High diversity of bacterial mercuric reductase genes from surface and sub-surface floodplain soil (Oak Ridge, USA). - Related Documents




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46601.0000High diversity of bacterial mercuric reductase genes from surface and sub-surface floodplain soil (Oak Ridge, USA). DNA was extracted from different depth soils (0-5, 45-55 and 90-100 cm below surface) sampled at Lower East Fork Poplar Creek floodplain (LEFPCF), Oak Ridge (TN, USA). The presence of merA genes, encoding the mercuric reductase, the key enzyme in detoxification of mercury in bacteria, was examined by PCR targeting Actinobacteria, Firmicutes or beta/gamma-Proteobacteria. beta/gamma-Proteobacteria merA genes were successfully amplified from all soils, whereas Actinobacteria were amplified only from surface soil. merA clone libraries were constructed and sequenced. beta/gamma-Proteobacteria sequences revealed high diversity in all soils, but limited vertical similarity. Less than 20% of the operational taxonomic units (OTU) (DNA sequences > or = 95% identical) were shared between the different soils. Only one of the 62 OTU was > or = 95% identical to a GenBank sequence, highlighting that cultivated bacteria are not representative of what is found in nature. Fewer merA sequences were obtained from the Actinobacteria, but these were also diverse, and all were different from GenBank sequences. A single clone was most closely related to merA of alpha-Proteobacteria. An alignment of putative merA genes of genome sequenced mainly marine alpha-Proteobacteria was used for design of merA primers. PCR amplification of soil alpha-Proteobacteria isolates and sequencing revealed that they were very different from the genome-sequenced bacteria (only 62%-66% identical at the amino-acid level), although internally similar. In light of the high functional diversity of mercury resistance genes and the limited vertical distribution of shared OTU, we discuss the role of horizontal gene transfer as a mechanism of bacterial adaptation to mercury.200718043664
335910.9996Marine bacteria harbor the sulfonamide resistance gene sul4 without mobile genetic elements. Marine bacteria are possible reservoirs of antibiotic-resistance genes (ARGs) originating not only from clinical and terrestrial hot spots but also from the marine environment. We report here for the first time a higher rate of the sulfonamide-resistance gene sul4 in marine bacterial isolates compared with other sul genes. Among four sulfonamide-resistance genes (sul1, sul2, sul3, and sul4), sul4 was most abundant (45%) in 74 sulfonamide-resistant marine isolates by PCR screening. The order of abundance was sul4 (33 isolates) >sul2 (6 isolates) >sul3 (5 isolates) >sul1 (1 isolate). Whole-genome sequencing of 23 isolates of sul4-expressing α- and γ-proteobacteria and bacilli revealed that sul4 was not accompanied by known mobile genetic elements. This suggests that sul4 in these marine isolates is clonally transferred and not horizontally transferable. Folate metabolism genes formed a cluster with sul4, suggesting that the cluster area plays a role in folate metabolism, at which sul4 functions as a dihydropteroate synthase. Thus, sul4 might be expressed in marine species and function in folate synthesis, but it is not a transferable ARG.202337779713
609920.9996Culture-dependent and independent studies of microbial diversity in highly copper-contaminated Chilean marine sediments. Cultivation and molecular-based approaches were used to study microbial diversity in two Chilean marine sediments contaminated with high (835 ppm) and very high concentrations of copper (1,533 ppm). The diversity of cultivable bacteria resistant to copper was studied at oxic and anoxic conditions, focusing on sulfate-, thiosulfate-, and iron-reducing bacteria. For both sediments, the cultivable bacteria isolated at oxic conditions were mostly affiliated to the genus Bacillus, while at anoxic conditions the majority of the cultivable bacteria found were closely related to members of the genera Desulfovibrio, Sphingomonas, and Virgibacillus. Copper resistance was between 100 and 400 ppm, with the exception of a strain affiliated to members of the genus Desulfuromonas, which was resistant up to 1,000 ppm of copper. In parallel, cloning and sequencing of 16S rRNA was performed to study the total bacterial diversity in the sediments. A weak correlation was observed between the isolated strains and the 16S rRNA operational taxonomic units detected. The presence of copper resistance genes (copA, cusA, and pcoA) was tested for all the strains isolated; only copA was detected in a few isolates, suggesting that other copper resistance mechanisms could be used by the bacteria in those highly copper-contaminated sediments.201322976340
48230.9995Natural hot spots for gain of multiple resistances: arsenic and antibiotic resistances in heterotrophic, aerobic bacteria from marine hydrothermal vent fields. Microorganisms are responsible for multiple antibiotic resistances that have been associated with resistance/tolerance to heavy metals, with consequences to public health. Many genes conferring these resistances are located on mobile genetic elements, easily exchanged among phylogenetically distant bacteria. The objective of the present work was to isolate arsenic-, antimonite-, and antibiotic-resistant strains and to determine the existence of plasmids harboring antibiotic/arsenic/antimonite resistance traits in phenotypically resistant strains, in a nonanthropogenically impacted environment. The hydrothermal Lucky Strike field in the Azores archipelago (North Atlantic, between 11°N and 38°N), at the Mid-Atlantic Ridge, protected under the OSPAR Convention, was sampled as a metal-rich pristine environment. A total of 35 strains from 8 different species were isolated in the presence of arsenate, arsenite, and antimonite. ACR3 and arsB genes were amplified from the sediment's total DNA, and 4 isolates also carried ACR3 genes. Phenotypic multiple resistances were found in all strains, and 7 strains had recoverable plasmids. Purified plasmids were sequenced by Illumina and assembled by EDENA V3, and contig annotation was performed using the "Rapid Annotation using the Subsystems Technology" server. Determinants of resistance to copper, zinc, cadmium, cobalt, and chromium as well as to the antibiotics β-lactams and fluoroquinolones were found in the 3 sequenced plasmids. Genes coding for heavy metal resistance and antibiotic resistance in the same mobile element were found, suggesting the possibility of horizontal gene transfer and distribution of theses resistances in the bacterial population.201525636836
356940.9995Identification of a new ribosomal protection type of tetracycline resistance gene, tet(36), from swine manure pits. Previously, only one ribosome protection type of a tetracycline resistance gene, tetQ, had been identified in Bacteroides spp. During an investigation of anaerobic bacteria present in swine feces and manure storage pits, a tetracycline-resistant Bacteroides strain was isolated. Subsequent analysis showed that this new Bacteroides strain, Bacteroides sp. strain 139, did not contain tetQ but contained a previously unidentified tetracycline resistance gene. Sequence analysis showed that the tetracycline resistance gene from Bacteroides sp. strain 139 encoded a protein (designated Tet 36) that defines a new class of ribosome protection types of tetracycline resistance. Tet 36 has 60% amino acid identity over 640 aa to TetQ and between 31 and 49% amino acid identity to the nine other ribosome protection types of tetracycline resistance genes. The tet(36) region was not observed to transfer from Bacteroides sp. strain 139 to another Bacteroides sp. under laboratory conditions. Yet tet(36) was found in other genera of bacteria isolated from the same swine manure pits and from swine feces. Phylogenetic analysis of the tet(36)-containing isolates indicated that tet(36) was present not only in the Cytophaga-Flavobacter-Bacteroides group to which Bacteroides sp. strain 139 belongs but also in gram-positive genera and gram-negative proteobacteria, indicating that horizontal transfer of tet(36) is occurring between these divergent phylogenetic groups in the farm environment.200312839793
596150.9995Characterization of novel antibiotic resistance genes identified by functional metagenomics on soil samples. The soil microbial community is highly complex and contains a high density of antibiotic-producing bacteria, making it a likely source of diverse antibiotic resistance determinants. We used functional metagenomics to search for antibiotic resistance genes in libraries generated from three different soil samples, containing 3.6 Gb of DNA in total. We identified 11 new antibiotic resistance genes: 3 conferring resistance to ampicillin, 2 to gentamicin, 2 to chloramphenicol and 4 to trimethoprim. One of the clones identified was a new trimethoprim resistance gene encoding a 26.8 kDa protein closely resembling unassigned reductases of the dihydrofolate reductase group. This protein, Tm8-3, conferred trimethoprim resistance in Escherichia coli and Sinorhizobium meliloti (γ- and α-proteobacteria respectively). We demonstrated that this gene encoded an enzyme with dihydrofolate reductase activity, with kinetic constants similar to other type I and II dihydrofolate reductases (K(m) of 8.9 µM for NADPH and 3.7 µM for dihydrofolate and IC(50) of 20 µM for trimethoprim). This is the first description of a new type of reductase conferring resistance to trimethoprim. Our results indicate that soil bacteria display a high level of genetic diversity and are a reservoir of antibiotic resistance genes, supporting the use of this approach for the discovery of novel enzymes with unexpected activities unpredictable from their amino acid sequences.201121281423
359360.9995Genes homologous to glycopeptide resistance vanA are widespread in soil microbial communities. The occurrence of d-Ala : d-Lac ligase genes homologous to glycopeptide resistance vanA was studied in samples of agricultural (n=9) and garden (n=3) soil by culture-independent methods. Cloning and sequencing of nested degenerate PCR products obtained from soil DNA revealed the occurrence of d-Ala : d-Ala ligase genes unrelated to vanA. In order to enhance detection of vanA-homologous genes, a third PCR step was added using primers targeting vanA in soil Paenibacillus. Sequencing of 25 clones obtained by this method allowed recovery of 23 novel sequences having 86-100% identity with vanA in enterococci. Such sequences were recovered from all agricultural samples as well as from two garden samples with no history of organic fertilization. The results indicated that soil is a rich and assorted reservoir of genes closely related to those conferring glycopeptide resistance in clinical bacteria.200616734783
615670.9995Diversity of arsenite transporter genes from arsenic-resistant soil bacteria. A PCR approach was developed to assess the occurrence and diversity of arsenite transporters in arsenic-resistant bacteria. For this purpose, three sets of degenerate primers were designed for the specific amplification of approximately 750bp fragments from arsB and two subsets of ACR3 (designated ACR3(1) and ACR3(2)) arsenite carrier gene families. These primers were used to screen a collection of 41 arsenic-resistant strains isolated from two soil samples with contrasting amounts of arsenic. PCR results showed that 70.7% of the isolates contained a gene related to arsB or ACR3, with three of them carrying both arsB and ACR3-like genes. Phylogenetic analysis of the protein sequences deduced from the amplicons indicated a prevalence of arsB in Firmicutes and Gammaproteobacteria, while ACR3(1) and ACR3(2) were mostly present in Actinobacteria and Alphaproteobacteria, respectively. In addition to validating the use of degenerate primers for the identification of arsenite transporter genes in a taxonomically wide range of bacteria, the study describes a novel collection of strains displaying interesting features of resistance to arsenate, arsenite and antimonite, and the ability to oxidize arsenite.200717258434
336080.9995Gentamicin resistance genes in environmental bacteria: prevalence and transfer. A comprehensive multiphasic survey of the prevalence and transfer of gentamicin resistance (Gm(r)) genes in different non-clinical environments has been performed. We were interested to find out whether Gm(r) genes described from clinical isolates can be detected in different environmental habitats and whether hot spots can be identified. Furthermore, this study aimed to evaluate the impact of selective pressure on the abundance and mobility of resistance genes. The study included samples from soils, rhizospheres, piggery manure, faeces from cattle, laying and broiler chickens, municipal and hospital sewage water, and coastal water. Six clusters of genes coding for Gm-modifying enzymes (aac(3)-I, aac(3)-II/VI, aac(3)-III/IV, aac(6')-II/Ib, ant(2'')-I, aph(2'')-I) were identified based on a database comparison and primer systems for each gene cluster were developed. Gm-resistant bacteria isolated from the different environments had a different taxonomic composition. In only 34 of 207 isolates, mainly originating from sewage, faeces and coastal water polluted with wastewater, were known Gm(r) genes corresponding to five of the six clusters detected. The strains belonged to genera in which the genes had previously been detected (Enterobacteriaceae, Pseudomonas, Acinetobacter) but also to phylogenetically distant bacteria, such as members of the CFB group, alpha- and beta-Proteobacteria. Gm(r) genes located on mobile genetic elements (MGE) could be captured in exogenous isolations into recipients belonging to alpha-, beta- and gamma-Proteobacteria from all environments except for soil. A high proportion of the MGE, conferring Gm resistance isolated from sewage, were identified as IncPbeta plasmids. Molecular detection of Gm(r) genes, and broad host range plasmid-specific sequences (IncP-1, IncN, IncW and IncQ) in environmental DNA indicated a habitat-specific dissemination. A high abundance and diversity of Gm(r) genes could be shown for samples from faeces (broilers, layers, cattle), from sewage, from seawater, collected close to a wastewater outflow, and from piggery manure. In the latter samples all six clusters of Gm(r) genes could be detected. The different kinds of selective pressure studied here seemed to enhance the abundance of MGE, while an effect on Gm(r) genes was not obvious.200219709289
336390.9995Spreading antibiotic resistance through spread manure: characteristics of a novel plasmid type with low %G+C content. Bioactive amounts of antibiotics as well as resistant bacteria reach the soil through manure fertilization. We investigated plasmids that may stimulate the environmental spread and interspecies transfer of antibiotic resistance. After treatment of two soils with manure, either with or without the sulfonamide antibiotic sulfadiazine, a significant increase in copies of the sulfonamide resistance gene sul2 was detected by qPCR. All sul2 carrying plasmids, captured in Escherichia coli from soil, belonged to a novel class of self-transferable replicons. Manuring and sulfadiazine significantly increased the abundance of this replicon type in a chemically fertilized but not in an annually manured soil, as determined by qPCR targeting a transfer gene. Restriction patterns and antibiograms showed a considerable diversity within this novel plasmid group. Analysis of three complete plasmid sequences revealed a conserved 30 kbp backbone with only 36% G+C content, comprised of transfer and maintenance genes with moderate homology to plasmid pIPO2 and a replication module (rep and oriV) of other descent. The plasmids differed in composition of the 27.0-28.3 kbp accessory region, each of which carried ISCR2 and several resistance genes. Acinetobacter spp. was identified as a potential host of such LowGC-type plasmids in manure and soil.200919055690
3122100.9995Hybrid sequence-based analysis reveals the distribution of bacterial species and genes in the oral microbiome at a high resolution. Bacteria in the oral microbiome are poorly identified owing to the lack of established culture methods for them. Thus, this study aimed to use culture-free analysis techniques, including bacterial single-cell genome sequencing, to identify bacterial species and investigate gene distribution in saliva. Saliva samples from the same individual were classified as inactivated or viable and then analyzed using 16S rRNA sequencing, metagenomic shotgun sequencing, and bacterial single-cell sequencing. The results of 16S rRNA sequencing revealed similar microbiota structures in both samples, with Streptococcus being the predominant genus. Metagenomic shotgun sequencing showed that approximately 80 % of the DNA in the samples was of non-bacterial origin, whereas single-cell sequencing showed an average contamination rate of 10.4 % per genome. Single-cell sequencing also yielded genome sequences for 43 out of 48 wells for the inactivated samples and 45 out of 48 wells for the viable samples. With respect to resistance genes, four out of 88 isolates carried cfxA, which encodes a β-lactamase, and four isolates carried erythromycin resistance genes. Tetracycline resistance genes were found in nine bacteria. Metagenomic shotgun sequencing provided complete sequences of cfxA, ermF, and ermX, whereas other resistance genes, such as tetQ and tetM, were detected as fragments. In addition, virulence factors from Streptococcus pneumoniae were the most common, with 13 genes detected. Our average nucleotide identity analysis also suggested five single-cell-isolated bacteria as potential novel species. These data would contribute to expanding the oral microbiome data resource.202438708423
3704110.9995Antibiotic resistance in bacteria isolated from the deep terrestrial subsurface. Various natural environments have been examined for the presence of antibiotic-resistant bacteria and/or novel resistance mechanisms, but little is known about resistance in the terrestrial deep subsurface. This study examined two deep environments that differ in their known period of isolation from surface environments and the bacteria therein. One hundred fifty-four strains of bacteria were isolated from sediments located 170-259 m below land surface at the US Department of Energy Savannah River Site (SRS) in South Carolina and Hanford Site (HS) in Washington. Analyses of 16S rRNA gene sequences showed that both sets of strains were phylogenetically diverse and could be assigned to several genera in three to four phyla. All of the strains were screened for resistance to 13 antibiotics by plating on selective media and 90% were resistant to at least one antibiotic. Eighty-six percent of the SRS and 62% of the HS strains were resistant to more than one antibiotic. Resistance to nalidixic acid, mupirocin, or ampicillin was noted most frequently. The results indicate that antibiotic resistance is common among subsurface bacteria. The somewhat higher frequencies of resistance and multiple resistance at the SRS may, in part, be due to recent surface influence, such as exposure to antibiotics used in agriculture. However, the HS strains have never been exposed to anthropogenic antibiotics but still had a reasonably high frequency of resistance. Given their long period of isolation from surface influences, it is possible that they possess some novel antibiotic resistance genes and/or resistance mechanisms.200918677528
3600120.9994Uncultured soil bacteria are a reservoir of new antibiotic resistance genes. Antibiotic resistance genes are typically isolated by cloning from cultured bacteria or by polymerase chain reaction (PCR) amplification from environmental samples. These methods do not access the potential reservoir of undiscovered antibiotic resistance genes harboured by soil bacteria because most soil bacteria are not cultured readily, and PCR detection of antibiotic resistance genes depends on primers that are based on known genes. To explore this reservoir, we isolated DNA directly from soil samples, cloned the DNA and selected for clones that expressed antibiotic resistance in Escherichia coli. We constructed four libraries that collectively contain 4.1 gigabases of cloned soil DNA. From these and two previously reported libraries, we identified nine clones expressing resistance to aminoglycoside antibiotics and one expressing tetracycline resistance. Based on the predicted amino acid sequences of the resistance genes, the resistance mechanisms include efflux of tetracycline and inactivation of aminoglycoside antibiotics by phosphorylation and acetylation. With one exception, all the sequences are considerably different from previously reported sequences. The results indicate that soil bacteria are a reservoir of antibiotic resistance genes with greater genetic diversity than previously accounted for, and that the diversity can be surveyed by a culture-independent method.200415305923
4662130.9994Characterization of a multiresistant mosaic plasmid from a fish farm Sediment Exiguobacterium sp. isolate reveals aggregation of functional clinic-associated antibiotic resistance genes. The genus Exiguobacterium can adapt readily to, and survive in, diverse environments. Our study demonstrated that Exiguobacterium sp. strain S3-2, isolated from marine sediment, is resistant to five antibiotics. The plasmid pMC1 in this strain carries seven putative resistance genes. We functionally characterized these resistance genes in Escherichia coli, and genes encoding dihydrofolate reductase and macrolide phosphotransferase were considered novel resistance genes based on their low similarities to known resistance genes. The plasmid G+C content distribution was highly heterogeneous. Only the G+C content of one block, which shared significant similarity with a plasmid from Exiguobacterium arabatum, fit well with the mean G+C content of the host. The remainder of the plasmid was composed of mobile elements with a markedly lower G+C ratio than the host. Interestingly, five mobile elements located on pMC1 showed significant similarities to sequences found in pathogens. Our data provided an example of the link between resistance genes in strains from the environment and the clinic and revealed the aggregation of antibiotic resistance genes in bacteria isolated from fish farms.201424362420
3123140.9994The Raw Milk Microbiota from Semi-Subsistence Farms Characteristics by NGS Analysis Method. The aim of this study was to analyze the microbiome of raw milk obtained from three semi-subsistence farms (A, B, and C) located in the Kuyavian-Pomeranian Voivodeship in Poland. The composition of drinking milk was assessed on the basis of 16S rRNA gene sequencing using the Ion Torrent platform. Based on the conducted research, significant changes in the composition of the milk microbiome were found depending on its place of origin. Bacteria belonging to the Bacillus (17.0%), Corynebacterium (12.0%) and Escherichia-Shigella (11.0%) genera were dominant in the milk collected from farm A. In the case of the milk from farm B, the dominant bacteria belonged to the Acinetobacter genus (21.0%), whereas in the sample from farm C, Escherichia-Shigella (24.8%) and Bacillus (10.3%) dominated the microbiome. An analysis was performed using the PICRUSt tool (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) in order to generate a profile of genes responsible for bacterial metabolism. The conducted analysis confirmed the diversity of the profile of genes responsible for bacterial metabolism in all the tested samples. On the other hand, simultaneous analysis of six KEGG Orthologs (KO), which participated in beta-lactam resistance responsible for antibiotic resistance of bacteria, demonstrated that there is no significant relationship between the predicted occurrence of these orthologs and the place of existence of microorganisms. Therefore, it can be supposed that bacterial resistance to beta-lactam antibiotics occurs regardless of the environmental niche, and that the antibiotic resistance maintained in the population is a factor that shapes the functional structure of the microbial consortia.202134443615
3601150.9994R factors mediate resistance to mercury, nickel, and cobalt. Fifty-five clinical isolates and laboratory stocks of Escherichia coli and Salmonella were studied for resistance to each of ten metals. Eleven clinical isolates carrying R factors were resistant to mercury, and, in each case, the resistance was mediated by a previously undefined R-factor gene. The gene was phenotypically expressed within 2 to 4 minutes after entry into sensitive bacteria, but the basis for the resistance remains undefined. Fourteen strains, 12 infected with R factors, were resistant to cobalt and nickel, but these resistances were mediated by R-factor genes in only two strains; separate R-factor genes mediated the resistances to nickel and cobalt. These and other results indicate that the genetic composition of R factors is greater than that originally defined.19675337360
3614160.9994Structure and diversity of arsenic resistant bacteria in an old tin mine area of Thailand. The microbial community structure in Thailand soils contaminated with low and high levels of arsenic was determined by denaturing gradient gel electrophoresis (DGGE). Band pattern analysis indicated that the bacterial community was not significantly different in the two soils. Phylogenetic analysis obtained by excising and sequencing six bands indicated that the soils were dominated by Arthobacter koreensis and proteobacteria. Two hundred and sixty-two bacterial isolates were obtained from arsenic contaminated soils. The majority of the As resistant isolates were gram-negative bacteria. MIC studies indicated that all of the tested bacteria had greater resistance to arsenate than arsenite. Some strains were capable of growing in medium containing up to 1,500 mg/l arsenite and arsenate. Correlations analysis of resistance patterns of arsenite resistance indicated that the isolated bacteria could be categorized into 13 groups, with a maximum similarity value of 100%. All strains were also evaluated for resistance to eight antibiotics. The antibiotic resistance patterns divided the strains into 100 unique groups, indicating that the strains were very diverse. Isolates from each antibiotic resistance group were characterized in more detail by using the repetitive extragenic palindromic-PCR (rep-PCR) DNA fingerprinting technique with ERIC primers. PCR products were analyzed by agarose gel electrophoresis. The genetic relatedness of 100 bacterial fingerprints, determined by using Pearson product moment similarity coefficient, showed that the isolates could be divided into four clusters, with similarity values ranging from 5-99%. While many isolates were genetically diverse, others were clonal in nature Additionally, the arsenic-resistant isolates were examined for the presence of arsenic resistance (ars) genes by using PCR, and 30% of the isolates were found to carry an arsenate reductase encoded by the arsC gene.201020134249
486170.9994Detection of heavy metal ion resistance genes in gram-positive and gram-negative bacteria isolated from a lead-contaminated site. Resistance to a range of heavy metal ions was determined for lead-resistant and other bacteria which had been isolated from a battery-manufacturing site contaminated with high concentration of lead. Several Gram-positive (belonging to the genera Arthrobacter and Corynebacterium) and Gram-negative (Alcaligenes species) isolates were resistant to lead, mercury, cadmium, cobalt, zinc and copper, although the levels of resistance to the different metal ions were specific for each isolate. Polymerase chain reaction, DNA-DNA hybridization and DNA sequencing were used to explore the nature of genetic systems responsible for the metal resistance in eight of the isolates. Specific DNA sequences could be amplified from the genomic DNA of all the isolates using primers for sections of the mer (mercury resistance determinant on the transposon Tn501) and pco (copper resistance determinant on the plasmid pRJ1004) genetic systems. Positive hybridizations with mer and pco probes indicated that the amplified segments were highly homologous to these genes. Some of the PCR products were cloned and partially sequenced, and the regions sequenced were highly homologous to the appropriate regions of the mer and pco determinants. These results demonstrate the wide distribution of mercury and copper resistance genes in both Gram-positive and Gram-negative isolates obtained from this lead-contaminated soil. In contrast, the czc (cobalt, zinc and cadmium resistance) and chr (chromate resistance) genes could not be amplified from DNAs of some isolates, indicating the limited contribution, if any, of these genetic systems to the metal ion resistance of these isolates.19979342884
3712180.9994Enumeration and characterization of culturable arsenate resistant bacteria in a large estuary. Arsenic is a toxic element that exists in two major inorganic forms, arsenate and arsenite. A number of bacteria have been shown to resist arsenic exposure, and even more bacteria appear to possess the genes for arsenic resistance. In this study, the numbers of culturable arsenate-resistant bacteria present in water at three coastal sites in the Lake Pontchartrain estuary, Louisiana, was determined. Despite insignificant (less than 1.33 microM) levels of arsenic in this system, 20-50% of the viable count of bacteria showed appreciable arsenate resistance, suggesting that arsenic-resistant bacteria are common and widespread. A diverse array of arsenate-resistant isolates was obtained, with 16S rRNA sequence analysis indicating 37 different bacterial strains, representing six major bacterial groups. Many of these isolates were affiliated with groups of bacteria that have been poorly characterized in terms of arsenic resistance, such as the Betaproteobacteria or Flavobacteria. Some isolates were capable of tolerating very high (> 100 mM) levels of arsenate, although arsenite resistance was generally much lower. The results suggest that arsenic-resistant bacteria are common, even in environments with insignificant arsenic contamination, and that many different groups of aquatic bacteria show appreciable arsenic resistance.200516261862
5962190.9994Functional screening of antibiotic resistance genes from human gut microbiota reveals a novel gene fusion. The human gut microbiota has a high density of bacteria that are considered a reservoir for antibiotic resistance genes (ARGs). In this study, one fosmid metagenomic library generated from the gut microbiota of four healthy humans was used to screen for ARGs against seven antibiotics. Eight new ARGs were obtained: one against amoxicillin, six against d-cycloserine, and one against kanamycin. The new amoxicillin resistance gene encodes a protein with 53% identity to a class D β-lactamase from Riemerella anatipestifer RA-GD. The six new d-cycloserine resistance genes encode proteins with 73-81% identity to known d-alanine-d-alanine ligases. The new kanamycin resistance gene encodes a protein of 274 amino acids with an N-terminus (amino acids 1-189) that has 42% identity to the 6'-aminoglycoside acetyltransferase [AAC(6')] from Enterococcus hirae and a C-terminus (amino acids 190-274) with 35% identity to a hypothetical protein from Clostridiales sp. SSC/2. A functional study on the novel kanamycin resistance gene showed that only the N-terminus conferred kanamycin resistance. Our results showed that functional metagenomics is a useful tool for the identification of new ARGs.201222845886