Distinct anal microbiome is correlated with anal cancer precursors in MSM with HIV.

OBJECTIVES: Anal cancer risk is elevated in MSM with HIV (MSMWH). Anal high-risk human papillomavirus (hr-HPV) infection is necessary but insufficient to develop high-grade squamous intraepithelial lesion (HSIL), the anal cancer precursor, suggesting additional factors. We sought to determine whether the microbiome of the anal canal is distinct by comparing it with the microbiome of stool. We also sought to determine whether changes in the anal microbiome are associated with HSIL among MSMWH. DESIGN: Cross-sectional comparison of the microbiome of the anal canal with the microbiome of stool in MSMWH and cross-sectional comparison of the anal microbiome of MSMWH with anal HSIL with the anal microbiome of MSMWH without anal HSIL. METHODS: Sterile swabs were used to sample the anus of MSMWH for microbiome and HPV testing, followed by high-resolution anoscopy. Stool samples were mailed from home. 16S sequencing was used for bacterial identification. Measures of alpha diversity, beta diversity, and differential abundance analysis were used to compare samples. RESULTS: One hundred sixty-six anal samples and 103 matching stool samples were sequenced. Beta diversity showed clustering of stool and anal samples. Of hr-HPV-positive MSMWH, 31 had HSIL and 13 had no SIL. Comparison of the microbiome between these revealed 28 different species. The highest-fold enrichment among MSMWH/hr-HPV/HSIL included pro-inflammatory and carcinogenic Prevotella, Parasuterella, Hungatella, Sneathia, and Fusobacterium species. The anti-inflammatory Anaerostipes caccae showed the greatest reduction among MSMWH/hr-HPV/HSIL. CONCLUSION: The anal microbiome is distinct from stool. A pro-inflammatory and carcinogenic environment may be associated with anal HSIL.

Development and characterization of a bacteriophage cocktail with high lytic efficacy against field-isolated Salmonella enterica.

Salmonella spp. is a prevalent pathogen that causes great public health concern worldwide. Bacteriophage-based cocktails have arisen as an alternative to antibiotics to inhibit the growth of Salmonella. However, the bactericidal effect of bacteriophage cocktails in vivo largely differs from their observed effect in vitro. This is partly because in vitro developments of cocktails do not always consider the bacterial diversity nor the environmental conditions where bacteriophages will have to replicate. Here, we isolated and sequenced 47 bacteriophages that showed variable degrees of lytic activity against 258 Salmonella isolates from a commercial broiler company in Brazil. Three of these bacteriophages were characterized and selected to assemble a cocktail. In vitro quantitative assays determined the cocktail to be highly effective against multiple serovars of Salmonella, including Minnesota and Heidelberg. Remarkably, the in vitro lytic activity of the cocktail was retained or improved in conditions that more closely resembled the chicken gut, such as anaerobiosis, 42°C, and Salmonella mono-strain biofilms. Analysis of bacterial cross-resistance between the 3 bacteriophages composing the cocktail revealed limited or no generation of cross-resistance. Our results highlight the relevance of an optimized flux of work to develop bacteriophage cocktails against Salmonella with high lytic efficacy and strong potential to be applied in vivo in commercial broiler farms.

A comprehensive transcription factor and DNA-binding motif resource for the construction of gene regulatory networks in Botrytis cinerea and Trichoderma atroviride.

Botrytis cinerea and Trichoderma atroviride are two relevant fungi in agricultural systems. To gain insights into these organisms' transcriptional gene regulatory networks (GRNs), we generated a manually curated transcription factor (TF) dataset for each of them, followed by a GRN inference utilizing available sequence motifs describing DNA-binding specificity and global gene expression data. As a proof of concept of the usefulness of this resource to pinpoint key transcriptional regulators, we employed publicly available transcriptomics data and a newly generated dual RNA-seq dataset to build context-specific Botrytis and Trichoderma GRNs under two different biological paradigms: exposure to continuous light and Botrytis-Trichoderma confrontation assays. Network analysis of fungal responses to constant light revealed striking differences in the transcriptional landscape of both fungi. On the other hand, we found that the confrontation of both microorganisms elicited a distinct set of differentially expressed genes with changes in T. atroviride exceeding those in B. cinerea. Using our regulatory network data, we were able to determine, in both fungi, central TFs involved in this interaction response, including TFs controlling a large set of extracellular peptidases in the biocontrol agent T. atroviride. In summary, our work provides a comprehensive catalog of transcription factors and regulatory interactions for both organisms. This catalog can now serve as a basis for generating novel hypotheses on transcriptional regulatory circuits in different experimental contexts.

Metabolic Differentiation of Co-occurring Accumulibacter Clades Revealed through Genome-Resolved Metatranscriptomics.

Natural microbial communities consist of closely related taxa that may exhibit phenotypic differences and inhabit distinct niches. However, connecting genetic diversity to ecological properties remains a challenge in microbial ecology due to the lack of pure cultures across the microbial tree of life. "Candidatus Accumulibacter phosphatis" (Accumulibacter) is a polyphosphate-accumulating organism that contributes to the enhanced biological phosphorus removal (EBPR) biotechnological process for removing excess phosphorus from wastewater and preventing eutrophication from downstream receiving waters. Distinct Accumulibacter clades often coexist in full-scale wastewater treatment plants and laboratory-scale enrichment bioreactors and have been hypothesized to inhabit distinct ecological niches. However, since individual strains of the Accumulibacter lineage have not been isolated in pure culture to date, these predictions have been made solely on genome-based comparisons and enrichments with varying strain compositions. Here, we used genome-resolved metagenomics and metatranscriptomics to explore the activity of coexisting Accumulibacter strains in an engineered bioreactor environment. We obtained four high-quality genomes of Accumulibacter strains that were present in the bioreactor ecosystem, one of which is a completely contiguous draft genome scaffolded with long Nanopore reads. We identified core and accessory genes to investigate how gene expression patterns differed among the dominating strains. Using this approach, we were able to identify putative pathways and functions that may confer distinct functions to Accumulibacter strains and provide key functional insights into this biotechnologically significant microbial lineage. IMPORTANCE "Candidatus Accumulibacter phosphatis" is a model polyphosphate-accumulating organism that has been studied using genome-resolved metagenomics, metatranscriptomics, and metaproteomics to understand the EBPR process. Within the Accumulibacter lineage, several similar but diverging clades are defined by the shared sequence identity of the polyphosphate kinase (ppk1) locus. These clades are predicted to have key functional differences in acetate uptake rates, phage defense mechanisms, and nitrogen-cycling capabilities. However, such hypotheses have largely been made based on gene content comparisons of sequenced Accumulibacter genomes, some of which were obtained from different systems. Here, we performed time series genome-resolved metatranscriptomics to explore gene expression patterns of coexisting Accumulibacter clades in the same bioreactor ecosystem. Our work provides an approach for elucidating ecologically relevant functions based on gene expression patterns between closely related microbial populations.

Redox stress response and UV tolerance in the acidophilic iron-oxidizing bacteria Leptospirillum ferriphilum and Acidithiobacillus ferrooxidans.

The oxidative stress response represents a sum of antioxidative mechanisms that are essential for determining the adaptation and abundance of microorganisms in the environment. Leptospirillum ferriphilum and Acidithiobacillus ferrooxidans are chemolithotrophic bacteria that obtain their energy from the oxidation of ferrous ion. Both microorganisms are important for bioleaching of sulfidic ores and both are tolerant to high levels of heavy metals and other factors that can induce oxidative stress. In this work, we compared the tolerance and response of L. ferriphilum and At. ferrooxidans to Fe3+, H2O2, K2CrO4, and UV-C radiation. We evaluated growth, generation of reactive oxygen species (ROS), oxidative damage to lipid membranes and DNA, and the activity of antioxidative proteins in cells exposed to these stressors. L. ferriphilum had higher cell density, lower ROS content and less lipid and DNA damage than At. ferrooxidans. Consistent with this, the activity levels of thioredoxin and superoxide dismutase in L. ferriphilum were upregulated and higher than in At. ferrooxidans. This indicated that L. ferriphilum has a higher capacity to respond to oxidative stress and to manage redox homeostasis. This capacity could largely contribute to the high abundance of this species in natural and anthropogenic sites.

Integrated Omic Analyses Provide Evidence that a "Candidatus Accumulibacter phosphatis" Strain Performs Denitrification under Microaerobic Conditions.

The ability of "Candidatus Accumulibacter phosphatis" to grow and remove phosphorus from wastewater under cycling anaerobic and aerobic conditions has also been investigated as a metabolism that could lead to simultaneous removal of nitrogen and phosphorus by a single organism. However, although phosphorus removal under cyclic anaerobic and anoxic conditions has been demonstrated, clarifying the role of "Ca. Accumulibacter phosphatis" in this process has been challenging, since (i) experimental research describes contradictory findings, (ii) none of the published "Ca. Accumulibacter phosphatis" genomes show the existence of a complete respiratory pathway for denitrification, and (iii) some genomes lacking a complete respiratory pathway have genes for assimilatory nitrate reduction. In this study, we used an integrated omics analysis to elucidate the physiology of a "Ca. Accumulibacter phosphatis" strain enriched in a reactor operated under cyclic anaerobic and microaerobic conditions. The reactor's performance suggested the ability of the enriched "Ca. Accumulibacter phosphatis" strain (clade IC) to simultaneously use oxygen and nitrate as electron acceptors under microaerobic conditions. A draft genome of this organism was assembled from metagenomic reads ("Ca. Accumulibacter phosphatis" UW-LDO-IC) and used as a reference to examine transcript abundance throughout one reactor cycle. The genome of UW-LDO-IC revealed the presence of a full pathway for respiratory denitrification. The observed transcript abundance patterns showed evidence of coregulation of the denitrifying genes along with a cbb 3 cytochrome, which has been characterized as having high affinity for oxygen. Furthermore, we identified an FNR-like binding motif upstream of the coregulated genes, suggesting transcription-level regulation of both denitrifying and respiratory pathways in UW-LDO-IC. Taking the results together, the omics analysis provides strong evidence that "Ca. Accumulibacter phosphatis" UW-LDO-IC uses oxygen and nitrate simultaneously as electron acceptors under microaerobic conditions. IMPORTANCE "Candidatus Accumulibacter phosphatis" is widely found in full-scale wastewater treatment plants, where it has been identified as the key organism for biological removal of phosphorus. Since aeration can account for 50% of the energy use during wastewater treatment, microaerobic conditions for wastewater treatment have emerged as a cost-effective alternative to conventional biological nutrient removal processes. Our report provides strong genomics-based evidence not only that "Ca. Accumulibacter phosphatis" is the main organism contributing to phosphorus removal under microaerobic conditions but also that this organism simultaneously respires nitrate and oxygen in this environment, consequently removing nitrogen and phosphorus from the wastewater. Such activity could be harnessed in innovative designs for cost-effective and energy-efficient optimization of wastewater treatment systems.

Genome-Enabled Insights into the Ecophysiology of the Comammox Bacterium "Candidatus Nitrospira nitrosa".

The recently discovered comammox bacteria have the potential to completely oxidize ammonia to nitrate. These microorganisms are part of the Nitrospira genus and are present in a variety of environments, including biological nutrient removal (BNR) systems. However, the physiological traits within and between comammox and nitrite-oxidizing bacterium (NOB)-like Nitrospira species have not been analyzed in these ecosystems. In this study, we identified Nitrospira strains dominating the nitrifying community of a sequencing batch reactor (SBR) performing BNR under microaerobic conditions. We recovered metagenome-derived draft genomes from two Nitrospira strains: (i) Nitrospira sp. strain UW-LDO-01, a comammox-like organism classified as "Candidatus Nitrospira nitrosa," and (ii) Nitrospira sp. strain UW-LDO-02, a nitrite-oxidizing strain belonging to the Nitrospira defluvii species. A comparative genomic analysis of these strains with other Nitrospira-like genomes identified genomic differences in "Ca. Nitrospira nitrosa" mainly attributed to each strain's niche adaptation. Traits associated with energy metabolism also differentiate comammox from NOB-like genomes. We also identified several transcriptionally regulated adaptive traits, including stress tolerance, biofilm formation, and microaerobic metabolism, which might explain survival of Nitrospira under multiple environmental conditions. Overall, our analysis expanded our understanding of the genetic functional features of "Ca. Nitrospira nitrosa" and identified genomic traits that further illuminate the phylogenetic diversity and metabolic plasticity of the Nitrospira genus. IMPORTANCENitrospira-like bacteria are among the most diverse and widespread nitrifiers in natural ecosystems and the dominant nitrite oxidizers in wastewater treatment plants (WWTPs). The recent discovery of comammox-like Nitrospira strains, capable of complete oxidation of ammonia to nitrate, raises new questions about specific traits responsible for the functional versatility and adaptation of this genus to a variety of environments. The availability of new Nitrospira genome sequences from both nitrite-oxidizing and comammox bacteria offers a way to analyze traits in different Nitrospira functional groups. Our comparative genomics analysis provided new insights into the adaptation of Nitrospira strains to specific lifestyles and environmental niches.

Candidatus Accumulibacter phosphatis clades enriched under cyclic anaerobic and microaerobic conditions simultaneously use different electron acceptors.

Lab- and pilot-scale simultaneous nitrification, denitrification and phosphorus removal-sequencing batch reactors were operated under cyclic anaerobic and micro-aerobic conditions. The use of oxygen, nitrite, and nitrate as electron acceptors by Candidatus Accumulibacter phosphatis during the micro-aerobic stage was investigated. A complete clade-level characterization of Accumulibacter in both reactors was performed using newly designed qPCR primers targeting the polyphosphate kinase gene (ppk1). In the lab-scale reactor, limited-oxygen conditions led to an alternated dominance of Clade IID and IC over the other clades. Results from batch tests when Clade IC was dominant (i.e., >92% of Accumulibacter) showed that this clade was capable of using oxygen, nitrite and nitrate as electron acceptors for P uptake. A more heterogeneous distribution of clades was found in the pilot-scale system (Clades IIA, IIB, IIC, IID, IA, and IC), and in this reactor, oxygen, nitrite and nitrate were also used as electron acceptors coupled to phosphorus uptake. However, nitrite was not an efficient electron acceptor in either reactor, and nitrate allowed only partial P removal. The results from the Clade IC dominated reactor indicated that either organisms in this clade can simultaneously use multiple electron acceptors under micro-aerobic conditions, or that the use of multiple electron acceptors by Clade IC is due to significant microdiversity within the Accumulibacter clades defined using the ppk1 gene.

Ammonia-oxidizing microbial communities in reactors with efficient nitrification at low-dissolved oxygen.

Ammonia-oxidizing microbial communities involved in ammonia oxidation under low dissolved oxygen (DO) conditions (<0.3 mg/L) were investigated using chemostat reactors. One lab-scale reactor (NS_LowDO) was seeded with sludge from a full-scale wastewater treatment plant (WWTP) not adapted to low-DO nitrification, while a second reactor (JP_LowDO) was seeded with sludge from a full-scale WWTP already achieving low-DO nitrifiaction. The experimental evidence from quantitative PCR, rDNA tag pyrosequencing, and fluorescence in situ hybridization (FISH) suggested that ammonia-oxidizing bacteria (AOB) in the Nitrosomonas genus were responsible for low-DO nitrification in the NS_LowDO reactor, whereas in the JP_LowDO reactor nitrification was not associated with any known ammonia-oxidizing prokaryote. Neither reactor had a significant population of ammonia-oxidizing archaea (AOA) or anaerobic ammonium oxidation (anammox) organisms. Organisms isolated from JP_LowDO were capable of autotrophic and heterotrophic ammonia utilization, albeit without stoichiometric accumulation of nitrite or nitrate. Based on the experimental evidence we propose that Pseudomonas, Xanthomonadaceae, Rhodococcus, and Sphingomonas are involved in nitrification under low-DO conditions.

Mathematical tools to optimize the design of oligonucleotide probes and primers.

The identification and quantification of specific organisms in mixed microbial communities often relies on the ability to design oligonucleotide probes and primers with high specificity and sensitivity. The design of these oligonucleotides (or "oligos" for short) shares many of the same principles in spite of their widely divergent applications. Three common molecular biology technologies that require oligonucleotide design are polymerase chain reaction (PCR), fluorescence in situ hybridization (FISH), and DNA microarrays. This article reviews techniques and software available for the design and optimization of oligos with the goal of targeting a specific group of organisms within mixed microbial communities. Strategies for enhancing specificity without compromising sensitivity are described, as well as design tools well suited for this purpose.