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48100.9891Characterization and structure prediction of partial length protein sequences of pcoA, pcoR and chrB genes from heavy metal resistant bacteria from the Klip River, South Africa. The Klip River has suffered from severe anthropogenic effects from industrial activities such as mining. Long-term exposure to heavy metal pollution has led to the development of heavy metal resistant strains of Pseudomonas sp. KR23, Lysinibacillus sp. KR25, and E. coli KR29. The objectives of this study were to characterize the genetics of copper and chromate resistance of the isolates. Copper and chromate resistance determinants were cloned and sequenced. Open reading frames (ORFs) related to the genes CopA and CopR were identified in E. coli KR29, PcoA in Lysinibacillus sp. KR25 and none related to chromate resistance were detected. The 3D-models predicted by I-TASSER disclose that the PcoA proteins consist of β-sheets, which form a part of the cupredoxin domain of the CopA copper resistance family of genes. The model for PcoR_29 revealed the presence of a helix turn helix; this forms part of a DNA binding protein, which is part of a heavy metal transcriptional regulator. The bacterial strains were cured using ethidium bromide. The genes encoding for heavy metal resistance and antibiotic resistance were found to be located on the chromosome for both Pseudomonas sp. (KR23) and E. coli (KR29). For Lysinibacillus (KR25) the heavy metal resistance determinants are suspected to be located on a mobile genetic element, which was not detected using gel electrophoresis.201525837632
609210.9891Colony-forming analysis of bacterial community succession in deglaciated soils indicates pioneer stress-tolerant opportunists. We investigated the response of bacterial communities inhabiting two deglaciated soils (10 and 100 years post-deglaciation) to two stimuli: (i) physical disruption (mixing), and (ii) disruption plus nutrient addition. PCR/DGGE analysis of 16S rRNA genes extracted from soil during a 168-h incubation period following the stimuli revealed that more bacterial phylotypes were stimulated in the 10-y soil than in the 100-y soil. In addition to 10-y and 100-y soils, two additional soils (46 and 70 y) were further differentiated using colony-forming curve (CFC) analysis during a 168-h incubation period, which revealed that younger soils contained a higher proportion of rapidly colonizing bacteria than successively older soils. "Eco-collections" of CFC isolates that represented colonies that formed "fast" (during the first 24 h) and "slow" (final 36 h) were harvested from 10-y and 100-y soils and differentiated according to response to three stress parameters: (i) tolerance to nutrient limitation, (ii) tolerance to temperature change, and (iii) resistance to antibiotics. The tested parameters distinguished "fast" from "slow" bacteria regardless of the age of the soil from which they were isolated. Specifically, eco-collections of "fast" bacteria exhibited greater nutrient- and temperature-stress tolerance as well as more frequent antibiotic resistance than "slow" bacteria. Further DGGE analysis showed that several eco-collection phylotype bands matched (electrophoretically) those of soil phylotypes enriched by mixing and nutrient stimulus. Overall, the results of this study indicated that the succession of colony-forming bacteria was differentiated by bacterial opportunism and temporal response to stimuli. Furthermore, although stress tolerance strategies are associated with opportunistic bacteria regardless of successional age, it appears that the proportion of opportunistic bacteria distinguishes early vs late succession forefield bacterial populations.200415692851
873920.9891LCT-EF258 with S17I Mutation in DprA Exhibits Horizontal Gene Transfer Deficiency After Spaceflight. BACKGROUND: Space is a special environment in which microgravity and cosmic rays are the primary factors that induce gene mutations of microorganisms. In our previous studies, a single point mutation in the gene dprA was found in an Enterococcus faecium strain of LCT-EF258 after spaceflight. DNA processing protein A (DprA) plays a prominent role in the horizontal transfer of genes among bacteria (such as Streptococcus pneumoniae, Helicobacter pylori, Bacillus subtilis, and Rhodobacter capsulatus). However, the function of DprA in E. faecium remains unknown. Furthermore, E. faecium could acquire antibiotic resistance through the horizontal transfer of antibiotic resistance genes, but it is unclear whether dprA mutants could affect this process in E. faecium.METHODS: In this study, we constructed a plasmid containing the vancomycin resistance gene vanA and then transferred the gene vanA into the dprA-mutant strain LCT-EF258 and the control strain LCT-EF90 using the electroporation technique. We then used Discovery Studio(TM) software to construct the 3D protein structure.RESULTS: The results showed that the horizontal transfer efficiency of the vancomycin resistance gene vanA in the dprA-mutant E. faecium decreased. And the hydrophobic core of the mutant DprA became stable and the binding affinity between the mutant DprA and ssDNA reduced.DISCUSSION: This study is an exploration of bacterial gene mutation after spaceflight. The dprA mutant could affect the ability of E. faecium to acquire exogenous resistance gene vanA, which offered us an interesting path to block the dissemination of resistance genes between strains.Yu Y, Chang D, Guo Q, Wang J, Liu C. LCT-EF258 with S171 mutation in DprA exhibits horizontal gene transfer deficiency after spaceflight. Aerosp Med Hum Perform. 2019; 90(2):116-122.201930670121
514130.9889Flavobacterium flabelliforme sp. nov. and Flavobacterium geliluteum sp. nov., Two Multidrug-Resistant Psychrotrophic Species Isolated From Antarctica. Despite unfavorable Antarctic conditions, such as cold temperatures, freeze-thaw cycles, high ultraviolet radiation, dryness and lack of nutrients, microorganisms were able to adapt and surprisingly thrive in this environment. In this study, eight cold-adapted Flavobacterium strains isolated from a remote Antarctic island, James Ross Island, were studied using a polyphasic taxonomic approach to determine their taxonomic position. Phylogenetic analyses based on the 16S rRNA gene and 92 core genes clearly showed that these strains formed two distinct phylogenetic clusters comprising three and five strains, with average nucleotide identities significantly below 90% between both proposed species as well as between their closest phylogenetic relatives. Phenotyping revealed a unique pattern of biochemical and physiological characteristics enabling differentiation from the closest phylogenetically related Flavobacterium spp. Chemotaxonomic analyses showed that type strains P4023(T) and P7388(T) were characterized by the major polyamine sym-homospermidine and a quinone system containing predominantly menaquinone MK-6. In the polar lipid profile phosphatidylethanolamine, an ornithine lipid and two unidentified lipids lacking a functional group were detected as major lipids. These characteristics along with fatty acid profiles confirmed that these species belong to the genus Flavobacterium. Thorough genomic analysis revealed the presence of numerous cold-inducible or cold-adaptation associated genes, such as cold-shock proteins, proteorhodopsin, carotenoid biosynthetic genes or oxidative-stress response genes. Genomes of type strains surprisingly harbored multiple prophages, with many of them predicted to be active. Genome-mining identified biosynthetic gene clusters in type strain genomes with a majority not matching any known clusters which supports further exploratory research possibilities involving these psychrotrophic bacteria. Antibiotic susceptibility testing revealed a pattern of multidrug-resistant phenotypes that were correlated with in silico antibiotic resistance prediction. Interestingly, while typical resistance finder tools failed to detect genes responsible for antibiotic resistance, genomic prediction confirmed a multidrug-resistant profile and suggested even broader resistance than tested. Results of this study confirmed and thoroughly characterized two novel psychrotrophic Flavobacterium species, for which the names Flavobacterium flabelliforme sp. nov. and Flavobacterium geliluteum sp. nov. are proposed.202134745033
349740.9887Biomarkers of antibiotic resistance genes during seasonal changes in wastewater treatment systems. To evaluate the seasonal distribution of antibiotic resistance genes (ARGs) and explore the reason for their patterns in different seasons and different systems, two wastewater treatment systems were selected and analyzed using high-throughput qPCR. Linear discriminant analysis (LDA) effect size (LEfSe) was used to discover the differential ARGs (biomarkers) and estimate the biomarkers' effect size. We found that the total absolute abundances of ARGs in inflows and excess sludge samples had no obvious seasonal fluctuations, while those in winter outflow samples decreased in comparison with the inflow samples. Eleven differentially abundant ARGs (biomarker genes, BmGs) (aadA5-02, aac-6-II, cmlA1-01, cmlA1-02, blaOXA10-02, aadA-02, tetX, aadA1, ereA, qacEΔ1-01, and blaTEM) in summer samples and 10 BmGs (tet-32, tetA-02, aacC2, vanC-03, aac-6-I1, tetE, ermB, mefA, tnpA - 07, and sul2) in winter samples were validated. According to 16S rRNA gene sequencing, the relative abundance of bacteria at the phylum level exhibited significant seasonal changes in outflow water (OW), and biomarker bacteria (BmB) were discovered at the family (or genus) level. Synechococcus and vadinCA02 are BmB in summer, and Trichococcus, Lactococcus, Pelosinus, Janthinobacterium, Nitrosomonadaceae and Sterolibacterium are BmB in winter. In addition, BmB have good correlations with BmGs in the same season, which indicates that bacterial community changes drive different distributions of ARGs during seasonal changes and that LEfSe is an acute and effective method for finding significantly different ARGs and bacteria between two or more classes. In conclusion, this study demonstrated the seasonal changes of BmGs and BmB at two wastewater treatment systems.201829169020
865450.9886Metagenomic and Metatranscriptomic Study of Microbial Metal Resistance in an Acidic Pit Lake. Cueva de la Mora (CM) is an acidic, meromictic pit lake in the Iberian Pyrite Belt characterized by extremely high metal(loid) concentrations and strong gradients in oxygen, metal, and nutrient concentrations. We hypothesized that geochemical variations with depth would result in differences in community composition and in metal resistance strategies among active microbial populations. We also hypothesized that metal resistance gene (MRG) expression would correlate with toxicity levels for dissolved metal species in the lake. Water samples were collected in the upper oxic layer, chemocline, and deep anoxic layer of the lake for shotgun metagenomic and metatranscriptomic sequencing. Metagenomic analyses revealed dramatic differences in the composition of the microbial communities with depth, consistent with changing geochemistry. Based on relative abundance of taxa identified in each metagenome, Eukaryotes (predominantly Coccomyxa) dominated the upper layer, while Archaea (predominantly Thermoplasmatales) dominated the deep layer, and a combination of Bacteria and Eukaryotes were abundant at the chemocline. We compared metal resistance across communities using a curated list of protein-coding MRGs with KEGG Orthology identifiers (KOs) and found that there were broad differences in the metal resistance strategies (e.g., intracellular metal accumulation) expressed by Eukaryotes, Bacteria, and Archaea. Although normalized abundances of MRG and MRG expression were generally higher in the deep layer, expression of metal-specific genes was not strongly related to variations in specific metal concentrations, especially for Cu and As. We also compared MRG potential and expression in metagenome assembled genomes (MAGs) from the deep layer, where metal concentrations are highest. Consistent with previous work showing differences in metal resistance mechanisms even at the strain level, MRG expression patterns varied strongly among MAG populations from the same depth. Some MAG populations expressed very few MRG known to date, suggesting that novel metal resistance strategies remain to be discovered in uncultivated acidophiles.202032899650
611460.9886Uranium and other heavy metal resistance and accumulation in bacteria isolated from uranium mine wastes. Ten bacterial strains isolated from uranium mine wastes were characterized in terms of their uranium and other metal resistance and accumulation. 16S rRNA gene sequence analysis identified the strains as members of genera Bacillus, Serratia, and Arthrobacter. Strains were able to utilize various carbon sources, particularly aromatic hydrocarbons, grow at broad pH and temperature ranges and produce non specific acid phosphatase relevant for metal phosphate precipitation in contaminated environment. The isolates exhibited high uranium and other heavy metals (Ni, Co, Cu and Cd) resistance and accumulation capacities. Particularly, Arthrobacter sp. J001 and Bacillus sp. J003 were superior in terms of U resistance at low pH (pH 4.0) along with metals and actinides (U and Th) removal with maximum cell loading of 1088 μmol U, 1293 μmol Th, 425 μmol Cu, 305 μmol Cd, 377 μmol Zn, 250 μmol Ni g(-1) cell dry wt. Genes encoding P(1B)-type ATPases (Cu-CPx and Zn-CPx) and ABC transporters (nik) as catalytic tools for maintaining cellular metal homeostasis were detected within several Bacillus spp., with possible incidence of horizontal gene transfer for the later gene showing phylogenetic lineage to α Proteobacteria members. The study provides evidence on intrinsic abilities of indigenous bacteria from U-mine suitable for survival and cleaning up of contaminated mine sites.201222375546
723970.9886Full-scale mesophilic biogas plants using manure as C-source: bacterial community shifts along the process cause changes in the abundance of resistance genes and mobile genetic elements. The application of manure, typically harboring bacteria carrying resistance genes (RGs) and mobile genetic elements (MGEs), as co-substrate in biogas plants (BGPs) might be critical when digestates are used as fertilizers. In the present study, the relative abundance of RGs and MGEs in total community (TC-) DNA from manure, fermenters and digestate samples taken at eight full-scale BGPs co-fermenting manure were determined by real-time PCR. In addition, the bacterial community composition of all digestates as well as manure and fermenter material from one BGP (BGP3) was characterized by 454-pyrosequencing of 16S rRNA amplicons from TC-DNA. Compared to respective input manures, relative abundances determined for sul1, sul2, tet(M), tet(Q), intI1, qacEΔ1, korB and traN were significantly lower in fermenters, whereas relative abundances of tet(W) were often higher in fermenters. The bacterial communities in all digestates were dominated by Firmicutes and Bacteroidetes while Proteobacteria were low in abundance and no Enterobacteriaceae were detected. High-throughput sequencing revealed shifts in bacterial communities during treatment for BGP3. Although in comparison to manure, digestate bacteria had lower relative abundances of RGs and MGEs except for tet(W), mesophilic BGPs seem not to be effective for prevention of the spread of RGs and MGEs via digestates into arable soils.201626772986
637980.9886Shotgun metagenome guided exploration of anthropogenically driven resistomic hotspots within Lonar soda lake of India. Anthropogenic activities mediated antibiotic resistance genes (ARGs) in the pristine aquatic bodies (lakes) is raising concern worldwide. Long read shotgun sequencing was used to assess taxonomic diversity, distribution of ARGs and metal resistance genes (MRGs) and mobile genetic elements (MGEs) in six sites within hypersaline Lonar soda lake (India) prone to various anthropogenic activities. Proteobacteria and Euryarchaeota were dominant phyla under domain Bacteria and Archaea respectively. Higher abundance of Bacteroidetes was pragmatic at sites 18LN5 and 18LN6. Functional analysis indicated 26 broad-spectrum ARGs types, not reported earlier in this ecosystem. Abundant ARG types identified were multidrug efflux, glycopepetide, bacitracin, tetracycline and aminogylcoside resistance. Sites 18LN1 and 18LN5 depicted 167 and 160 different ARGs subtypes respectively and rpoB2, bcrA, tetA(48), mupA, ompR, patA, vanR and multidrug ABC transporter genes were present in all samples. The rpoB2 gene was dominant in 18LN1, whereas bcrA gene in 18LN2-18LN6 sites. Around 24 MRGs types were detected with higher abundance of arsenic in 18LN1 and copper in 18LN2-18LN6, signifying metal contamination linked to MRGs. The bacterial taxa Pseudomonas, Thioalkalivibrio, Burkholderia, Clostridium, Paenibacillus, Bacillus and Streptomyces were significantly associated with ARGs. This study highlights the resistomic hotspots in the lake for deploying policies for conservation efforts.202032155479
774190.9885Microbial diversity of a full-scale UASB reactor applied to poultry slaughterhouse wastewater treatment: integration of 16S rRNA gene amplicon and shotgun metagenomic sequencing. The 16S rRNA gene amplicon and whole-genome shotgun metagenomic (WGSM) sequencing approaches were used to investigate wide-spectrum profiles of microbial composition and metabolic diversity from a full-scale UASB reactor applied to poultry slaughterhouse wastewater treatment. The data were generated by using MiSeq 2 × 250 bp and HiSeq 2 × 150 bp Illumina sequencing platforms for 16S amplicon and WGSM sequencing, respectively. Each approach revealed a distinct microbial community profile, with Pseudomonas and Psychrobacter as predominant genus for the WGSM dataset and Clostridium and Methanosaeta for the 16S rRNA gene amplicon dataset. The virome characterization revealed the presence of two viral families with Bacteria and Archaea as host, Myoviridae, and Siphoviridae. A wide functional diversity was found with predominance of genes involved in the metabolism of acetone, butanol, and ethanol synthesis; and one-carbon metabolism (e.g., methanogenesis). Genes related to the acetotrophic methanogenesis pathways were more abundant than methylotrophic and hydrogenotrophic, corroborating the taxonomic results that showed the prevalence of the acetotrophic genus Methanosaeta. Moreover, the dataset indicated a variety of metabolic genes involved in sulfur, nitrogen, iron, and phosphorus cycles, with many genera able to act in all cycles. BLAST analysis against Antibiotic Resistance Genes Database (ARDB) revealed that microbial community contained 43 different types of antibiotic resistance genes, some of them were associated with growth chicken promotion (e.g., bacitracin, tetracycline, and polymyxin).201728229558
7639100.9885Structural and Functional Changes of Groundwater Bacterial Community During Temperature and pH Disturbances. In this study, we report the characteristics of a microbial community in sampled groundwater and elucidate the effects of temperature and pH disturbances on bacterial structure and nitrogen-cycling functions. The predominant phyla of candidate OD1, candidate OP3, and Proteobacteria represented more than half of the total bacteria, which clearly manifested as a "low nucleic acid content (LNA) bacteria majority" type via flow cytometric fingerprint. The results showed that LNA bacteria were more tolerant to rapid changes in temperature and pH, compared to high nucleic acid content (HNA) bacteria. A continuous temperature increase test demonstrated that the LNA bacterial group was less competitive than the HNA bacterial group in terms of maintaining their cell intactness and growth potential. In contrast, the percentage of intact LNA bacteria was maintained at nearly 70% with pH decrease, despite a 50% decrease in total intact cells. Next-generation sequencing results revealed strong resistance and growth potential of phylum Proteobacteria when the temperature increased or the pH decreased in groundwater, especially for subclasses α-, β-, and γ-Proteobacteria. In addition, relative abundance of nitrogen-related functional genes by qPCR showed no difference in nitrifiers or denitrifiers within 0.45 μm-captured and 0.45 μm-filterable bacteria due to phylogenetic diversity. One exception was the monophyletic anammox bacteria that belong to the phylum Planctomycetes, which were mostly captured on a 0.45-μm filter. Furthermore, we showed that both temperature increase and pH decrease could enhance the denitrification potential, whereas the nitrification and anammox potentials were weakened.201930706112
3612110.9884Copper resistance in Desulfovibrio strain R2. A sulfate-reducing bacterium, designated as strain R2, was isolated from wastewater of a ball-bearing manufacturing facility in Tomsk, Western Siberia. This isolate was resistant up to 800 mg Cu/l in the growth medium. By comparison, Cu-resistance of reference cultures of sulfate-reducing bacteria ranged from 50 to 75 mg Cu/l. Growth experiments with strain R2 showed that Cu was an essential trace element and, on one hand, enhanced growth at concentrations up to 10 mg/l but, on the other hand, the growth rate decreased and lag-period extended at copper concentrations of >50 mg/l. Phenotypic characteristics and a 1078 bp nucleotide sequence of the 16S rDNA placed strain R2 within the genus Desulfovibrio. Desulfovibrio R2 carried at least one plasmid of approximately of 23.1 kbp. A 636 bp fragment of the pcoR gene of the pco operon that encodes Cu resistance was amplified by PCR from plasmid DNA of strain R2. The pco genes are involved in Cu-resistance in some enteric and aerobic soil bacteria. Desulfovibrio R2 is a prospective strain for bioremediation purposes and for developing a homologous system for transformation of Cu-resistance in sulfate-reducing bacteria.200312755486
8484120.9884Deciphering the acidophilia and acid resistance in Acetilactobacillus jinshanensis dominating baijiu fermentation through multi-omics analysis. Lactic acid bacteria (LAB) are pivotal in constructing the intricate bio-catalytic networks underlying traditional fermented foods such as Baijiu. However, LAB and their metabolic mechanisms are partially understood in Moutai flavor Baijiu fermentation. Here, we found that Acetilactobacillus jinshanensis became the· dominant species with relative abundance reaching 92%, where the acid accumulated rapidly and peaked at almost 30 g/kg in Moutai flavor Baijiu. After separation, purification, and cultivation, A. jinshanensis exhibited pronounced acidophilia and higher acid resistance compared to other LAB. Further integrated multi-omics analysis revealed that fatty acid synthesis, cell membrane integrity, pHi and redox homeostasis maintenance, protein and amide syntheses were possibly crucial acid-resistant mechanisms in A. jinshanensis. Structural proteomics indicated that the surfaces of A. jinshanensis proteases contained more positively charged amino acid residues to maintain protein stability in acidic environments. The genes HSP20 and acpP were identified as acid-resistant genes for A. jinshanensis by heterologous expression analysis. These findings not only enhance our understanding of LAB in Baijiu, providing a scientific basis for acid regulation for production process, but also offer valuable insights for studying core species in other fermentation systems.202539448165
6347130.9884Bifidobacterium adolescentis is resistant to pyrazinamide, isoniazid, and streptomycin. The current study aims to understand the resistance of Bifidobacterium adolescentis to different anti-tubercular drugs (first-line oral tuberculosis drugs). The bacteria were grown with anti-tubercular drugs such as isoniazid, pyrazinamide, and streptomycin to better understand the resistance phenomena. It was found that even at tenfold higher concentrations, growth rates remained unchanged. In addition, a small number of bacteria were found to aggregate strongly, a property that protects against the toxicity of the drug. Further FE-SEM (Field Emission Scanning Electron Microscopy) analysis revealed that some bacteria became excessively long, elongated, and protruded on the surface. Size scattering analysis confirmed the presence of bifidobacteria in the size range of 1.0-100 μm. After whole genome sequence analysis, certain mutations were found in the relevant gene. In vitro, foam formation and growth in the presence of H(2)O(2) and HPLC (High Performance Liquid Chromatography) studies provide additional evidence for the presence of catalase. According to RAST (Rapid Annotation Using Subsystems Technology) annotation and CARD (Comprehensive Antibiotic Resistance Database analysis), there were not many components in the genome that were resistant to antibiotics. Whole genome sequence (WGS) analysis does not show the presence of bacteriocins and antibiotic resistance genes, but few hypothetical proteins were observed. 3D structure and docking studies suggest their interaction with specific ligands.202439609447
7240140.9884Effects of industrial effluents containing moderate levels of antibiotic mixtures on the abundance of antibiotic resistance genes and bacterial community composition in exposed creek sediments. Environmental discharges of very high (mg/L) antibiotic levels from pharmaceutical production contributed to the selection, spread and persistence of antibiotic resistance. However, the effects of less antibiotic-polluted effluents (μg/L) from drug-formulation on exposed aquatic microbial communities are still scarce. Here we analyzed formulation effluents and sediments from the receiving creek collected at the discharge site (DW0), upstream (UP) and 3000 m downstream of discharge (DW3000) during winter and summer season. Chemical analyses indicated the largest amounts of trimethoprim (up to 5.08 mg/kg) and azithromycin (up to 0.39 mg/kg) at DW0, but sulfonamides accumulated at DW3000 (total up to 1.17 mg/kg). Quantitative PCR revealed significantly increased relative abundance of various antibiotic resistance genes (ARGs) against β-lactams, macrolides, sulfonamides, trimethoprim and tetracyclines in sediments from DW0, despite relatively high background levels of some ARGs already at UP site. However, only sulfonamide (sul2) and macrolide ARG subtypes (mphG and msrE) were still elevated at DW3000 compared to UP. Sequencing of 16S rRNA genes revealed pronounced changes in the sediment bacterial community composition from both DW sites compared to UP site, regardless of the season. Numerous taxa with increased relative abundance at DW0 decreased to background levels at DW3000, suggesting die-off or lack of transport of effluent-originating bacteria. In contrast, various taxa that were more abundant in sediments than in effluents increased in relative abundance at DW3000 but not at DW0, possibly due to selection imposed by high sulfonamide levels. Network analysis revealed strong correlation between some clinically relevant ARGs (e.g. bla(GES), bla(OXA), ermB, tet39, sul2) and taxa with elevated abundance at DW sites, and known to harbour opportunistic pathogens, such as Acinetobacter, Arcobacter, Aeromonas and Shewanella. Our results demonstrate the necessity for improved management of pharmaceutical and rural waste disposal for mitigating the increasing problems with antibiotic resistance.202031855637
7132150.9883Impact of blending for direct potable reuse on premise plumbing microbial ecology and regrowth of opportunistic pathogens and antibiotic resistant bacteria. Little is known about how introducing recycled water intended for direct potable reuse (DPR) into distribution systems and premise plumbing will affect water quality at the point of use, particularly with respect to effects on microbial communities and regrowth. The examination of potential growth of opportunistic pathogens (OPs) and spread of antibiotic resistance genes (ARGs), each representing serious and growing public health concerns, by introducing DPR water has not previously been evaluated. In this study, the impact of blending purified DPR water with traditional drinking water sources was investigated with respect to treatment techniques, blending location, and blending ratio. Water from four U.S. utility partners was treated in bench- and pilot-scale treatment trains to simulate DPR with blending. Water was incubated in simulated premise plumbing rigs made of PVC pipe containing brass coupons to measure regrowth of total bacteria (16S rRNA genes, heterotrophic plate count), OPs (Legionella spp., Mycobacterium spp., Pseudomonas aeruginosa), ARGs (qnrA, vanA), and an indicator of horizontal gene transfer and multi-drug resistance (intI1). The microbial community composition was profiled and the resistome (i.e., all ARGs present) was characterized in select samples using next generation sequencing. While regrowth of total bacteria (16S rRNA genes) from the start of the incubation through week eight consistently occurred across tested scenarios (Wilcoxon, p ≤ 0.0001), total bacteria were not more abundant in the water or biofilm of any DPR scenario than in the corresponding conventional potable condition (p ≥ 0.0748). Regrowth of OP marker genes, qnrA, vanA, and intI1 were not significantly greater in water or biofilm for any DPR blends treated with advanced oxidation compared to corresponding potable water (p ≥ 0.1047). This study of initial bacteria colonizing pipes after introduction of blended DPR water revealed little evidence (i.e., one target in one water type) of exacerbated regrowth of total bacteria, OPs, or ARGs in premise plumbing.201930594092
8678160.9883Metagenomics-Guided Discovery of Potential Bacterial Metallothionein Genes from the Soil Microbiome That Confer Cu and/or Cd Resistance. Metallothionein (MT) genes are valuable genetic materials for developing metal bioremediation tools. Currently, a limited number of prokaryotic MTs have been experimentally identified, which necessitates the expansion of bacterial MT diversity. In this study, we conducted a metagenomics-guided analysis for the discovery of potential bacterial MT genes from the soil microbiome. More specifically, we combined resistance gene enrichment through diversity loss, metagenomic mining with a dedicated MT database, evolutionary trace analysis, DNA chemical synthesis, and functional genomic validation to identify novel MTs. Results showed that Cu stress induced a compositional change in the soil microbiome, with an enrichment of metal-resistant bacteria in soils with higher Cu concentrations. Shotgun metagenomic sequencing was performed to obtain the gene pool of environmental DNA (eDNA), which was subjected to a local BLAST search against an MT database for detecting putative MT genes. Evolutional trace analysis led to the identification of 27 potential MTs with conserved cysteine/histidine motifs different from those of known prokaryotic MTs. Following chemical synthesis of these 27 potential MT genes and heterologous expression in Escherichia coli, six of them were found to improve the hosts' growth substantially and enhanced the hosts' sorption of Cu, Cd, and Zn, among which MT5 led to a 13.7-fold increase in Cd accumulation. Furthermore, four of them restored Cu and/or Cd resistance in two metal-sensitive E. coli strains.IMPORTANCE The metagenomics-guided procedure developed here bypasses the difficulties encountered in classic PCR-based approaches and led to the discovery of novel MT genes, which may be useful in developing bioremediation tools. The procedure used here expands our knowledge on the diversity of bacterial MTs in the environment and may also be applicable to identify other functional genes from eDNA.202032111593
7720170.9883Traditionally produced tempeh harbors more diverse bacteria with more putative health-promoting properties than industrially produced tempeh. In recent years, there has been a significant shift towards industrialization in food production, resulting in the implementation of higher hygiene standards globally. Our study focused on examining the impact of hygiene standards on tempeh, a popular Rhizopus-based fermented soybean product native to Indonesia, and now famous around the world. We observed that tempeh produced with standardized hygiene measures exhibited a microbiome with comparable bacterial abundances but a markedly different community structure and function than traditionally produced tempeh. In detail, we found a decreased bacterial abundance of lactobacilli and enterobacteria, bacterial diversity, different indicator taxa, and significantly changed community structure in industrial tempeh. A similar picture was found for functional analysis: the quantity of bacterial genes was similar but qualitative changes were found for genes associated with human health. The resistome of tempeh varied based on its microbiome composition. The higher number of antimicrobial resistance genes in tempeh produced without standardized hygiene measures mainly belong to multidrug efflux pumps known to occur in plant-based food. Our findings were confirmed by functional insights into genomes and metagenome-assembled genomes from the dominant bacteria, e.g. Leuconostoc, Limosilactobacillus, Lactobacillus, Enterococcus, Paenibacillus, Azotobacter and Enterobacter. They harboured an impressive spectrum of genes important for human health, e.g. for production of vitamin B(1), B(7), B(12), and K, iron and zinc transport systems and short chain fatty acid production. In conclusion, industrially produced tempeh harbours a less diverse microbiome than the traditional one. Although this ensures production at large scales as well as biosafety, in the long-term it can lead to potential effects for human gut health.202439614549
6100180.9883Characterization of copper-resistant bacteria and bacterial communities from copper-polluted agricultural soils of central Chile. BACKGROUND: Copper mining has led to Cu pollution in agricultural soils. In this report, the effects of Cu pollution on bacterial communities of agricultural soils from Valparaiso region, central Chile, were studied. Denaturing gradient gel electrophoresis (DGGE) of the 16S rRNA genes was used for the characterization of bacterial communities from Cu-polluted and non-polluted soils. Cu-resistant bacterial strains were isolated from Cu-polluted soils and characterized. RESULTS: DGGE showed a similar high number of bands and banding pattern of the bacterial communities from Cu-polluted and non-polluted soils. The presence of copA genes encoding the multi-copper oxidase that confers Cu-resistance in bacteria was detected by PCR in metagenomic DNA from the three Cu-polluted soils, but not in the non-polluted soil. The number of Cu-tolerant heterotrophic cultivable bacteria was significantly higher in Cu-polluted soils than in the non-polluted soil. Ninety two Cu-resistant bacterial strains were isolated from three Cu-polluted agricultural soils. Five isolated strains showed high resistance to copper (MIC ranged from 3.1 to 4.7 mM) and also resistance to other heavy metals. 16S rRNA gene sequence analyses indicate that these isolates belong to the genera Sphingomonas, Stenotrophomonas and Arthrobacter. The Sphingomonas sp. strains O12, A32 and A55 and Stenotrophomonas sp. C21 possess plasmids containing the Cu-resistance copA genes. Arthrobacter sp. O4 possesses the copA gene, but plasmids were not detected in this strain. The amino acid sequences of CopA from Sphingomonas isolates (O12, A32 and A55), Stenotrophomonas strain (C21) and Arthrobacter strain (O4) are closely related to CopA from Sphingomonas, Stenotrophomonas and Arthrobacter strains, respectively. CONCLUSIONS: This study suggests that bacterial communities of agricultural soils from central Chile exposed to long-term Cu-pollution have been adapted by acquiring Cu genetic determinants. Five bacterial isolates showed high copper resistance and additional resistance to other heavy metals. Detection of copA gene in plasmids of four Cu-resistant isolates indicates that mobile genetic elements are involved in the spreading of Cu genetic determinants in polluted environments.201222950448
7953190.9882Rapid impact of phenanthrene and arsenic on bacterial community structure and activities in sand batches. The impact of both organic and inorganic pollution on the structure of soil microbial communities is poorly documented. A short-time batch experiment (6 days) was conducted to study the impact of both types of pollutants on the taxonomic, metabolic and functional diversity of soil bacteria. For this purpose sand spiked with phenanthrene (500 mg kg(-1) sand) or arsenic (arsenite 0.66 mM and arsenate 12.5 mM) was supplemented with artificial root exudates and was inoculated with bacteria originated from an aged PAH and heavy-metal-polluted soil. The bacterial community was characterised using bacterial strain isolation, TTGE fingerprinting and proteomics. Without pollutant, or with phenanthrene or arsenic, there were no significant differences in the abundance of bacteria and the communities were dominated by Pseudomonas and Paenibacillus genera. However, at the concentrations used, both phenanthrene or arsenic were toxic as shown by the decrease in mineralisation activities. Using community-level physiological profiles (Biolog Ecoplates™) or differential proteomics, we observed that the pollutants had an impact on the community physiology, in particular phenanthrene induced a general cellular stress response with changes in the central metabolism and membrane protein synthesis. Real-time PCR quantification of functional genes and transcripts revealed that arsenic induced the transcription of functional arsenic resistance and speciation genes (arsB, ACR3 and aioA), while no transcription of PAH-degradation genes (PAH-dioxygenase and catechol-dioxygenase) was detected with phenanthrene. Altogether, in our tested conditions, pollutants do not have a major effect on community abundance or taxonomic composition but rather have an impact on metabolic and functional bacterial properties.201424189653