In vitro development of resistance against antipseudomonal agents: comparison of novel ß-lactam/ß-lactamase inhibitor combinations and other ß-lactam agents.

We subjected seven P. aeruginosa isolates to a 10-day serial passaging against five antipseudomonal agents to evaluate resistance levels post-exposure and putative resistance mechanisms in terminal mutants were analyzed by whole-genome sequencing analysis. Meropenem (mean, 38-fold increase), cefepime (14.4-fold), and piperacillin-tazobactam (52.9-fold) terminal mutants displayed high minimum inhibitory concentration (MIC) values compared to those obtained after exposure to ceftolozane-tazobactam (11.4-fold) and ceftazidime-avibactam (5.7-fold). Fewer isolates developed elevated MIC values for other ß-lactams and agents belonging to other classes when exposed to meropenem in comparison to other agents. Alterations in nalC and nalD, involved in the upregulation of the efflux pump system MexAB-OprM, were common and observed more frequently in isolates exposed to ceftazidime-avibactam and meropenem. These alterations, along with ones in mexR and amrR, provided resistance to most ß-lactams and levofloxacin but not imipenem. The second most common gene altered was mpl, which is involved in the recycling of the cell wall peptidoglycan. These alterations were mainly noted in isolates exposed to ceftolozane-tazobactam and piperacillin-tazobactam but also in one cefepime-exposed isolate. Alterations in other genes known to be involved in ß-lactam resistance (ftsI, oprD, phoP, pepA, and cplA) and multiple genes involved in lipopolysaccharide biosynthesis were also present. The data generated here suggest that there is a difference in the mechanisms selected for high-level resistance between newer ß-lactam/ß-lactamase inhibitor combinations and older agents. Nevertheless, the isolates exposed to all agents displayed elevated MIC values for other ß-lactams (except imipenem) and quinolones tested mainly due to alterations in the MexAB-OprM regulators that extrude these agents.

Genome-Resolved Metagenomics and Metatranscriptomics Reveal Insights into the Ecology and Metabolism of Anaerobic Microbial Communities in PCB-Contaminated Sediments.

Growth of organohalide-respiring bacteria such as Dehalococcoides mccartyi on halogenated organics (e.g., polychlorinated biphenyls (PCBs)) at contaminated sites or in enrichment culture requires interaction and support from other microbial community members. To evaluate naturally occurring interactions between Dehalococcoides and key supporting microorganisms (e.g., production of H2, acetate, and corrinoids) in PCB-contaminated sediments, metagenomic and metatranscriptomic sequencing was conducted on DNA and RNA extracted from sediment microcosms, showing evidence of both Dehalococcoides growth and PCB dechlorination. Using a genome-resolved approach, 160 metagenome-assembled genomes (MAGs), including three Dehalococcoides MAGs, were recovered. A novel reductive dehalogenase gene, distantly related to the chlorophenol dehalogenase gene cprA (pairwise amino acid identity: 23.75%), was significantly expressed. Using MAG gene expression data, 112 MAGs were assigned functional roles (e.g., corrinoid producers, acetate/H2 producers, etc.). A network coexpression analysis of all 160 MAGs revealed correlations between 39 MAGs and the Dehalococcoides MAGs. The network analysis also showed that MAGs assigned with functional roles that support Dehalococcoides growth (e.g., corrinoid assembly, and production of intermediates required for corrinoid synthesis) displayed significant coexpression correlations with Dehalococcoides MAGs. This work demonstrates the power of genome-resolved metagenomic and metatranscriptomic analyses, which unify taxonomy and function, in investigating the ecology of dehalogenating microbial communities.

Aerobic Bioaugmentation to Decrease Polychlorinated Biphenyl (PCB) Emissions from Contaminated Sediments to Air.

We conducted experiments to determine whether bioaugmentation with aerobic, polychlorinated biphenyl (PCB)-degrading microorganisms can mitigate polychlorinated biphenyl (PCB) emissions from contaminated sediment to air. Paraburkholderia xenovorans strain LB400 was added to bioreactors containing PCB-contaminated site sediment. PCB mass in both the headspace and aqueous bioreactor compartments was measured using passive samplers over 35 days. Time-series measurements of all 209 PCB congeners revealed a 57% decrease in total PCB mass accumulated in the vapor phase of bioaugmented treatments relative to non-bioaugmented controls, on average. A comparative congener-specific analysis revealed preferential biodegradation of lower-chlorinated PCBs (LC-PCBs) by LB400. Release of the most abundant congener (PCB 4 [2,2'-dichlorobiphenyl]) decreased by over 90%. Simulations with a PCB reactive transport model closely aligned with experimental observations. We also evaluated the effect of the phytogenic biosurfactant, saponin, on PCB bioavailability and biodegradation by LB400. Time-series qPCR measurements of biphenyl dioxygenase (bphA) genes showed that saponin better maintained bphA abundance, compared to the saponin-free treatment. These findings indicate that an active population of bioaugmented, aerobic PCB-degrading microorganisms can effectively lower PCB emissions and may therefore contribute to minimizing PCB inhalation exposure in communities surrounding PCB-contaminated sites.

Combined read- and assembly-based metagenomics to reconstruct a Dehalococcoides mccartyi genome from PCB-contaminated sediments and evaluate functional differences among organohalide-respiring consortia in the presence of different halogenated contaminants.

Microbial communities that support respiration of halogenated organic contaminants by Dehalococcoides sp. facilitate full-scale bioremediation of chlorinated ethenes and demonstrate the potential to aid in bioremediation of halogenated aromatics like polychlorinated biphenyls (PCBs). However, it remains unclear if Dehalococcoides-containing microbial community dynamics observed in sediment-free systems quantitatively resemble that of sediment environments. To evaluate that possibility we assembled, annotated, and analyzed a Dehalococcoides sp. metagenome-assembled genome (MAG) from PCB-contaminated sediments. Phylogenetic analysis of reductive dehalogenase gene (rdhA) sequences within the MAG revealed that pcbA1 and pcbA4/5-like rdhA were absent, while several candidate PCB dehalogenase genes and potentially novel rdhA sequences were identified. Using a compositional comparative metagenomics approach, we quantified Dehalococcoides-containing microbial community structure shifts in response to halogenated organics and the presence of sediments. Functional level analysis revealed significantly greater abundances of genes associated with cobamide remodeling and horizontal gene transfer in tetrachloroethene-fed cultures as compared to halogenated aromatic-exposed consortia with or without sediments, despite little evidence of statistically significant differences in microbial community taxonomic structure. Our findings support the use of a generalizable comparative metagenomics workflow to evaluate Dehalococcoides-containing consortia in sediments and sediment-free environments to eludicate functions and microbial interactions that facilitate bioremediation of halogenated organic contaminants.

Metagenomes, Metagenome-Assembled Genomes, and Metatranscriptomes from Polychlorinated Biphenyl-Contaminated Sediment Microcosms.

We present a comprehensive data set that describes an anaerobic microbial consortium native to polychlorinated biphenyl (PCB)-contaminated sediments. Obtained from sediment microcosms incubated for 200 days, the data set includes 4 metagenomes, 4 metatranscriptomes (in duplicate), and 62 metagenome-assembled genomes and captures microbial community interactions, structure, and function relevant to anaerobic PCB biodegradation.

Natural Biodegradation of Vinyl Chloride and cis-Dichloroethene in Aerobic and Suboxic Conditions.

Chlorinated ethene (CE) groundwater contamination is commonly treated through anaerobic biodegradation (i.e., reductive dechlorination) either as part of an engineered system or through natural attenuation. Aerobic biodegradation has also been recognized as a potentially significant pathway for the removal of the lower CEs cis-1,2-dichloroethene (cDCE) and vinyl chloride (VC). However, the role of aerobic biodegradation under low oxygen conditions typical of contaminated groundwater is unclear. Bacteria capable of aerobic VC biodegradation appear to be common in the environment, while aerobic biodegradation of cDCE is less common and little is known regarding the organisms responsible. In this study, we investigate the role of aerobic cDCE and VC biodegradation in a mixed contaminant plume (including CEs, BTEX, and ketones) at Naval Air Station North Island, Installation Restoration Site 9. Sediment and groundwater collected from the plume source area, mid-plume, and shoreline were used to prepare microcosms under fully aerobic (8 mg/L dissolved oxygen (DO)) and suboxic (< 1 mg/L DO) conditions. In the shoreline microcosms, VC and cDCE were rapidly degraded under suboxic conditions (100% and 77% removal in < 62 days). In the suboxic VC microcosms, biodegradation was associated with a > 5 order of magnitude increase in the abundance of functional gene etnE, part of the aerobic VC utilization pathway. VC and cDCE were degraded more slowly under fully aerobic conditions (74% and 30% removal) in 110 days. High-throughput 16S rRNA and etnE sequencing suggest the presence of novel VC- and cDCE-degrading bacteria. These results suggest that natural aerobic biodegradation of cDCE and VC is occurring at the site and provide new evidence that low (< 1 mg/L) DO levels play a significant role in natural attenuation of cDCE and VC.

Microbial communities in polychlorinated biphenyl (PCB)-contaminated wastewater lagoon sediments: PCB congener, quantitative PCR, and 16S rRNA gene amplicon sequencing datasets.

The potential for aerobic and anaerobic microbial natural attenuation of PCBs in freshwater sediments is described by PCB congener, quantitative PCR, and 16S rRNA gene amplicon sequencing datasets generated, in duplicate, from 27 sediment samples collected from a PCB-contaminated freshwater lagoon (54 samples total). Sediment samples were subjected to a hexane PCB extraction protocol and the concentrations of 209 PCB congeners were determined in hexane extracts by gas chromatography with a tandem mass spectrometry detection. DNA was extracted from sediments sediment samples and used for qPCR and 16S rRNA amplicon sequencing. The abundance of 16S rRNA genes (i.e., Dehalococcoides and putative dechlorinating Chloroflexi) and functional genes (i.e., reductive dehalogenase (rdhA) and biphenyl dioxygenase (bphA)) associated with aerobic and anaerobic PCB biodegradation, along with the total 16S rRNA genes abundance, was determined by SYBR green qPCR. The microbial community composition and structure in all sediment samples was obtained by 16S rRNA gene amplicon sequencing. Primers targeting the 16S rRNA gene V4 region were used to produce 16S rRNA gene amplicons that were sequencing with the high-throughput Illumina MiSeq platform and sequencing chemistry. The 16S rRNA gene sequencing dataset along with PCB congener and qPCR datasets included as metadata, could be reused in meta-analyses that aim to determine microbial community interactions in contaminated environments, and uncover relationships between microbial community structure and environmental variable (e.g., PCB congener concentrations).

Growth of Dehalococcoides spp. and increased abundance of reductive dehalogenase genes in anaerobic PCB-contaminated sediment microcosms.

Polychlorinated biphenyls (PCBs) contaminate 19% of US Superfund sites and represent a serious risk to human and environmental health. One promising strategy to remediate PCB-contaminated sediments utilizes organohalide-respiring bacteria (OHRB) that dechlorinate PCBs.However, functional genes that act as biomarkers for PCB dechlorination processes (i.e., reductive dehalogenase genes) are poorly understood. Here, we developed anaerobic sediment microcosms that harbor an OHRB community dominated by the genus Dehalococcoides. During the 430-day microcosm incubation, Dehalococcoides 16S rRNA sequences increased two orders of magnitude to 107 copies/g of sediment, and at the same time, PCB118 decreased by as much as 70%. In addition, the OHRB community dechlorinated a range of penta- and tetra-chlorinated PCB congeners including PCBs 66, 70 + 74 + 76, 95, 90 + 101, and PCB110 without exogenous electron donor. We quantified candidate reductive dehalogenase (RDase) genes over a 430-day incubation period and found rd14, a reductive dehalogenase that belongs to Dehalococcoides mccartyi strain CG5, was enriched to 107 copies/g of sediment. At the same time, pcbA5 was enriched to only 105 copies/g of sediment. A survey for additional RDase genes revealed sequences similar to strain CG5's rd4 and rd8. In addition to demonstrating the PCB dechlorination potential of native microbial communities in contaminated freshwater sediments, our results suggest candidate functional genes with previously unexplored potential could serve as biomarkers of PCB dechlorination processes.

PCB dechlorination hotspots and reductive dehalogenase genes in sediments from a contaminated wastewater lagoon.

Polychlorinated biphenyls (PCBs) are a class of persistent organic pollutants that are distributed worldwide. Although industrial PCB production has stopped, legacy contamination can be traced to several different commercial mixtures (e.g., Aroclors in the USA). Despite their persistence, PCBs are subject to naturally occurring biodegradation processes, although the microbes and enzymes involved are poorly understood. The biodegradation potential of PCB-contaminated sediments in a wastewater lagoon located in Virginia (USA) was studied. Total PCB concentrations in sediments ranged from 6.34 to 12,700 mg/kg. PCB congener profiles in sediment sample were similar to Aroclor 1248; however, PCB congener profiles at several locations showed evidence of dechlorination. The sediment microbial community structure varied among samples but was dominated by Proteobacteria and Firmicutes. The relative abundance of putative dechlorinating Chloroflexi (including Dehalococcoides sp.) was 0.01-0.19% among the sediment samples, with Dehalococcoides sp. representing 0.6-14.8% of this group. Other possible PCB dechlorinators present included the Clostridia and the Geobacteraceae. A PCR survey for potential PCB reductive dehalogenase genes (RDases) yielded 11 sequences related to RDase genes in PCB-respiring Dehalococcoides mccartyi strain CG5 and PCB-dechlorinating D. mccartyi strain CBDB1. This is the first study to retrieve potential PCB RDase genes from unenriched PCB-contaminated sediments.