Is Long-Term Heavy Metal Exposure Driving Carriage of Antibiotic Resistance in Environmental Opportunistic Pathogens: A Comprehensive Phenomic and Genomic Assessment Using Serratia sp. SRS-8-S-2018.

The carriage of both, heavy metal and antibiotic resistance appears to be a common trait in bacterial communities native to long-term contaminated habitats, including the Savannah River Site (SRS). There is widespread soil contamination at the SRS; a United States Department of Energy (DOE) facility with long-term contamination from past industrial and nuclear weapons production activities. To further evaluate the genomic and metabolic traits that underpin metal and antibiotic resistance, a robust mercury (Hg) and uranium (U)-resistant strain- SRS-8-S-2018, was isolated. Minimum inhibitory concentration of this strain revealed resistance to Hg (10 µg/ml) and U (5 mM), the two main heavy metal contaminants at the SRS. Metabolic assessment of strain SRS-8-S-2018 using Biolog metabolic fingerprinting analysis revealed preference for carbohydrate utilization followed by polymers, amino acids, carboxy acids, and esters; this physiological activity diminished when Hg stress was provided at 1 and 3 µg/ml and completely ceased at 5 µg/ml Hg, indicating that continued release of Hg will have negative metabolic impacts to even those microorganisms that possess high resistance ability. Development of antibiotic resistance in strain SRS-8-S-2018 was evaluated at a functional level using phenomics, which confirmed broad resistance against 70.8% of the 48 antibiotics tested. Evolutionary and adaptive traits of strain SRS-8-S-2018 were further assessed using genomics, which revealed the strain to taxonomically affiliate with Serratia marcescens species, possessing a genome size of 5,323,630 bp, 5,261 proteins (CDS), 55 genes for transfer RNA (tRNA), and an average G + C content of 59.48. Comparative genomics with closest taxonomic relatives revealed 360 distinct genes in SRS-8-S-2018, with multiple functions related to both, antibiotic and heavy metal resistance, which likely facilitates the strain's survival in a metalliferous soil habitat. Comparisons drawn between the environmentally isolated Serratia SRS-8-S-2018 with 31 other strains revealed a closer functional association with medically relevant isolates suggesting that propensity of environmental Serratia isolates in acquiring virulence traits, as a function of long-term exposure to heavy metals, which is facilitating development, recruitment and proliferation of not only metal resistant genes (MRGs) but antibiotic resistant genes (ARGs), which can potentially trigger future bacterial pathogen outbreaks emanating from contaminated environmental habitats.

Characterization of Bacterial and Fungal Assemblages From Historically Contaminated Metalliferous Soils Using Metagenomics Coupled With Diffusion Chambers and Microbial Traps.

The majority of environmental microbiomes are not amenable to cultivation under standard laboratory growth conditions and hence remain uncharacterized. For environmental applications, such as bioremediation, it is necessary to isolate microbes performing the desired function, which may not necessarily be the fast growing or the copiotroph microbiota. Toward this end, cultivation and isolation of microbial strains using diffusion chambers (DC) and/or microbial traps (MT) have both been recently demonstrated to be effective strategies because microbial enrichment is facilitated by soil nutrients and not by synthetically defined media, thus simulating their native habitat. In this study, DC/MT chambers were established using soils collected from two US Department of Energy (DOE) sites with long-term history of heavy metal contamination, including mercury (Hg). To characterize the contamination levels and nutrient status, soils were first analyzed for total mercury (THg), methylmercury (MeHg), total carbon (TC), total nitrogen (TN), and total phosphorus (TP). Multivariate statistical analysis on these measurements facilitated binning of soils under high, medium and low levels of contamination. Bacterial and fungal microbiomes that developed within the DC and MT chambers were evaluated using comparative metagenomics, revealing Chthoniobacter, Burkholderia and Bradyrhizobium spp., as the predominant bacteria while Penicillium, Thielavia, and Trichoderma predominated among fungi. Many of these core microbiomes were also retrieved as axenic isolates. Furthermore, canonical correspondence analysis (CCA) of biogeochemical measurements, metal concentrations and bacterial communities revealed a positive correlation of Chthoniobacter/Bradyrhizobium spp., to THg whereas Burkholderia spp., correlated with MeHg. Penicillium spp., correlated with THg whereas Trichoderma spp., and Aspergillus spp., correlated with MeHg, from the MT approach. This is the first metagenomics-based assessment, isolation and characterization of soil-borne bacterial and fungal communities colonizing the diffusion chambers (DC) and microbial traps (MT) established with long-term metal contaminated soils. Overall, this study provides proof-of-concept for the successful application of DC/MT based assessment of mercury resistant (HgR) microbiomes in legacy metal-contaminated soils, having complex contamination issues. Overall, this study brings out the significance of microbial communities and their relevance in context to heavy metal cycling for better stewardship and restoration of such historically contaminated systems.

Corrigendum: Genomic Characterization of a Mercury Resistant Arthrobacter sp. H-02-3 Reveals the Presence of Heavy Metal and Antibiotic Resistance Determinants.

[This corrects the article DOI: 10.3389/fmicb.2019.03039.].

Metagenomic Evaluation of Bacterial and Fungal Assemblages Enriched within Diffusion Chambers and Microbial Traps Containing Uraniferous Soils.

Despite significant technological advancements in the field of microbial ecology, cultivation and subsequent isolation of the vast majority of environmental microorganisms continues to pose challenges. Isolation of the environmental microbiomes is prerequisite to better understand a myriad of ecosystem services they provide, such as bioremediation of contaminants. Towards this end, in this culturomics study, we evaluated the colonization of soil bacterial and fungal communities within diffusion chambers (DC) and microbial traps (MT) established using uraniferous soils collected from a historically contaminated soil from Aiken, USA. Microbial assemblages were compared between the DC and MT relative to the native soils using amplicon based metagenomic and bioinformatic analysis. The overall rationale of this study is that DC and MT growth chambers provide the optimum conditions under which desired microbiota, identified in a previous study to serve as the "core" microbiomes, will proliferate, leading to their successful isolation. Specifically, the core microbiomes consisted of assemblages of bacteria (Burkholderia spp.) and fungi (Penicillium spp.), respectively. The findings from this study further supported previous data such that the abundance and diversity of the desired "core" microbiomes significantly increased as a function of enrichments over three consecutive generations of DC and MT, respectively. Metagenomic analysis of the DC/MT generations also revealed that enrichment and stable populations of the desired "core" bacterial and fungal microbiomes develop within the first 20 days of incubation and the practice of subsequent transfers for second and third generations, as is standard in previous studies, may be unnecessary. As a cost and time cutting measure, this study recommends running the DC/MT chambers for only a 20-day time period, as opposed to previous studies, which were run for months. In summation, it was concluded that, using the diffusion chamber-based enrichment techniques, growth of desired microbiota possessing environmentally relevant functions can be achieved in a much shorter time frame than has been previously shown.

Metagenomics-Guided Survey, Isolation, and Characterization of Uranium Resistant Microbiota from the Savannah River Site, USA.

Despite the recent advancements in culturomics, isolation of the majority of environmental microbiota performing critical ecosystem services, such as bioremediation of contaminants, remains elusive. Towards this end, we conducted a metagenomics-guided comparative assessment of soil microbial diversity and functions present in uraniferous soils relative to those that grew in diffusion chambers (DC) or microbial traps (MT), followed by isolation of uranium (U) resistant microbiota. Shotgun metagenomic analysis performed on the soils used to establish the DC/MT chambers revealed Proteobacterial phyla and Burkholderia genus to be the most abundant among bacteria. The chamber-associated growth conditions further increased their abundances relative to the soils. Ascomycota was the most abundant fungal phylum in the chambers relative to the soils, with Penicillium as the most dominant genus. Metagenomics-based taxonomic findings completely mirrored the taxonomic composition of the retrieved isolates such that the U-resistant bacteria and fungi mainly belonged to Burkholderia and Penicillium species, thus confirming that the chambers facilitated proliferation and subsequent isolation of specific microbiota with environmentally relevant functions. Furthermore, shotgun metagenomic analysis also revealed that the gene classes for carbohydrate metabolism, virulence, and respiration predominated with functions related to stress response, membrane transport, and metabolism of aromatic compounds were also identified, albeit at lower levels. Of major note was the successful isolation of a potentially novel Penicillium species using the MT approach, as evidenced by whole genome sequence analysis and comparative genomic analysis, thus enhancing our overall understanding on the uranium cycling microbiota within the tested uraniferous soils.

Genomic Characterization of a Mercury Resistant Arthrobacter sp. H-02-3 Reveals the Presence of Heavy Metal and Antibiotic Resistance Determinants

Nuclear production and industrial activities led to widespread contamination of the Department of Energy (DOE) managed Savannah River Site (SRS), located in South Carolina, United States. The H-02 wetland system was constructed in 2007 for the treatment of industrial and storm water runoff from the SRS Tritium Facility. Albeit at low levels, mercury (Hg) has been detected in the soils of the H-02 wetland ecosystem. In anoxic sediments, Hg is typically methylated by anaerobic microbiota, forming the highly neurotoxic methylmercury (MeHg), which biomagnifies across food webs. However, in surficial oxic wetland soils, microbially mediated demethylation and/or volatilization processes can transform Hg2+ into the less toxic Hg0 form which is released into the atmosphere, thus circumventing MeHg formation. To obtain a deeper understanding on bacterial Hg volatilization, a robust Hg-resistant (HgR) bacteria, called as strain H-02-3 was isolated from the H-02 soils. A draft genome sequence of this strain was obtained at a coverage of 700×, which assembled in 44 contigs with an N50 of 171,569 bp. The genomic size of the strain H-02-3 was 4,708,612 bp with a total number of 4,240 genes; phylogenomic analysis revealed the strain as an Arthrobacter species. Comparative genomics revealed the presence of 1100 unique genes in strain H-02-3, representing 26.7% of the total genome; many identified previously as metal resistance genes (MRGs). Specific to Hg-cycling, the presence of mercuric ion reductase (merA), the organomercurial lyase (merB), and the mercuric resistance operon regulatory protein, were identified. By inference, it can be proposed that the organomercurial lyase facilitates the demethylation of MeHg into Hg2+ which is then reduced to Hg0 by MerA in strain H-02-3. Furthermore, gene prediction using resistome analysis of strain H-02-3 revealed the presence of several antibiotic resistance genes (ARGs), that statistically correlated with the presence of metal resistant genes (MRGs), suggesting co-occurrence patterns of MRGs and ARGs in the strain. Overall, this study delineates environmentally beneficial traits that likely facilitates survival of Arthrobacter sp. H-02-3 within the H-02 wetland soil. Finally, this study also highlights the largely ignored public health risk associated with the co-development of ARGs and MRGs in bacteria native to historically contaminated soils.

Proteogenomic Analysis of Burkholderia Species Strains 25 and 46 Isolated from Uraniferous Soils Reveals Multiple Mechanisms to Cope with Uranium Stress.

Two Burkholderia spp. (strains SRS-25 and SRS-46) were isolated from high concentrations of uranium (U) from the U.S. Department of Energy (DOE)-managed Savannah River Site (SRS). SRS contains soil gradients that remain co-contaminated by heavy metals from previous nuclear weapons production activities. Uranium (U) is one of the dominant contaminants within the SRS impacted soils, which can be microbially transformed into less toxic forms. We established microcosms containing strains SRS-25 and SRS-46 spiked with U and evaluated the microbially-mediated depletion with concomitant genomic and proteomic analysis. Both strains showed a rapid depletion of U; draft genome sequences revealed SRS-25 genome to be of approximately 8,152,324 bp, a G + C content of 66.5, containing a total 7604 coding sequences with 77 total RNA genes. Similarly, strain SRS-46 contained a genome size of 8,587,429 bp with a G + C content of 67.1, 7895 coding sequences, with 73 total RNA genes, respectively. An in-depth, genome-wide comparisons between strains 25, 46 and a previously isolated strain from our research (Burkholderia sp. strain SRS-W-2-2016), revealed a common pool of 3128 genes; many were found to be homologues to previously characterized metal resistance genes (e.g., for cadmium, cobalt, and zinc), as well as for transporter, stress/detoxification, cytochromes, and drug resistance functions. Furthermore, proteomic analysis of strains with or without U stress, revealed the increased expression of 34 proteins from strain SRS-25 and 52 proteins from strain SRS-46; similar to the genomic analyses, many of these proteins have previously been shown to function in stress response, DNA repair, protein biosynthesis and metabolism. Overall, this comparative proteogenomics study confirms the repertoire of metabolic and stress response functions likely rendering the ecological competitiveness to the isolated strains for colonization and survival in the heavy metals contaminated SRS soil habitat.

Draft Genome Sequence of Pseudomonas sp. Strain B1, Isolated from a Contaminated Sediment.

The draft genome sequence of Pseudomonas sp. strain B1, isolated from a contaminated soil, is reported. The genome comprises 6,706,934 bases, 6,059 coding sequences, and 70 RNAs and has a G+C content of 60.3%. A suite of biodegradative genes, many located on genomic islands, were identified from strain B1, further enhancing our understanding of the versatile pseudomonads.

Draft Genome Sequence of Serratia sp. Strain S1B, Isolated from Mercury-Contaminated Soil.

We report here the draft genome sequence of Serratia sp. strain S1B, comprising 7,710,841 bases, 7,075 coding sequences, a G+C content of 45.9%, and 138 RNAs. Notably, a repertoire of biodegradative genes, several occurring on genomic islands, was also identified, which enhances our understanding of the environmental relevance of Serratia spp.

Physiological and Comparative Genomic Analysis of Arthrobacter sp. SRS-W-1-2016 Provides Insights on Niche Adaptation for Survival in Uraniferous Soils.

Arthrobacter sp. strain SRS-W-1-2016 was isolated on high concentrations of uranium (U) from the Savannah River Site (SRS) that remains co-contaminated by radionuclides, heavy metals, and organics. SRS is located on the northeast bank of the Savannah River (South Carolina, USA), which is a U.S. Department of Energy (DOE) managed ecosystem left historically contaminated from decades of nuclear weapons production activities. Predominant contaminants within the impacted SRS environment include U and Nickel (Ni), both of which can be transformed microbially into less toxic forms via metal complexation mechanisms. Strain SRS-W-1-2016 was isolated from the uraniferous SRS soils on high concentrations of U (4200 µM) and Ni (8500 µM), but rapid growth was observed at much lower concentrations of 500 µM U and 1000 µM Ni, respectively. Microcosm studies established with strain SRS-W-1-2016 revealed a rapid decline in the concentration of spiked U such that it was almost undetectable in the supernatant by 72 h of incubation. Conversely, Ni concentrations remained unchanged, suggesting that the strain removed U but not Ni under the tested conditions. To obtain a deeper understanding of the metabolic potential, a draft genome sequence of strain SRS-W-1-2016 was obtained at a coverage of 90×, assembling into 93 contigs with an N50 contig length of 92,788 bases. The genomic size of strain SRS-W-1-2016 was found to be 4,564,701 bases with a total number of 4327 putative genes. An in-depth, genome-wide comparison between strain SRS-W-1-2016 and its four closest taxonomic relatives revealed 1159 distinct genes, representing 26.7% of its total genome; many associating with metal resistance proteins (e.g., for cadmium, cobalt, and zinc), transporter proteins, stress proteins, cytochromes, and drug resistance functions. Additionally, several gene homologues coding for resistance to metals were identified in the strain, such as outer membrane efflux pump proteins, peptide/nickel transport substrate and ATP-binding proteins, a high-affinity nickel-transport protein, and the spoT gene, which was recently implicated in bacterial resistance towards U. Detailed genome mining analysis of strain SRS-W-1-2016 also revealed the presence of a plethora of secondary metabolite biosynthetic gene clusters likely facilitating resistance to antibiotics, biocides, and metals. Additionally, several gene homologous for the well-known oxygenase enzyme system were also identified, potentially functioning to generate energy via the breakdown of organic compounds and thus enabling the successful colonization and natural attenuation of contaminants by Arthrobacter sp. SRS-W-1-2016 at the SRS site.

Genome-centric evaluation of Burkholderia sp. strain SRS-W-2-2016 resistant to high concentrations of uranium and nickel isolated from the Savannah River Site (SRS), USA.

Savannah River Site (SRS), an approximately 800-km2 former nuclear weapons production facility located near Aiken, SC remains co-contaminated by heavy metals and radionuclides. To gain a better understanding on microbially-mediated bioremediation mechanisms, several bacterial strains resistant to high concentrations of Uranium (U) and Nickel (Ni) were isolated from the Steeds Pond soils located within the SRS site. One of the isolated strains, designated as strain SRS-W-2-2016, grew robustly on both U and Ni. To fully understand the arsenal of metabolic functions possessed by this strain, a draft whole genome sequence (WGS) was obtained, assembled, annotated and analyzed. Genome-centric evaluation revealed the isolate to belong to the Burkholderia genus with close affiliation to B. xenovorans LB400, an aggressive polychlorinated biphenyl-degrader. At a coverage of 90 ×, the genome of strain SRS-W-2-2016 consisted of 8,035,584 bases with a total number of 7071 putative genes assembling into 191 contigs with an N50 contig length of 134,675 bases. Several gene homologues coding for resistance to heavy metals/radionuclides were identified in strain SRS-W-2-2016, such as a suite of outer membrane efflux pump proteins similar to nickel/cobalt transporter regulators, peptide/nickel transport substrate and ATP-binding proteins, permease proteins, and a high-affinity nickel-transport protein. Also noteworthy were two separate gene fragments in strain SRS-W-2-2016 homologous to the spoT gene; recently correlated with bacterial tolerance to U. Additionally, a plethora of oxygenase genes were also identified in the isolate, potentially involved in the breakdown of organic compounds facilitating the strain's successful colonization and survival in the SRS co-contaminated soils. The WGS project of Burkholderia sp. strain SRS-W-2-2016 is available at DDBJ/ENA/GenBank under the accession #MSDV00000000.