Density-based binning of gene clusters to infer function or evolutionary history using GeneGrouper.

MOTIVATION: Identifying variant forms of gene clusters of interest in phylogenetically proximate and distant taxa can help to infer their evolutionary histories and functions. Conserved gene clusters may differ by only a few genes, but these small differences can in turn induce substantial phenotypes, such as by the formation of pseudogenes or insertions interrupting regulation. Particularly as microbial genomes and metagenomic assemblies become increasingly abundant, unsupervised grouping of similar, but not necessarily identical, gene clusters into consistent bins can provide a population-level understanding of their gene content variation and functional homology. RESULTS: We developed GeneGrouper, a command-line tool that uses a density-based clustering method to group gene clusters into bins. GeneGrouper demonstrated high recall and precision in benchmarks for the detection of the 23-gene Salmonella enterica LT2 Pdu gene cluster and four-gene Pseudomonas aeruginosa PAO1 Mex gene cluster among 435 genomes spanning mixed taxa. In a subsequent application investigating the diversity and impact of gene-complete and -incomplete LT2 Pdu gene clusters in 1130 S.enterica genomes, GeneGrouper identified a novel, frequently occurring pduN pseudogene. When investigated in vivo, introduction of the pduN pseudogene negatively impacted microcompartment formation. We next demonstrated the versatility of GeneGrouper by clustering distant homologous gene clusters and variable gene clusters found in integrative and conjugative elements. AVAILABILITY AND IMPLEMENTATION: GeneGrouper software and code are publicly available at https://pypi.org/project/GeneGrouper/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Quaternary Ammonium Compounds: A Chemical Class of Emerging Concern.

Quaternary ammonium compounds (QACs), a large class of chemicals that includes high production volume substances, have been used for decades as antimicrobials, preservatives, and antistatic agents and for other functions in cleaning, disinfecting, personal care products, and durable consumer goods. QAC use has accelerated in response to the COVID-19 pandemic and the banning of 19 antimicrobials from several personal care products by the US Food and Drug Administration in 2016. Studies conducted before and after the onset of the pandemic indicate increased human exposure to QACs. Environmental releases of these chemicals have also increased. Emerging information on adverse environmental and human health impacts of QACs is motivating a reconsideration of the risks and benefits across the life cycle of their production, use, and disposal. This work presents a critical review of the literature and scientific perspective developed by a multidisciplinary, multi-institutional team of authors from academia, governmental, and nonprofit organizations. The review evaluates currently available information on the ecological and human health profile of QACs and identifies multiple areas of potential concern. Adverse ecological effects include acute and chronic toxicity to susceptible aquatic organisms, with concentrations of some QACs approaching levels of concern. Suspected or known adverse health outcomes include dermal and respiratory effects, developmental and reproductive toxicity, disruption of metabolic function such as lipid homeostasis, and impairment of mitochondrial function. QACs' role in antimicrobial resistance has also been demonstrated. In the US regulatory system, how a QAC is managed depends on how it is used, for example in pesticides or personal care products. This can result in the same QACs receiving different degrees of scrutiny depending on the use and the agency regulating it. Further, the US Environmental Protection Agency's current method of grouping QACs based on structure, first proposed in 1988, is insufficient to address the wide range of QAC chemistries, potential toxicities, and exposure scenarios. Consequently, exposures to common mixtures of QACs and from multiple sources remain largely unassessed. Some restrictions on the use of QACs have been implemented in the US and elsewhere, primarily focused on personal care products. Assessing the risks posed by QACs is hampered by their vast structural diversity and a lack of quantitative data on exposure and toxicity for the majority of these compounds. This review identifies important data gaps and provides research and policy recommendations for preserving the utility of QAC chemistries while also seeking to limit adverse environmental and human health effects.

Heterotrophic denitrification: An overlooked factor that contributes to nitrogen removal in n-DAMO mixed culture.

Nitrate/nitrite-dependent anaerobic methane oxidation (n-DAMO) has been recognized as a sustainable process for simultaneous removal of nitrogen and methane. The metabolisms of denitrifying anaerobic methanotrophs, including Candidatus Methanoperedens and Candidatus Methylomirabilis, have been well studied. However, potential roles of heterotrophs co-existing with these anaerobic methanotrophs are generally overlooked. In this study, we pulse-fed methane and nitrate into an anaerobic laboratory sequencing batch bioreactor and enriched a mixed culture with stable nitrate removal rate (NRR) of ∼28 mg NO3--N L-1 d-1. Microbial community analysis indicates abundant heterotrophs, e.g., Arenimonas (5.3%-18.9%) and Fimbriimonadales ATM1 (6.4%), were enriched together with denitrifying anaerobic methanotrophs Ca. Methanoperedens (10.8%-13.2%) and Ca. Methylomirabilis (27.4%-34.3%). The results of metagenomics and batch tests suggested that the denitrifying anaerobic methanotrophs were capable of generating methane-derived intermediates (i.e., formate and acetate), which were employed by non-methanotrophic heterotrophs for denitrification and biomass growth. These findings offer new insights into the roles of heterotrophs in n-DAMO mixed culture, which may help to optimize n-DAMO process for nitrogen removal from wastewater.

Mobilizable antibiotic resistance genes are present in dust microbial communities.

The decades-long global trend of urbanization has led to a population that spends increasing amounts of time indoors. Exposure to microbes in buildings, and specifically in dust, is thus also increasing, and has been linked to various health outcomes and to antibiotic resistance genes (ARGs). These are most efficiently screened using DNA sequencing, but this method does not determine which microbes are viable, nor does it reveal whether their ARGs can actually disseminate to other microbes. We have thus performed the first study to: 1) examine the potential for ARG dissemination in indoor dust microbial communities, and 2) validate the presence of detected mobile ARGs in viable dust bacteria. Specifically, we integrated 166 dust metagenomes from 43 different buildings. Sequences were assembled, annotated, and screened for potential integrons, transposons, plasmids, and associated ARGs. The same dust samples were further investigated using cultivation and isolate genome and plasmid sequencing. Potential ARGs were detected in dust isolate genomes, and we confirmed their placement on mobile genetic elements using long-read sequencing. We found 183 ARGs, of which 52 were potentially mobile (associated with a putative plasmid, transposon or integron). One dust isolate related to Staphylococcus equorum proved to contain a plasmid carrying an ARG that was detected metagenomically and confirmed through whole genome and plasmid sequencing. This study thus highlights the power of combining cultivation with metagenomics to assess the risk of potentially mobile ARGs for public health.

Triclosan Tolerance Is Driven by a Conserved Mechanism in Diverse Pseudomonas Species.

Perturbation of natural microbial communities by antimicrobials, such as triclosan, can result in selection for antibiotic tolerance, which is of particular concern when pathogens are present. Members of the genus Pseudomonas are found in many natural microbial communities and frequently demonstrate increased abundance following triclosan exposure. The pathogen and well-studied model organism Pseudomonas aeruginosa exhibits high triclosan tolerance; however, it is unknown if all Pseudomonas species share this trait or if there are susceptible strains. We characterized the triclosan tolerance phenotypes of diverse Pseudomonas isolates obtained from triclosan-exposed built environments and identified both tolerant and sensitive strains. High tolerance is associated with carriage of the enoyl-acyl carrier reductase (ENR) isozyme gene fabV, compared to the lesser protective effects of efflux or presence of ENRs. Given its unique importance, we examined fabV distribution throughout Pseudomonas species using large-scale phylogenomic analyses. We find fabV presence or absence is largely invariant at the species level but demonstrates multiple gain and loss events in its evolutionary history. We further provide evidence of its presence on mobile genetic elements. Our results demonstrate the surprising variability in triclosan tolerance in Pseudomonas and confirm fabV to be a useful indicator for high triclosan tolerance in Pseudomonas These findings provide a framework for better monitoring of Pseudomonas in triclosan-exposed environments and interpreting effects on species and gene composition.IMPORTANCE Closely related species are typically assumed to demonstrate similar phenotypes driven by underlying conserved genotypes. When monitoring for the effect of antimicrobials on the types of species that may be selected for, this assumption may prove to be incorrect, and identification of additional genetic markers may be necessary. We isolated several phylogenetically diverse members of Pseudomonas from indoor environments and tested their phenotypic tolerance toward the commonly used antimicrobial triclosan. Although Pseudomonas isolates are broadly regarded to be highly triclosan tolerant, we demonstrate the presence of both triclosan-tolerant and -susceptible strains, separated by a difference in tolerance of nearly 3 orders of magnitude. Bioinformatic and experimental investigation demonstrated that the presence of the gene fabV was associated with high tolerance. We demonstrate that fabV is not evenly distributed in all Pseudomonas species and that its presence could be a useful predictor of high triclosan tolerance suitable for antimicrobial monitoring efforts involving triclosan.

Biological Mitigation of Antibiotic Resistance Gene Dissemination by Antioxidant-Producing Microorganisms in Activated Sludge Systems.

Antibiotic resistance is the principal mechanism of an evergrowing bacterial threat. Antibiotic residues in the environment are a major contributor to the spread of antibiotic resistance genes (ARGs). Subinhibitory concentrations of antibiotics cause bacteria to produce reactive oxygen species (ROS), which can lead to mutagenesis and horizontal gene transfer (HGT) of ARGs; however, little is known about the mitigation of ARG dissemination through ROS removal by antioxidants. In this study, we examine how antioxidant-producing microorganisms inoculated in replicate activated sludge systems can biologically mitigate the dissemination of ARGs. Through quantitative polymerase chain reaction (qPCR), we showed that antioxidant-producing microorganisms could decrease the persistence of the RP4 plasmid and alleviate enrichment of ARGs (sul1) and class 1 integrons (intl1). Metagenomic sequencing identified the most diverse resistome and the most mutated Escherichia coli ARGs in the reactor that contained antibiotics but no antioxidant-producing microorganisms, suggesting that antioxidant-producing microorganisms mitigated ARG enrichment and mutation. Host classification revealed that antioxidant-producing microorganisms decreased the diversity of ARG hosts by shaping the microbial community through competition and functional pathway changes. Conjugative experiments demonstrated that conjugative transfer of ARGs could be mitigated by coculture with antioxidant-producing microorganisms. Overall, this is a novel study that shows how ARG enrichment and HGT can be mitigated through bioaugmentation with antioxidant-producing microorganisms.

Structure and Functional Attributes of Bacterial Communities in Premise Plumbing Across the United States.

Microbes that thrive in premise plumbing can have potentially important effects on human health. Yet, how and why plumbing-associated microbial communities vary across broad spatial scales remain undetermined. We characterized the bacterial communities in 496 showerheads collected from across the continental United States. The overall community structure, determined by 16S rRNA gene amplicon sequencing, revealed high levels of bacterial diversity. Although a large fraction of the observed variation in community composition could not be explained, differences in bacterial community composition were associated with water supply (private well water vs public municipal water), water source (groundwater vs surface water), and associated differences in water chemistry (pH and chlorine). Most notably, showerheads in homes supplied with public water had higher abundances of Blastomonas, Mycobacterium, and Porphyrobacter, while Pseudorhodoplanes, Novosphingobium, and Nitrospira were more abundant in those receiving private well water. We conducted shotgun metagenomic analyses on 92 of these samples to assess differences in genomic attributes. Public water-sourced showerheads had communities enriched in genes related to lipid and xenobiotic metabolisms, virulence factors, and antibiotic resistance. In contrast, genes associated with oxidative stress and membrane transporters were over-represented in communities from private well water-sourced showerheads compared to those supplied by public water systems. These results highlight the broad diversity of bacteria found in premise plumbing across the United States and the role of the water source and treatment in shaping the microbial community structure and functional potential.

Association of sociocultural factors with initiation of the kidney transplant evaluation process.

Although research shows that minorities exhibit higher levels of medical mistrust, perceived racism, and discrimination in healthcare settings, the degree to which these underlying sociocultural factors preclude end-stage renal disease (ESRD) patients from initiating kidney transplant evaluation is unknown. We telephone surveyed 528 adult ESRD patients of black or white race referred for evaluation to a Georgia transplant center (N = 3) in 2014-2016. We used multivariable logistic regression to examine associations between sociocultural factors and evaluation initiation, adjusting for demographic, clinical, and socioeconomic characteristics. Despite blacks (n = 407) reporting higher levels of medical mistrust (40.0% vs 26.4%, P < .01), perceived racism (55.5% vs 18.2%, P < .01), and experienced discrimination (29.0% vs 15.7%, P < .01) than whites (n = 121), blacks were only slightly less likely than whites to initiate evaluation (49.6% vs 57.9%, P = .11). However, after adjustment, medical mistrust (odds ratio [OR]: 0.59; 95% confidence interval [CI]: 0.39, 0.91), experienced discrimination (OR: 0.62, 95% CI: 0.41, 0.95), and perceived racism (OR: 0.61; 95% CI: 0.40, 0.92) were associated with lower evaluation initiation. Results suggest that sociocultural disparities exist in early kidney transplant access and occur despite the absence of a significant racial disparity in evaluation initiation. Interventions to reduce disparities in transplantation access should target underlying sociocultural factors, not just race.

Adherence to Antidepressants in Underserved Communities: A Comparison of Electronic Monitoring and Self-report Measures.

Nonadherence to antidepressants is widespread and poses a significant barrier to optimal management and treatment of depression in community settings. The objective of this study was to compare self-reported and electronic monitoring of adherence to antidepressants and to examine the relationship of these measures with depressive symptoms in a medically underserved community. Adherence to antidepressants was measured in 38 primary care patients from the West Philadelphia area using self-report and electronic monitoring (Medication Event Monitoring System caps). Self-report and electronic monitoring of antidepressant adherence showed fair agreement at baseline, slight agreement at 6 weeks, and slight agreement at 12 weeks. Adherence to antidepressants as assessed by electronic monitors was significantly associated with depression remission at 12 weeks [adjusted odds ratio 18.6, 95% confidence interval (1.05, 330.56)]. Compared with electronic monitoring, self-reported adherence tended to overestimate medication adherence to antidepressants. Adherence assessed by electronic monitoring was associated with depression remission.

Impacts of indoor surface finishes on bacterial viability.

Microbes in indoor environments are constantly being exposed to antimicrobial surface finishes. Many are rendered non-viable after spending extended periods of time under low-moisture, low-nutrient surface conditions, regardless of whether those surfaces have been amended with antimicrobial chemicals. However, some microorganisms remain viable even after prolonged exposure to these hostile conditions. Work with specific model pathogens makes it difficult to draw general conclusions about how chemical and physical properties of surfaces affect microbes. Here, we explore the survival of a synthetic community of non-model microorganisms isolated from built environments following exposure to three chemically and physically distinct surface finishes. Our findings demonstrated the differences in bacterial survival associated with three chemically and physically distinct materials. Alkaline clay surfaces select for an alkaliphilic bacterium, Kocuria rosea, whereas acidic mold-resistant paint favors Bacillus timonensis, a Gram-negative spore-forming bacterium that also survives on antimicrobial surfaces after 24 hours of exposure. Additionally, antibiotic-resistant Pantoea allii did not exhibit prolonged retention on antimicrobial surfaces. Our controlled microcosm experiment integrates measurement of indoor chemistry and microbiology to elucidate the complex biochemical interactions that influence the indoor microbiome.

From one species to another: A review on the interaction between chemistry and microbiology in relation to cleaning in the built environment.

Since the advent of soap, personal hygiene practices have revolved around removal, sterilization, and disinfection-both of visible soil and microscopic organisms-for a myriad of cultural, aesthetic, or health-related reasons. Cleaning methods and products vary widely in their recommended use, effectiveness, risk to users or building occupants, environmental sustainability, and ecological impact. Advancements in science and technology have facilitated in-depth analyses of the indoor microbiome, and studies in this field suggest that the traditional "scorched-earth cleaning" mentality-that surfaces must be completely sterilized and prevent microbial establishment-may contribute to long-term human health consequences. Moreover, the materials, products, activities, and microbial communities indoors all contribute to, or remove, chemical species to the indoor environment. This review examines the effects of cleaning with respect to the interaction of chemistry, indoor microbiology, and human health.

Racial disparities in preemptive referral for kidney transplantation in Georgia.

BACKGROUND: Racial disparities persist in access to kidney transplantation. Racial differences in preemptive referral, or referral prior to dialysis start, may explain this discrepancy. METHODS: Patient-level data on kidney transplant referrals (2005-2012) from all Georgia transplant centers were linked to the United States Renal Data System to examine racial disparities in preemptive referral, waitlisting, and living donor transplant. Adjusted logistic regression and Cox proportional hazard models determined the associations between race (African American vs white) and preemptive referral, and placement on the waitlist and receipt of a living donor kidney, respectively. RESULTS: Among 7752 adults referred for transplant evaluation, 20.38% (n = 1580) were preemptively referred. The odds of African Americans being preemptively referred for transplant evaluation were 37% (OR = 0.63; [95% CI: 0.55 0.71]) lower than white patients. Among preemptively referred patients, there was no racial difference (African Americans compared to white patients. HR = 0.96; [95% CI: 0.88, 1.04]) in waitlisting. However, African Americans were 70% less likely than white patients to receive a living donor transplant (HR = 0.30; [95% CI: 0.21, 0.42]). CONCLUSION: Racial disparities in transplant receipt may be partially explained by disparities in preemptive referral. Interventions to reduce racial disparities in kidney transplant access may need to be targeted earlier in the disease process.

Process evaluation of the RaDIANT community study: a dialysis facility-level intervention to increase referral for kidney transplantation.

BACKGROUND: The Reducing Disparities in Access to kidNey Transplantation Community Study (RaDIANT) was an End-Stage Renal Disease (ESRD) Network 6-developed, dialysis facility-level randomized trial testing the effectiveness of a 1-year multicomponent education and quality improvement intervention in increasing referral for kidney transplant evaluation among selected Georgia dialysis facilities. METHODS: To assess implementation of the RaDIANT intervention, we conducted a process evaluation at the conclusion of the intervention period (January-December 2014). We administered a 20-item survey to the staff involved with transplant education in 67 dialysis facilities randomized to participate in intervention activities. Survey items assessed facility participation in the intervention (fidelity and reach), helpfulness and willingness to continue intervention activities (sustainability), suggestions for improving intervention components (sustainability), and factors that may have influenced participation and study outcomes (context). We defined high fidelity to the intervention as completing 11 or more activities, and high participation in an activity as having at least 75% participation across intervention facilities. RESULTS: Staff from 65 of the 67 dialysis facilities completed the questionnaire, and more than half (50.8%) reported high adherence (fidelity) to RaDIANT intervention requirements. Nearly two-thirds (63.1%) of facilities reported that RaDIANT intervention activities were helpful or very helpful, with 90.8% of facilities willing to continue at least one intervention component beyond the study period. Intervention components with high participation emphasized staff and patient-level education, including in-service staff orientations, patient and family education programs, and patient educational materials. Suggested improvements for intervention activities emphasized addressing financial barriers to transplantation, with financial education materials perceived as most helpful among RaDIANT educational materials. Variation in facility-level fidelity of the RADIANT intervention did not significantly influence the mean difference in proportion of patients referred pre- (2013) and post-intervention (2014). CONCLUSIONS: We found high fidelity to the RaDIANT multicomponent intervention at the majority of intervention facilities, with sustainability of select intervention components at intervention facilities and feasibility for dissemination across ESRD Networks. Future modification of the intervention should emphasize financial education regarding kidney transplantation and amend intervention components that facilities perceive as time-intensive or non-sustainable. TRIAL REGISTRATION: Clinicaltrials.gov number NCT02092727 . Registered 13 Mar 2014 (retrospectively registered).

Antimicrobial Chemicals Are Associated with Elevated Antibiotic Resistance Genes in the Indoor Dust Microbiome.

Antibiotic resistance is increasingly widespread, largely due to human influence. Here, we explore the relationship between antibiotic resistance genes and the antimicrobial chemicals triclosan, triclocarban, and methyl-, ethyl-, propyl-, and butylparaben in the dust microbiome. Dust samples from a mixed-use athletic and educational facility were subjected to microbial and chemical analyses using a combination of 16S rRNA amplicon sequencing, shotgun metagenome sequencing, and liquid chromatography tandem mass spectrometry. The dust resistome was characterized by identifying antibiotic resistance genes annotated in the Comprehensive Antibiotic Resistance Database (CARD) from the metagenomes of each sample using the Short, Better Representative Extract Data set (ShortBRED). The three most highly abundant antibiotic resistance genes were tet(W), blaSRT-1, and erm(B). The complete dust resistome was then compared against the measured concentrations of antimicrobial chemicals, which for triclosan ranged from 0.5 to 1970 ng/g dust. We observed six significant positive associations between the concentration of an antimicrobial chemical and the relative abundance of an antibiotic resistance gene, including one between the ubiquitous antimicrobial triclosan and erm(X), a 23S rRNA methyltransferase implicated in resistance to several antibiotics. This study is the first to look for an association between antibiotic resistance genes and antimicrobial chemicals in dust.

N-Terminal-oriented proteogenomics of the marine bacterium roseobacter denitrificans Och114 using N-Succinimidyloxycarbonylmethyl)tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP) labeling and diagonal chromatography.

Given the ease of whole genome sequencing with next-generation sequencers, structural and functional gene annotation is now purely based on automated prediction. However, errors in gene structure are frequent, the correct determination of start codons being one of the main concerns. Here, we combine protein N termini derivatization using (N-Succinimidyloxycarbonylmethyl)tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP Ac-OSu) as a labeling reagent with the COmbined FRActional DIagonal Chromatography (COFRADIC) sorting method to enrich labeled N-terminal peptides for mass spectrometry detection. Protein digestion was performed in parallel with three proteases to obtain a reliable automatic validation of protein N termini. The analysis of these N-terminal enriched fractions by high-resolution tandem mass spectrometry allowed the annotation refinement of 534 proteins of the model marine bacterium Roseobacter denitrificans OCh114. This study is especially efficient regarding mass spectrometry analytical time. From the 534 validated N termini, 480 confirmed existing gene annotations, 41 highlighted erroneous start codon annotations, five revealed totally new mis-annotated genes; the mass spectrometry data also suggested the existence of multiple start sites for eight different genes, a result that challenges the current view of protein translation initiation. Finally, we identified several proteins for which classical genome homology-driven annotation was inconsistent, questioning the validity of automatic annotation pipelines and emphasizing the need for complementary proteomic data. All data have been deposited to the ProteomeXchange with identifier PXD000337.

N-terminomics and proteogenomics, getting off to a good start.

Proteogenomics consists of the annotation or reannotation of protein-coding nucleic acid sequences based on the empirical observation of their gene products. While functional annotation of predicted genes is increasingly feasible given the multiplicity of genomes available for many branches of the tree of life, the accurate annotation of the translational start sites is still a point of contention. Extensive coverage of the proteome, including specifically the N-termini, is now possible, thanks to next-generation mass spectrometers able to record data from thousands of proteins at once. Efforts to increase the peptide coverage of protein sequences and to detect low abundance proteins are important to make proteomic and proteogenomic studies more comprehensive. In this review, we present the panoply of N-terminus-oriented strategies that have been developed over the last decade.

Shotgun proteomics suggests involvement of additional enzymes in dioxin degradation by Sphingomonas wittichii RW1.

Chlorinated congeners of dibenzo-p-dioxin and dibenzofuran are widely dispersed pollutants that can be treated using microorganisms, such as the Sphingomonas wittichii RW1 bacterium, able to transform some of them into non-toxic substances. The enzymes of the upper pathway for dibenzo-p-dioxin degradation in S. wittichii RW1 have been biochemically and genetically characterized, but its genome sequence indicated the existence of a tremendous potential for aromatic compound transformation, with 56 ring-hydroxylating dioxygenase subunits, 34 extradiol dioxygenases and 40 hydrolases. To further characterize this enzymatic arsenal, new methodological approaches should be employed. Here, a large shotgun proteomic survey was performed on cells grown on dibenzofuran, dibenzo-p-dioxin and 2-chlorodibenzo-p-dioxin, and compared with growth on acetate. Changes in the proteome were monitored over time. In total, 502 proteins were observed and quantified using a label-free mass spectrometry-based approach; all data were deposited to the ProteomeXchange (PXD000403). Our results confirmed the roles of the dioxin dioxygenase DxnA1A2, trihydroxybiphenyl dioxygenase DbfB, meta-cleavage product hydrolase DxnB and reductase RedA2, and corroborated the proposed involvement of the Swit_3046 dioxygenase and DxnB2 hydrolase. Trends across substrates and over the course of growth do not support concerted pathway regulation and suggest the involvement of an additional hydrolase and several TonB-dependent receptors.

Heterogeneous Antibiotic Resistance Gene Removal Impedes Evaluation of Constructed Wetlands for Effective Greywater Treatment.

Microorganisms carrying antimicrobial resistance genes are often found in greywater. As the reuse of greywater becomes increasingly needed, it is imperative to determine how greywater treatment impacts antimicrobial resistance genes (ARGs). Using qPCR and SmartChip™ qPCR, we characterized ARG patterns in greywater microbial communities before, during, and after treatment by a recirculating vertical flow constructed wetland. In parallel, we examined the impact of greywater-treated irrigation on soil, including the occurrence of emerging micropollutants and the taxonomic and ARG compositions of microbial communities. Most ARGs in raw greywater are removed efficiently during the winter season, while some ARGs in the effluents increase in summer. SmartChip™ qPCR revealed the presence of ARGs, such as tetracycline and beta-lactam resistance genes, in both raw and treated greywater, but most abundantly in the filter bed. It also showed that aminoglycoside and vancomycin gene abundances significantly increased after treatment. In the irrigated soil, the type of water (potable or treated greywater) had no specific impact on the total bacterial abundance (16S rRNA gene). No overlapping ARGs were found between treated greywater and greywater-irrigated soil. This study indicates ARG abundance and richness increased after treatment, possibly due to the concentration effects of the filter beds.

A synthetic biology approach to assemble and reboot clinically relevant Pseudomonas aeruginosa tailed phages.

The rise in the frequency of antibiotic resistance has made bacterial infections, specifically Pseudomonas aeruginosa, a cause for greater concern. Phage therapy is a promising solution that uses naturally isolated phages to treat bacterial infections. Ecological limitations, which stipulate a discrete host range and the inevitable evolution of resistance, may be overcome through a better understanding of phage biology and the utilization of engineered phages. In this study, we developed a synthetic biology approach to construct tailed phages that naturally target clinically relevant strains of Pseudomonas aeruginosa. As proof of concept, we successfully cloned and assembled the JG024 and DMS3 phage genomes in yeast using transformation-associated recombination cloning and rebooted these two phage genomes in two different strains of P. aeruginosa. We identified factors that affected phage reboot efficiency like the phage species or the presence of antiviral defense systems in the bacterial strain. We have successfully extended this method to two other phage species and observed that the method enables the reboot of phages that are naturally unable to infect the strain used for reboot. This research represents a critical step toward the construction of clinically relevant, engineered P. aeruginosa phages.IMPORTANCEPseudomonas aeruginosa is a bacterium responsible for severe infections and a common major complication in cystic fibrosis. The use of antibiotics to treat bacterial infections has become increasingly difficult as antibiotic resistance has become more prevalent. Phage therapy is an alternative solution that is already being used in some European countries, but its use is limited by the narrow host range due to the phage receptor specificity, the presence of antiviral defense systems in the bacterial strain, and the possible emergence of phage resistance. In this study, we demonstrate the use of a synthetic biology approach to construct and reboot clinically relevant P. aeruginosa tailed phages. This method enables a significant expansion of possibilities through the construction of engineered phages for therapy applications.

Proteogenomics for environmental microbiology.

Proteogenomics sensu stricto refers to the use of proteomic data to refine the annotation of genomes from model organisms. Because of the limitations of automatic annotation pipelines, a relatively high number of errors occur during the structural annotation of genes coding for proteins. Whether putative orphan sequences or short genes encoding low-molecular-weight proteins really exist is still frequently a mystery. Whether start codons are well defined is also an open debate. These problems are exacerbated for genomes of microorganisms belonging to poorly documented genera, as related sequences are not always available for homology-guided annotation. The functional annotation of a significant proportion of genes is also another well-known issue when annotating environmental microorganisms. High-throughput shotgun proteomics has recently greatly evolved, allowing the exploration of the proteome from any microorganism at an unprecedented depth. The structural and functional annotation process may be usefully complemented with experimental data. Indeed, proteogenomic mapping has been successfully performed for a wide variety of organisms. Specific approaches devoted to systematically establishing the N-termini of a large set of proteins are being developed. N-terminomics is giving rise to datasets of experimentally proven translational start codons as well as validated peptide signals for secreted proteins. By extension, combining genomic and proteomic data is becoming routine in many research projects. The proteomic analysis of organisms with unfinished genome sequences, the so-called composite proteomics, and the search for microbial biomarkers by bottom-up and top-down combined approaches are some examples of proteogenomic-flavored studies. They illustrate the advent of a new era of environmental microbiology where proteomics and genomics are intimately integrated to answer key biological questions.