitude of amino acid mutations, from which we could infer the likely geographical origins through bioinformatic analysis. This study demonstrated that long-term tracking of the temporal dynamics and sequencing of variants in wastewater would aid in identifying which factors contribute the most to SARS-CoV-2 dissemination within the local community, facilitating an appropriate public health response to control future outbreaks as we now live with endemic SARS-CoV-2.

Efficient in planta production of amidated antimicrobial peptides that are active against drug-resistant ESKAPE pathogens.

Antimicrobial peptides (AMPs) are promising next-generation antibiotics that can be used to combat drug-resistant pathogens. However, the high cost involved in AMP synthesis and their short plasma half-life render their clinical translation a challenge. To address these shortcomings, we report efficient production of bioactive amidated AMPs by transient expression of glycine-extended AMPs in Nicotiana benthamiana line expressing the mammalian enzyme peptidylglycine α-amidating mono-oxygenase (PAM). Cationic AMPs accumulate to substantial levels in PAM transgenic plants compare to nontransgenic N. benthamiana. Moreover, AMPs purified from plants exhibit robust killing activity against six highly virulent and antibiotic resistant ESKAPE pathogens, prevent their biofilm formation, analogous to their synthetic counterparts and synergize with antibiotics. We also perform a base case techno-economic analysis of our platform, demonstrating the potential economic advantages and scalability for industrial use. Taken together, our experimental data and techno-economic analysis demonstrate the potential use of plant chassis for large-scale production of clinical-grade AMPs.

Discovering, Characterizing, and Applying Acyl Homoserine Lactone-Quenching Enzymes to Mitigate Microbe-Associated Problems Under Saline Conditions.

Quorum quenching (QQ) is proposed as a new strategy for mitigating microbe-associated problems (e.g., fouling, biocorrosion). However, most QQ agents reported to date have not been evaluated for their quenching efficacies under conditions representative of seawater desalination plants, cooling towers or marine aquaculture. In this study, bacterial strains were isolated from Saudi Arabian coastal environments and screened for acyl homoserine lactone (AHL)-quenching activities. Five AHL quenching bacterial isolates from the genera Pseudoalteromonas, Pontibacillus, and Altererythrobacter exhibited high AHL-quenching activity at a salinity level of 58 g/L and a pH of 7.8 at 50°C. This result demonstrates the potential use of these QQ bacteria in mitigating microbe-associated problems under saline and alkaline conditions at high (>37°C) temperatures. Further characterizations of the QQ efficacies revealed two bacterial isolates, namely, Pseudoalteromonas sp. L11 and Altererythrobacter sp. S1-5, which could possess enzymatic QQ activity. The genome sequences of L11 and S1-5 with a homologous screening against reported AHL quenching genes suggest the existence of four possible QQ coding genes in each strain. Specifically, two novel AHL enzymes, AiiAS1-5 and EstS1-5 from Altererythrobacter sp. S1-5, both contain signal peptides and exhibit QQ activity over a broad range of pH, salinity, and temperature values. In particular, AiiAS1-5 demonstrated activity against a wide spectrum of AHL molecules. When tested against three bacterial species, namely, Aeromonas hydrophila, Pseudomonas aeruginosa, and Vibrio alginolyticus, AiiAS1-5 was able to inhibit the motility of all three species under saline conditions. The biofilm formation associated with P. aeruginosa was also significantly inhibited by AiiAS1-5. AiiAS1-5 also reduced the quorum sensing-mediated virulence traits of A. hydrophila, P. aeruginosa, and V. alginolyticus during the mid and late exponential phases of cell growth. The enzyme did not impose any detrimental effects on cell growth, suggesting a lower potential for the target bacterium to develop resistance over long-term exposure. Overall, this study suggested that some QQ enzymes obtained from the bacteria that inhabit saline environments under high temperatures have potential applications in the mitigation of microbe-associated problems.

Salinity-Mediated Increment in Sulfate Reduction, Biofilm Formation, and Quorum Sensing: A Potential Connection Between Quorum Sensing and Sulfate Reduction?

Biocorrosion in marine environment is often associated with biofilms of sulfate reducing bacteria (SRB). However, not much information is available on the mechanism underlying exacerbated rates of SRB-mediated biocorrosion under saline conditions. Using Desulfovibrio (D.) vulgaris and Desulfobacterium (Db.) corrodens as model SRBs, the enhancement effects of salinity on sulfate reduction, N-acyl homoserine lactone (AHL) production and biofilm formation by SRBs were demonstrated. Under saline conditions, D. vulgaris and Db. corrodens exhibited significantly higher specific sulfate reduction and specific AHL production rates as well as elevated rates of biofilm formation compared to freshwater medium. Salinity-induced enhancement traits were also confirmed at transcript level through reverse transcription quantitative polymerase chain reaction (RT-qPCR) approach, which showed salinity-influenced increase in the expression of genes associated with carbon metabolism, sulfate reduction, biofilm formation and histidine kinase signal transduction. In addition, by deploying quorum sensing (QS) inhibitors, a potential connection between sulfate reduction and AHL production under saline conditions was demonstrated, which is most significant during early stages of sulfate metabolism. The findings collectively revealed the interconnection between QS, sulfate reduction and biofilm formation among SRBs, and implied the potential of deploying quorum quenching approaches to control SRB-based biocorrosion in saline conditions.

Inactivation and Gene Expression of a Virulent Wastewater Escherichia coli Strain and the Nonvirulent Commensal Escherichia coli DSM1103 Strain upon Solar Irradiation.

This study examined the decay kinetics and molecular responses of two Escherichia coli strains upon solar irradiation. The first is E. coli PI-7, a virulent and antibiotic-resistant strain that was isolated from wastewater and carries the emerging NDM-1 antibiotic resistance gene. The other strain, E. coli DSM1103, displayed lower virulence and antibiotic resistance than E. coli PI-7. In a buffer solution, E. coli PI-7 displayed a longer lag phase prior to decay and a longer half-life compared with E. coli DSM1103 (6.64 ± 0.63 h and 2.85 ± 0.46 min vs 1.33 ± 0.52 h and 2.04 ± 0.36 min). In wastewater, both E. coli strains decayed slower than they did in buffer. Although solar irradiation remained effective in reducing the numbers of both strains by more than 5-log10 in <24 h, comparative genomics and transcriptomics revealed differences in the genomes and overall regulation of genes between the two E. coli strains. A wider arsenal of genes related to oxidative stress, cellular repair and protective mechanisms were upregulated in E. coli PI-7. Subpopulations of E. coli PI-7 expressed genes related to dormancy and persister cell formation during the late decay phase, which may have accounted for its prolonged persistence. Upon prolonged solar irradiation, both E. coli strains displayed upregulation of genes related to horizontal gene transfer and antibiotic resistance. Virulence functions unique to E. coli PI-7 were also upregulated. Our findings collectively indicated that, whereas solar irradiation is able to reduce total cell numbers, viable E. coli remained and expressed genes that enable survival despite solar treatment. There remains a need for heightened levels of concern regarding risks arising from the dissemination of E. coli that may remain viable in wastewater after solar irradiation.

A simple strategy for extracellular production of CotA laccase in Escherichia coli and decolorization of simulated textile effluent by recombinant laccase.

Laccases are green oxidases with a number of potential industrial applications. In this study, recombinant Bacillus subtilis CotA laccase was secreted by Escherichia coli via both the α-hemolysin secretion system and the YebF secretion system after microaerobic induction. Meanwhile, we discovered a much simpler approach for extracellular production of recombinant CotA laccase from E. coli, involving alternation of induction conditions to release recombinant CotA following intracellular expression. By optimizing the induction parameters, the extracellular yield of recombinant CotA laccase was improved from 157.4 to 2401.3 U/L after 24 h of induction. This strategy could be suitable for large-scale production of CotA laccase for industrial use. Recombinant CotA laccase was purified by Ni2+ affinity chromatography in a single step and showed similar biochemical properties to wild-type laccase. Purified as well as crude recombinant CotA laccase efficiently decolorized seven structurally different dyes. The decolorization capability of recombinant CotA laccase under harsh conditions was investigated by incubation of the enzyme with a simulated textile effluent (STE) with pH 11.6, 3.5 % salinity and peak absorbance of 10.42. Recombinant CotA laccase efficiently decolorized 77.0 % of STE after 48 h reaction, demonstrating the potential of this enzyme for industrial dye effluent treatment.

Quorum Sensing and the Use of Quorum Quenchers as Natural Biocides to Inhibit Sulfate-Reducing Bacteria.

Sulfate-reducing bacteria (SRB) are one of the main protagonist groups of biocorrosion in the seawater environment. Given their principal role in biocorrosion, it remains a crucial task to develop strategies to reduce the abundance of SRBs. Conventional approaches include the use of biocides and antibiotics, which can impose health, safety, and environmental concerns. This review examines an alternative approach to this problem. This is achieved by reviewing the role of quorum sensing (QS) in SRB populations and its impact on the biofilm formation process. Genome databases of SRBs are mined to look for putative QS systems and homologous protein sequences representative of autoinducer receptors or synthases. Subsequently, this review puts forward the potential use of quorum quenchers as natural biocides against SRBs and outlines the potential strategies for the implementation of this approach.

Isolation and Characterization of NDM-Positive Escherichia coli from Municipal Wastewater in Jeddah, Saudi Arabia.

The emergence of resistance to last-resort antibiotics is a public health concern of global scale. Besides direct person-to-person propagation, environmental pathways might contribute to the dissemination of antibiotic-resistant bacteria and antibiotic resistance genes (ARGs). Here, we describe the incidence of blaNDM-1, a gene conferring resistance to carbapenems, in the wastewater of the city of Jeddah, Saudi Arabia, over a 1-year period. blaNDM-1 was detected at concentrations ranging from 10(4) to 10(5) copies/m(3) of untreated wastewater during the entire monitoring period. These results indicate the ubiquity and high incidence of blaNDM-1 in the local wastewater. To track the bacteria carrying blaNDM-1, we isolated Escherichia coli PI7, a strain of sequence type 101 (ST101), from wastewater around the Hajj event in October 2013. Genome sequencing of this strain revealed an extensive repertoire of ARGs as well as virulence and invasive traits. These traits were further confirmed by antibiotic resistance profiling and in vitro cell internalization in HeLa cell cultures. Given that this strain remains viable even after a certain duration in the sewerage, and that Jeddah lacks a robust sanitary infrastructure to fully capture all generated sewage, the presence of this bacterium in the untreated wastewater represents a potential hazard to the local public health. To the best of our knowledge, this is the first report of a blaNDM-1-positive E. coli strain isolated from a nonnosocomial environment in Saudi Arabia and may set a priority concern for the need to establish improved surveillance for carbapenem-resistant E. coli in the country and nearby regions.