Robustness of cancer microbiome signals over a broad range of methodological variation.

In 2020, we identified cancer-specific microbial signals in The Cancer Genome Atlas (TCGA) [1]. Multiple peer-reviewed papers independently verified or extended our findings [2-12]. Given this impact, we carefully considered concerns by Gihawi et al. [13] that batch correction and database contamination with host sequences artificially created the appearance of cancer type-specific microbiomes. (1) We tested batch correction by comparing raw and Voom-SNM-corrected data per-batch, finding predictive equivalence and significantly similar features. We found consistent results with a modern microbiome-specific method (ConQuR [14]), and when restricting to taxa found in an independent, highly-decontaminated cohort. (2) Using Conterminator [15], we found low levels of human contamination in our original databases (~1% of genomes). We demonstrated that the increased detection of human reads in Gihawi et al. [13] was due to using a newer human genome reference. (3) We developed Exhaustive, a method twice as sensitive as Conterminator, to clean RefSeq. We comprehensively host-deplete TCGA with many human (pan)genome references. We repeated all analyses with this and the Gihawi et al. [13] pipeline, and found cancer type-specific microbiomes. These extensive re-analyses and updated methods validate our original conclusion that cancer type-specific microbial signatures exist in TCGA, and show they are robust to methodology.

Transformation of hydroxylated polychlorinated biphenyls by bacterial 2-hydroxybiphenyl 3-monooxygenase.

Monohydroxylated PCBs (OH-PCBs) are an (eco)toxicologically significant group of compounds, as they arise from the oxidation of polychlorinated biphenyls (PCBs) and, at the same time, may exert even more severe toxic effects than their parent PCB molecules. Despite having been widely detected in environmental samples, plants, and animals, information on the fate of OH-PCBs in the environment is scarce, including on the enzymatic machinery behind their degradation. To date, only a few bacterial taxa capable of OH-PCB transformation have been reported. In this study, we aimed to obtain a deeper insight into the transformation of OH-PCBs in soil bacteria and isolated a Pseudomonas sp. strain P1B16 based on its ability to use o-phenylphenol (2-PP) which, when exposed to the Delor 103-derived OH-PCB mixture, depleted a wide spectrum of mono-, di, and trichlorinated OH-PCBs. In the P1B16 genome, a region designated as hbp was identified, which bears a set of putative genes involved in the transformation of OH-PCBs, namely hbpA encoding for a putative flavin-dependent 2-hydroxybiphenyl monooxygenase, hbpC (2,3-dihydroxybiphenyl-1,2-dioxygenase), hbpD (2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase), and the transcriptional activator-encoding gene hbpR. The hbpA coding sequence was heterologously expressed, purified, and its substrate specificity was investigated towards the Delor 103-derived OH-PCB mixture, individual OH-PCBs, and multiple (chlorinated) phenolics. Apart from 2-PP and 2-chlorophenol, HbpA was also demonstrated to transform a range of OH-PCBs, including a 3-hydroxy-2,2',4',5,5'-pentachlorobiphenyl. Importantly, this is the first direct evidence of HbpA homologs being involved in the degradation of OH-PCBs. Moreover, using a P1B16-based biosensor strain, the specific induction of hbp genes by 2-PP, 3-phenylphenol, 4-phenylphenol, and the OH-PCB mixture was demonstrated. This study provides direct evidence on the specific enzymatic machinery responsible for the transformation of OH-PCBs in bacteria, with many implications in ecotoxicology, environmental restoration, and microbial ecology in habitats burdened with PCB contamination.

Signatures of HIV and Major Depressive Disorder in the Plasma Microbiome.

Inter-individual differences in the gut microbiome are linked to alterations in inflammation and blood-brain barrier permeability, which may increase the risk of depression in people with HIV (PWH). The microbiome profile of blood, which is considered by many to be typically sterile, remains largely unexplored. We aimed to characterize the blood plasma microbiome composition and assess its association with major depressive disorder (MDD) in PWH and people without HIV (PWoH). In this cross-sectional, observational cohort, we used shallow-shotgun metagenomic sequencing to characterize the plasma microbiome of 151 participants (84 PWH and 67 PWoH), all of whom underwent a comprehensive neuropsychiatric assessment. The microbial composition did not differ between PWH and PWoH or between participants with MDD and those without it. Using the songbird model, we computed the log ratio of the highest and lowest 30% of the ranked classes associated with HIV and MDD. We found that HIV infection and lifetime MDD were enriched in a set of differentially abundant inflammatory classes, such as Flavobacteria and Nitrospira. Our results suggest that the circulating plasma microbiome may increase the risk of MDD related to dysbiosis-induced inflammation in PWH. If confirmed, these findings may indicate new biological mechanisms that could be targeted to improve treatment of MDD in PWH.

Genomic analysis of Acinetobacter pittii CEP14 reveals its extensive biodegradation capabilities, including cometabolic degradation of cis-1,2-dichloroethene.

Halogenated organic compounds are naturally occurring in subsurface environments; however, accumulation of the degradative intermediate cis-1,2-dichloroethene (cDCE) at soil and groundwater sites contaminated with xenobiotic chlorinated ethenes is a global environmental and public health issue. Identifying microorganisms capable of cDCE degradation in these environments is of interest because of their potential application to bioremediation techniques. In this study, we sequenced, assembled, and analyzed the complete genome of Acinetobacter pittii CEP14, a strain isolated from chloroethene-contaminated groundwater, that has demonstrated the ability for aerobic cometabolic degradation of cDCE in the presence of n-hexane, phenol, and toluene. The A. pittii CEP14 genome consists of a 3.93 Mbp-long chromosome (GenBank accession no. CP084921) with a GC content of 38.9% and three plasmids (GenBank accession no. CP084922, CP084923, and CP084924). Gene function was assigned to 83.4% of the 3,930 coding DNA sequences. Functional annotation of the genome revealed that the CEP14 strain possessed all genetic elements to mediate the degradation of a range of aliphatic and aromatic compounds, including n-hexane and phenol. In addition, it harbors gene clusters involved in cytosol detoxification and oxidative stress resistance, which could play a role in the mitigation of toxic chemical intermediates that can arise during the degradation of cDCE. Gene clusters for heavy metal and antibiotic resistance were also identified in the genome of CEP14. These results suggest that CEP14 may be a versatile degrader of xenobiotic compounds and well-adapted to polluted environments, where a combination of heavy metal and organic compound pollution is often found.

A comparison of DNA/RNA extraction protocols for high-throughput sequencing of microbial communities.

One goal of microbial ecology researchers is to capture the maximum amount of information from all organisms in a sample. The recent COVID-19 pandemic, caused by the RNA virus SARS-CoV-2, has highlighted a gap in traditional DNA-based protocols, including the high-throughput methods the authors previously established as field standards. To enable simultaneous SARS-CoV-2 and microbial community profiling, the authors compared the relative performance of two total nucleic acid extraction protocols with the authors' previously benchmarked protocol. The authors included a diverse panel of environmental and host-associated sample types, including body sites commonly swabbed for COVID-19 testing. Here the authors present results comparing the cost, processing time, DNA and RNA yield, microbial community composition, limit of detection and well-to-well contamination between these protocols.

A comparison of DNA/RNA extraction protocols for high-throughput sequencing of microbial communities.

One goal among microbial ecology researchers is to capture the maximum amount of information from all organisms in a sample. The recent COVID-19 pandemic, caused by the RNA virus SARS-CoV-2, has highlighted a gap in traditional DNA-based protocols, including the high-throughput methods we previously established as field standards. To enable simultaneous SARS-CoV-2 and microbial community profiling, we compare the relative performance of two total nucleic acid extraction protocols and our previously benchmarked protocol. We included a diverse panel of environmental and host-associated sample types, including body sites commonly swabbed for COVID-19 testing. Here we present results comparing the cost, processing time, DNA and RNA yield, microbial community composition, limit of detection, and well-to-well contamination, between these protocols. Accession numbers: Raw sequence data were deposited at the European Nucleotide Archive (accession#: ERP124610) and raw and processed data are available at Qiita (Study ID: 12201). All processing and analysis code is available on GitHub ( github.com/justinshaffer/Extraction_test_MagMAX ). Methods summary: To allow for downstream applications involving RNA-based organisms such as SARS-CoV-2, we compared the two extraction protocols designed to extract DNA and RNA against our previously established protocol for extracting only DNA for microbial community analyses. Across 10 diverse sample types, one of the two protocols was equivalent or better than our established DNA-based protocol. Our conclusion is based on per-sample comparisons of DNA and RNA yield, the number of quality sequences generated, microbial community alpha- and beta-diversity and taxonomic composition, the limit of detection, and extent of well-to-well contamination.

Microbiome analyses of blood and tissues suggest cancer diagnostic approach.

Systematic characterization of the cancer microbiome provides the opportunity to develop techniques that exploit non-human, microorganism-derived molecules in the diagnosis of a major human disease. Following recent demonstrations that some types of cancer show substantial microbial contributions1-10, we re-examined whole-genome and whole-transcriptome sequencing studies in The Cancer Genome Atlas11 (TCGA) of 33 types of cancer from treatment-naive patients (a total of 18,116 samples) for microbial reads, and found unique microbial signatures in tissue and blood within and between most major types of cancer. These TCGA blood signatures remained predictive when applied to patients with stage Ia-IIc cancer and cancers lacking any genomic alterations currently measured on two commercial-grade cell-free tumour DNA platforms, despite the use of very stringent decontamination analyses that discarded up to 92.3% of total sequence data. In addition, we could discriminate among samples from healthy, cancer-free individuals (n = 69) and those from patients with multiple types of cancer (prostate, lung, and melanoma; 100 samples in total) solely using plasma-derived, cell-free microbial nucleic acids. This potential microbiome-based oncology diagnostic tool warrants further exploration.

Complete genome sequence of Pseudomonas alcaliphila JAB1 (=DSM 26533), a versatile degrader of organic pollutants.

In this study, following its isolation from contaminated soil, the genomic sequence of Pseudomonas alcaliphila strain JAB1 (=DSM 26533), a biphenyl-degrading bacterium, is reported and analyzed in relation to its extensive degradative capabilities. The P. alcaliphila JAB1 genome (GenBank accession no. CP016162) consists of a single 5.34 Mbp-long chromosome with a GC content of 62.5%. Gene function was assigned to 3816 of the 4908 predicted genes. The genome harbors a bph gene cluster, permitting degradation of biphenyl and many congeners of polychlorinated biphenyls (PCBs), a ben gene cluster, enabling benzoate and its derivatives to be degraded, and phe gene cluster, which permits phenol degradation. In addition, P. alcaliphila JAB1 is capable of cometabolically degrading cis-1,2-dichloroethylene (cDCE) when grown on phenol. The strain carries both catechol and protocatechuate branches of the ß-ketoadipate pathway, which is used to funnel the pollutants to the central metabolism. Furthermore, we propose that clustering of MALDI-TOF MS spectra with closest phylogenetic relatives should be used when taxonomically classifying the isolated bacterium; this, together with 16S rRNA gene sequence and chemotaxonomic data analyses, enables more precise identification of the culture at the species level.

Secondary compound hypothesis revisited: Selected plant secondary metabolites promote bacterial degradation of cis-1,2-dichloroethylene (cDCE).

Cis-1,2-dichloroethylene (cDCE), which is a common hazardous compound, often accumulates during incomplete reductive dechlorination of higher chlorinated ethenes (CEs) at contaminated sites. Simple monoaromatics, such as toluene and phenol, have been proven to induce biotransformation of cDCE in microbial communities incapable of cDCE degradation in the absence of other carbon sources. The goal of this microcosm-based laboratory study was to discover non-toxic natural monoaromatic secondary plant metabolites (SPMEs) that could enhance cDCE degradation in a similar manner to toluene and phenol. Eight SPMEs were selected on the basis of their monoaromatic molecular structure and widespread occurrence in nature. The suitability of the SPMEs chosen to support bacterial growth and to promote cDCE degradation was evaluated in aerobic microbial cultures enriched from cDCE-contaminated soil in the presence of each SPME tested and cDCE. Significant cDCE depletions were achieved in cultures enriched on acetophenone, phenethyl alcohol, p-hydroxybenzoic acid and trans-cinnamic acid. 16S rRNA gene sequence analysis of each microbial community revealed ubiquitous enrichment of bacteria affiliated with the genera Cupriavidus, Rhodococcus, Burkholderia, Acinetobacter and Pseudomonas. Our results provide further confirmation of the previously stated secondary compound hypothesis that plant metabolites released into the rhizosphere can trigger biodegradation of environmental pollutants, including cDCE.

Differential Impacts of Willow and Mineral Fertilizer on Bacterial Communities and Biodegradation in Diesel Fuel Oil-Contaminated Soil.

Despite decades of research there is limited understanding of how vegetation impacts the ability of microbial communities to process organic contaminants in soil. Using a combination of traditional and molecular assays, we examined how phytoremediation with willow and/or fertilization affected the microbial community present and active in the transformation of diesel contaminants. In a pot study, willow had a significant role in structuring the total bacterial community and resulted in significant decreases in diesel range organics (DRO). However, stable isotope probing (SIP) indicated that fertilizer drove the differences seen in community structure and function. Finally, analysis of the total variance in both pot and SIP experiments indicated an interactive effect between willow and fertilizer on the bacterial communities. This study clearly demonstrates that a willow native to Alaska accelerates DRO degradation, and together with fertilizer, increases aromatic degradation by shifting microbial community structure and the identity of active naphthalene degraders.

Sludge minimization in municipal wastewater treatment by polyhydroxyalkanoate (PHA) production.

An innovative approach has been recently proposed in order to link polyhydroxyalkanoates (PHA) production with sludge minimization in municipal wastewater treatment, where (1) a sequencing batch reactor (SBR) is used for the simultaneous municipal wastewater treatment and the selection/enrichment of biomass with storage ability and (2) the acidogenic fermentation of the primary sludge is used to produce a stream rich in volatile fatty acids (VFAs) as the carbon source for the following PHA accumulation stage. The reliability of the proposed process has been evaluated at lab scale by using substrate synthetic mixtures for both stages, simulating a low-strength municipal wastewater and the effluent from primary sludge fermentation, respectively. Six SBR runs were performed under the same operating conditions, each time starting from a new activated sludge inoculum. In every SBR run, despite the low VFA content (10% chemical oxygen demand, COD basis) of the substrate synthetic mixture, a stable feast-famine regime was established, ensuring the necessary selection/enrichment of the sludge and soluble COD removal to 89%. A good process reproducibility was observed, as also confirmed by denaturing gradient gel electrophoresis (DGGE) analysis of the microbial community, which showed that a high similarity after SBR steady-state had been reached. The main variation factors of the storage properties among different runs were uncontrolled changes of settling properties which in turn caused variations of both sludge retention time and specific organic loading rate. In the following accumulation batch tests, the selected/enriched consortium was able to accumulate PHA with good rate (63 mg CODPHA g CODXa(-1) h(-1)) and yield (0.23 CODPHA CODΔS(-1)) in spite that the feeding solution was different from the acclimation one. Even though the PHA production performance still requires optimization, the proposed process has a good potential especially if coupled to minimization of both primary sludge (by its use as the VFA source for the PHA accumulation, via previous fermentation) and excess secondary sludge (by its use as the biomass source for the PHA accumulation).

Development of an attached-growth process for the on-site bioremediation of an aquifer polluted by chlorinated solvents.

A procedure for the design of an aerobic cometabolic process for the on-site degradation of chlorinated solvents in a packed bed reactor was developed using groundwater from an aquifer contaminated by trichloroethylene (TCE) and 1,1,2,2-tetrachloroethane (TeCA). The work led to the selection of butane among five tested growth substrates, and to the development and characterization from the site's indigenous biomass of a suspended-cell consortium capable to degrade TCE (first order constant: 96 L gprotein(-1) day(-1) at 30 °C and 4.3 L gprotein(-1) day(-1) at 15 °C) with a 90 % mineralization of the organic chlorine. The consortium immobilization had strong effects on the butane and TCE degradation rates. The microbial community structure was slightly changed by a temperature shift from 30 to 15 °C, but remarkably affected by biomass adhesion. Given the higher TCE normalized degradation rate (0.59 day(-1) at 15 °C) and attached biomass concentration (0.13 gprotein Lbioreactor(-1) at 15 °C) attained, the porous ceramic carrier Biomax was selected as the best option for the packed bed reactor process. The low TeCA degradation rate exhibited by the developed consortium suggested the inclusion of a chemical pre-treatment based on the TeCA to TCE conversion via ß-elimination, a very fast reaction at alkaline pH. To the best of the authors' knowledge, this represents the first attempt to develop a procedure for the development of a packed bed reactor process for the aerobic cometabolism of chlorinated solvents.

Feed frequency in a sequencing batch reactor strongly affects the production of polyhydroxyalkanoates (PHAs) from volatile fatty acids.

The production of polyhydroxyalkanoates (PHAs) by activated sludge selected in a sequencing batch reactor (SBR) has been investigated. Several SBR runs were performed at the same applied organic load rate (OLR), hydraulic retention time (HRT) and feed concentration (8.5 g COD L(-1) of volatile fatty acids, VFAs) under aerobic conditions. The effect of the feeding time was only evaluated with a cycle length of 8h; for this particular cycle length, an increase in the storage response was observed by increasing the rate at which the substrate was fed into the reactor (at a fixed feeding frequency). Furthermore, a significantly stronger effect was observed by decreasing the cycle length from 8h to 6h and then to 2h, changing the feed frequency or changing the organic load given per cycle (all of the other conditions remained the same): the length of the feast phase decreased from 26 to 20.0 and then to 19.7% of the overall cycle length, respectively, due to an increase in the substrate removal rate. This removal rate was high and similar for the runs with cycle lengths of 2h and 6h in the SBR. This result was due to an increase in the selective pressure and the specific storage properties of the selected biomass. The highest polymer productivity after long-term accumulation batch tests was 1.7 g PHA L(-1)d(-1), with PHA content in the biomass of approximately 50% on a COD basis under nitrogen limitation. The DGGE profiles showed that the good storage performance correlated to the development of Lampropedia hyalina, which was only observed in the SBR runs characterized by a shorter cycle length.

Trichloroethylene aerobic cometabolism by suspended and immobilized butane-growing microbial consortia: a kinetic study.

A kinetic study of butane uptake and trichloroethylene (TCE) aerobic cometabolism was conducted by two suspended-cell (15 and 30°C) and two attached-cell (15 and 30°C) consortia obtained from the indigenous biomass of a TCE-contaminated aquifer. The shift from suspended to attached cells resulted in an increase of butane (15 and 30°C) and TCE (15°C) biodegradation rates, and a significant decrease of butane inhibition on TCE biodegradation. The TCE 15°C maximum specific biodegradation rate was equal to 0.011 mg(TCE ) mg(protein)(-1) d(-1) with suspended cells and 0.021 mg(TCE) mg(protein)(-1) d(-1) with attached cells. The type of mutual butane/TCE inhibition depended on temperature and biomass conditions. On the basis of a continuous-flow simulation, a packed-bed PFR inoculated with the 15 or 30°C attached-cell consortium could attain a 99.96% conversion of the studied site's average TCE concentration with a 0.4-0.5-day hydraulic residence time, with a low effect of temperature on the TCE degradation performances.

Aerobic/anaerobic/aerobic sequenced biodegradation of a mixture of chlorinated ethenes, ethanes and methanes in batch bioreactors.

A novel aerobic/anaerobic/aerobic treatment was implemented in batch reactors containing aquifer materials from a site contaminated by tetrachloroethylene (PCE), trichloroethylene (TCE), vinyl chloride (VC), 1,1,2-trichloroethane (1,1,2-TCA) and chloroform (CF). Consortia grown aerobically on methane, propane, n-pentane and n-hexane completely biodegraded the chlorinated solvent mixture, via aerobic cometabolism of VC, CF, TCE and 1,1,2-TCA, followed by PCE reductive dechlorination (RD) to 1,2-cis-dichlorothylene (cis-DCE) or TCE, and cis-DCE/TCE cometabolism in a further aerobic phase. n-Hexane was the best substrate. No electron donor was supplied for RD, which likely utilized cellular material produced during the aerobic phase. Chloride release was stoichiometric with chlorinated solvent biodegradation. According to the Lepidium sativum ecotoxicity test, a decreased toxicity was observed with propane, n-pentane and n-hexane, but not methane. A kinetic study of PCE RD allowed to estimate the PCE maximum specific rate (0.57 ± 0.07 mg mg(protein)(-1) day(-1)) and half-saturation constant (6.7 ± 1.5 mg L(-1)).

A physicochemical-biotechnological approach for an integrated valorization of olive mill wastewater.

An integrated physicochemical-biotechnological approach for a multipurpose valorization of olive mill wastewaters was studied. More than 60% of the wastewater natural polyphenols were recovered through a solid phase extraction procedure, by employing Amberlite XAD16 resin as the adsorbent and ethanol as the biocompatible desorbing phase. Thereafter, the dephenolized effluent was fed to a mesophilic anaerobic acidogenic packed-bed biofilm reactor for the bioconversion of the organic leftover into volatile fatty acids (VFAs). A VFAs concentration of 19 gCODL(-1) was obtained, representing more than 70% of the COD occurring in the anaerobic effluent. The biotechnological process was assessed by means of bio-molecular analyses, which showed that the reactor packed bed was mostly colonized by bacteria of the Firmicutes phylogenetic group. The biorefinery scheme developed in this study allowed the obtainment of 1.59 g of polyphenols per liter of wastewater treated and 2.72 gCODL(-1) day(-1) of VFAs.