Crucifer Lesion-Associated Xanthomonas Strains Show Multi-Resistance to Heavy Metals and Antibiotics. - Related Documents




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451301.0000Crucifer Lesion-Associated Xanthomonas Strains Show Multi-Resistance to Heavy Metals and Antibiotics. Copper resistance in phytopathogens is a major challenge to crop production globally and is known to be driven by excessive use of copper-based pesticides. However, recent studies have shown co-selection of multiple heavy metal and antibiotic resistance genes in bacteria exposed to heavy metal and xenobiotics, which may impact the epidemiology of plant, animal, and human diseases. In this study, multi-resistance to heavy metals and antibiotics were evaluated in local Xanthomonas campestris pv. campestris (Xcc) and co-isolated Xanthomonas melonis (Xmel) strains from infected crucifer plants in Trinidad. Resistance to cobalt, cadmium, zinc, copper, and arsenic (V) was observed in both Xanthomonas species up to 25 mM. Heavy metal resistance (HMR) genes were found on a small plasmid-derived locus with ~ 90% similarity to a Stenotrophomonas spp. chromosomal locus and a X. perforans pLH3.1 plasmid. The co-occurrence of mobile elements in these regions implies their organization on a composite transposon-like structure. HMR genes in Xcc strains showed the lowest similarity to references, and the cus and ars operons appear to be unique among Xanthomonads. Overall, the similarity of HMR genes to Stenotrophomonas sp. chromosomal genomes suggest their origin in this genus or a related organism and subsequent spread through lateral gene transfer events. Further resistome characterization revealed the presence of small multidrug resistance (SMR), multidrug resistance (MDR) efflux pumps, and bla (Xcc) genes for broad biocide resistance in both species. Concurrently, resistance to antibiotics (streptomycin, kanamycin, tetracycline, chloramphenicol, and ampicillin) up to 1000 µg/mL was confirmed.202438598029
451410.9996Phenotypic and resistome analysis of antibiotic and heavy metal resistance in the Antarctic bacterium Pseudomonas sp. AU10. Resistance to antibiotics and heavy metals in Antarctic bacteria has been investigated due to anthropogenic impact on the continent. However, there is still much to learn about the genetic determinants of resistance in native bacteria. In this study, we investigated antibiotic, heavy metal, and metalloid resistance in Pseudomonas sp. AU10, isolated from King George Island (Antarctica), and analyzed its genome to look for all the associated genetic determinants (resistome). We found that AU10 displayed resistance to Cr(VI), Cu(II), Mn(II), Fe(II), and As(V), and produced an exopolysaccharide with high Cr(VI)-biosorption capacity. Additionaly, the strain showed resistance to aminopenicillins, cefotaxime, aztreonam, azithromycin, and intermediate resistance to chloramphenicol. Regarding the resistome, we did not find resistance genes in AU10's natural plasmid or in a prophage context. Only a copper resistance cluster indicated possible horizontal acquisition. The mechanisms of resistance found were mostly efflux systems, several sequestering proteins, and a few enzymes, such as an AmpC β-lactamase or a chromate reductase, which would account for the observed phenotypic profile. In contrast, the presence of a few gene clusters, including the terZABCDE operon for tellurite resistance, did not correlate with the expected phenotype. Despite the observed resistance to multiple antibiotics and heavy metals, the lack of resistance genes within evident mobile genetic elements is suggestive of the preserved nature of AU10's Antarctic habitat. As Pseudomonas species are good bioindicators of human impact in Antarctic environments, we consider that our results could help refine surveillance studies based on monitoring resistances and associated resistomes in these populations.202337783937
370920.9996Potential of tellurite resistance in heterotrophic bacteria from mining environments. Untreated mining wastes and improper disposal of high-tech devices generate an environmental increase of bioavailable metalloids, exerting stress on autochthonous microbial populations. Tellurium is a metalloid, an element with raising economic importance; nevertheless, its interaction with living organisms is not yet fully understood. Here we characterized aerobic heterotrophic bacteria, isolated from high metal-content mining residues, able to resist/reduce tellurite into tellurium structures and to determine the presence of confirmed tellurite resistance genetic determinants in resistant strains. We identified over 50 tellurite-resistant strains, among 144 isolates, eight strains reduced tellurite to tellurium at different rates, with the concomitant production of tellurium deposits. Most tellurite resistance genes were found in strains from Bacillales, with the prevalence of genes of the ter operon. This work demonstrated that bacterial isolates, from environments with a persistent selective pressure, are potential candidates for uncovering strategies for tellurite resistance and/or production of valuable Te-containing materials.202235784792
450930.9996Distribution of triclosan-resistant genes in major pathogenic microorganisms revealed by metagenome and genome-wide analysis. The substantial use of triclosan (TCS) has been aimed to kill pathogenic bacteria, but TCS resistance seems to be prevalent in microbial species and limited knowledge exists about TCS resistance determinants in a majority of pathogenic bacteria. We aimed to evaluate the distribution of TCS resistance determinants in major pathogenic bacteria (N = 231) and to assess the enrichment of potentially pathogenic genera in TCS contaminated environments. A TCS-resistant gene (TRG) database was constructed and experimentally validated to predict TCS resistance in major pathogenic bacteria. Genome-wide in silico analysis was performed to define the distribution of TCS-resistant determinants in major pathogens. Microbiome analysis of TCS contaminated soil samples was also performed to investigate the abundance of TCS-resistant pathogens. We experimentally confirmed that TCS resistance could be accurately predicted using genome-wide in silico analysis against TRG database. Predicted TCS resistant phenotypes were observed in all of the tested bacterial strains (N = 17), and heterologous expression of selected TCS resistant genes from those strains conferred expected levels of TCS resistance in an alternative host Escherichia coli. Moreover, genome-wide analysis revealed that potential TCS resistance determinants were abundant among the majority of human-associated pathogens (79%) and soil-borne plant pathogenic bacteria (98%). These included a variety of enoyl-acyl carrier protein reductase (ENRs) homologues, AcrB efflux pumps, and ENR substitutions. FabI ENR, which is the only known effective target for TCS, was either co-localized with other TCS resistance determinants or had TCS resistance-associated substitutions. Furthermore, microbiome analysis revealed that pathogenic genera with intrinsic TCS-resistant determinants exist in TCS contaminated environments. We conclude that TCS may not be as effective against the majority of bacterial pathogens as previously presumed. Further, the excessive use of this biocide in natural environments may selectively enrich for not only TCS-resistant bacterial pathogens, but possibly for additional resistance to multiple antibiotics.201829420585
360140.9996R 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
450050.9996Mosaic tetracycline resistance genes encoding ribosomal protection proteins. First reported in 2003, mosaic tetracycline resistance genes are a subgroup of the genes encoding ribosomal protection proteins (RPPs). They are formed when two or more RPP-encoding genes recombine resulting in a functional chimera. To date, the majority of mosaic genes are derived from sections of three RPP genes, tet(O), tet(W) and tet(32), with others comprising tet(M) and tet(S). In this first review of mosaic genes, we report on their structure, diversity and prevalence, and suggest that these genes may be responsible for an under-reported contribution to tetracycline resistance in bacteria.201627494928
466260.9996Characterization 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
447170.9996Update on acquired tetracycline resistance genes. This mini-review summarizes the changes in the field of bacterial acquired tetracycline resistance (tet) and oxytetracycline (otr) genes identified since the last major review in 2001. Thirty-eight acquired tetracycline resistant (Tc(r)) genes are known of which nine are new and include five genes coding for energy-dependent efflux proteins, two genes coding for ribosomal protection proteins, and two genes coding for tetracycline inactivating enzymes. The number of inactivating enzymes has increased from one to three, suggesting that work needs to be done to determine the role these enzymes play in bacterial resistance to tetracycline. In the same time period, 66 new genera have been identified which carry one or more of the previously described 29 Tc(r) genes. Included in the new genera is, for the first time, an obligate intracellular pathogen suggesting that this sheltered group of bacteria is capable of DNA exchange with non-obligate intracellular bacteria. The number of genera carrying ribosomal protection genes increased dramatically with the tet(M) gene now identified in 42 genera as compared with 24 and the tet(W) gene found in 17 new genera as compared to two genera in the last major review. New conjugative transposons, carrying different ribosomal protection tet genes, have been identified and an increase in the number of antibiotic resistance genes linked to tet genes has been found. Whether these new elements may help to spread the tet genes they carry to a wider bacterial host range is discussed.200515837373
438080.9996Comparative genome analysis of ciprofloxacin-resistant Pseudomonas aeruginosa reveals genes within newly identified high variability regions associated with drug resistance development. The alarming rise of ciprofloxacin-resistant Pseudomonas aeruginosa has been reported in several clinical studies. Though the mutation of resistance genes and their role in drug resistance has been researched, the process by which the bacterium acquires high-level resistance is still not well understood. How does the genomic evolution of P. aeruginosa affect resistance development? Could the exposure of antibiotics to the bacteria enrich genomic variants that lead to the development of resistance, and if so, how are these variants distributed through the genome? To answer these questions, we performed 454 pyrosequencing and a whole genome analysis both before and after exposure to ciprofloxacin. The comparative sequence data revealed 93 unique resistance strain variation sites, which included a mutation in the DNA gyrase subunit A gene. We generated variation-distribution maps comparing the wild and resistant types, and isolated 19 candidates from three discrete resistance-associated high variability regions that had available transposon mutants, to perform a ciprofloxacin exposure assay. Of these region candidates with transposon disruptions, 79% (15/19) showed a reduction in the ability to gain high-level resistance, suggesting that genes within these high variability regions might enrich for certain functions associated with resistance development.201323808957
370190.9996Genetic Determinants for Metal Tolerance and Antimicrobial Resistance Detected in Bacteria Isolated from Soils of Olive Tree Farms. Copper-derived compounds are often used in olive tree farms. In a previous study, a collection of bacterial strains isolated from olive tree farms were identified and tested for phenotypic antimicrobial resistance and heavy metal tolerance. The aim of this work was to study the genetic determinants of resistance and to evaluate the co-occurrence of metal tolerance and antibiotic resistance genes. Both metal tolerance and antibiotic resistance genes (including beta-lactamase genes) were detected in the bacterial strains from Cu-treated soils. A high percentage of the strains positive for metal tolerance genes also carried antibiotic resistance genes, especially for genes involved in resistances to beta-lactams and tetracycline. Significant associations were detected between genes involved in copper tolerance and genes coding for beta-lactamases or tetracycline resistance mechanisms. A significant association was also detected between zntA (coding for a Zn(II)-translocating P-type ATPase) and tetC genes. In conclusion, bacteria from soils of Cu-treated olive farms may carry both metal tolerance and antibiotic resistance genes. The positive associations detected between metal tolerance genes and antibiotic resistance genes suggests co-selection of such genetic traits by exposure to metals.202032756388
3710100.9996Tolerance to various toxicants by marine bacteria highly resistant to mercury. Bacteria highly resistant to mercury isolated from seawater and sediment samples were tested for growth in the presence of different heavy metals, pesticides, phenol, formaldehyde, formic acid, and trichloroethane to investigate their potential for growth in the presence of a variety of toxic xenobiotics. We hypothesized that bacteria resistant to high concentrations of mercury would have potential capacities to tolerate or possibly degrade a variety of toxic materials and thus would be important in environmental pollution bioremediation. The mercury-resistant bacteria were found to belong to Pseudomonas, Proteus, Xanthomonas, Alteromonas, Aeromonas, and Enterobacteriaceae. All these environmental bacterial strains tolerant to mercury used in this study were capable of growth at a far higher concentration (50 ppm) of mercury than previously reported. Likewise, their ability to grow in the presence of toxic xenobiotics, either singly or in combination, was superior to that of bacteria incapable of growth in media containing 5 ppm mercury. Plasmid-curing assays done in this study ascertained that resistance to mercury antibiotics, and toxic xenobiotics is mediated by chromosomally borne genes and/or transposable elements rather than by plasmids.200312876655
3596110.9995Association of mercury resistance with antibiotic resistance in the gram-negative fecal bacteria of primates. Gram-negative fecal bacterial from three longitudinal Hg exposure experiments and from two independent survey collections were examined for their carriage of the mercury resistance (mer) locus. The occurrence of antibiotic resistance was also assessed in both mercury-resistant (Hgr) and mercury-susceptible (Hgs) isolates from the same collections. The longitudinal studies involved exposure of the intestinal flora to Hg released from amalgam "silver" dental restorations in six monkeys. Hgr strains were recovered before the installation of amalgams, and frequently these became the dominant strains while amalgams were installed. Such persistent Hgr strains always carried the same mer locus throughout the experiments. In both the longitudinal and survey collections, certain mer loci were preferentially associated with one genus, whereas other mer loci were recovered from many genera. In general, strains with any mer locus were more likely to be multiresistant than were strains without mer loci; this clustering tendency was also seen for antibiotic resistance genes. However, the association of antibiotic multiresistance with mer loci was not random; regardless of source, certain mer loci occurred in highly multiresistant strains (with as many as seven antibiotic resistances), whereas other mer loci were found in strains without any antibiotic resistance. The majority of highly multiresistant Hgr strains also carried genes characteristic of an integron, a novel genetic element which enables the formation of tandem arrays of antibiotic resistance genes. Hgr strains lacking antibiotic resistance showed no evidence of integron components.19979361435
4529120.9995Evolution of gentamicin and arsenite resistance acquisition in Ralstonia pickettii water isolates. Ralstonia pickettii are ubiquitous in water environments. Members of this species are frequently, but not always, resistant to both gentamicin and arsenite. Gentamicin and arsenite co-resistance and the putative molecular mechanisms were investigated. A group of 37 R. pickettii strains isolated from drinking water and hospital wastewater were characterized for gentamicin and arsenite resistance phenotypes, the number and size of plasmids, and screened for genetic elements associated with arsenite tolerance, Integrative and Conjugative Elements (ICEs), among other. The genomes of three representative strains were compared. Most gentamicin resistant (GR) isolates (32/33) were resistant to arsenite, and harbored ICE- and ars operon-related genes. These genetic elements were not detected in any of the five arsenite susceptible strains, regardless of the GR (n = 1) or gentamicin susceptibility (GS) (n = 4) phenotype. The comparison of the genomes of two GR (one resistant and one susceptible to arsenite) and one GS strains suggested that these phenotypes correspond to three phylogroups, distinguished by presence of some genes only in GR isolates, in addition to point mutations in functional genes. The presence of ICEs and ars operon-related genes suggest that arsenite resistance might have been acquired by GR lineages.202133197514
4658130.9995Class 1 integrons potentially predating the association with tn402-like transposition genes are present in a sediment microbial community. Integrons are genetic elements that contribute to lateral gene transfer in bacteria as a consequence of possessing a site-specific recombination system. This system facilitates the spread of genes when they are part of mobile cassettes. Most integrons are contained within chromosomes and are confined to specific bacterial lineages. However, this is not the case for class 1 integrons, which were the first to be identified and are one of the single biggest contributors to multidrug-resistant nosocomial infections, carrying resistance to many antibiotics in diverse pathogens on a global scale. The rapid spread of class 1 integrons in the last 60 years is partly a result of their association with a specific suite of transposition functions, which has facilitated their recruitment by plasmids and other transposons. The widespread use of antibiotics has acted as a positive selection pressure for bacteria, especially pathogens, which harbor class 1 integrons and their associated antibiotic resistance genes. Here, we have isolated bacteria from soil and sediment in the absence of antibiotic selection. Class 1 integrons were recovered from four different bacterial species not known to be human pathogens or commensals. All four integrons lacked the transposition genes previously considered to be a characteristic of this class. At least two of these integrons were located on a chromosome, and none of them possessed antibiotic resistance genes. We conclude that novel class 1 integrons are present in a sediment environment in various bacteria of the beta-proteobacterial class. These data suggest that the dispersal of this class may have begun before the "antibiotic era."200616885440
4664140.9995Comprehensive screening of genomic and metagenomic data reveals a large diversity of tetracycline resistance genes. Tetracyclines are broad-spectrum antibiotics used to prevent or treat a variety of bacterial infections. Resistance is often mediated through mobile resistance genes, which encode one of the three main mechanisms: active efflux, ribosomal target protection or enzymatic degradation. In the last few decades, a large number of new tetracycline-resistance genes have been discovered in clinical settings. These genes are hypothesized to originate from environmental and commensal bacteria, but the diversity of tetracycline-resistance determinants that have not yet been mobilized into pathogens is unknown. In this study, we aimed to characterize the potential tetracycline resistome by screening genomic and metagenomic data for novel resistance genes. By using probabilistic models, we predicted 1254 unique putative tetracycline resistance genes, representing 195 gene families (<70 % amino acid sequence identity), whereof 164 families had not been described previously. Out of 17 predicted genes selected for experimental verification, 7 induced a resistance phenotype in an Escherichia coli host. Several of the predicted genes were located on mobile genetic elements or in regions that indicated mobility, suggesting that they easily can be shared between bacteria. Furthermore, phylogenetic analysis indicated several events of horizontal gene transfer between bacterial phyla. Our results also suggested that acquired efflux pumps originate from proteobacterial species, while ribosomal protection genes have been mobilized from Firmicutes and Actinobacteria. This study significantly expands the knowledge of known and putatively novel tetracycline resistance genes, their mobility and evolutionary history. The study also provides insights into the unknown resistome and genes that may be encountered in clinical settings in the future.202033125315
3599150.9995Distribution of the pco Gene Cluster and Associated Genetic Determinants among Swine Escherichia coli from a Controlled Feeding Trial. Copper is used as an alternative to antibiotics for growth promotion and disease prevention. However, bacteria developed tolerance mechanisms for elevated copper concentrations, including those encoded by the pco operon in Gram-negative bacteria. Using cohorts of weaned piglets, this study showed that the supplementation of feed with copper concentrations as used in the field did not result in a significant short-term increase in the proportion of pco-positive fecal Escherichia coli. The pco and sil (silver resistance) operons were found concurrently in all screened isolates, and whole-genome sequencing showed that they were distributed among a diversity of unrelated E. coli strains. The presence of pco/sil in E. coli was not associated with elevated copper minimal inhibitory concentrations (MICs) under a variety of conditions. As found in previous studies, the pco/sil operons were part of a Tn7-like structure found both on the chromosome or on plasmids in the E. coli strains investigated. Transfer of a pco/sil IncHI2 plasmid from E. coli to Salmonella enterica resulted in elevated copper MICs in the latter. Escherichia coli may represent a reservoir of pco/sil genes transferable to other organisms such as S. enterica, for which it may represent an advantage in the presence of copper. This, in turn, has the potential for co-selection of resistance to antibiotics.201830340352
4657160.9995Discovery of the fourth mobile sulfonamide resistance gene. BACKGROUND: Over the past 75 years, human pathogens have acquired antibiotic resistance genes (ARGs), often from environmental bacteria. Integrons play a major role in the acquisition of antibiotic resistance genes. We therefore hypothesized that focused exploration of integron gene cassettes from microbial communities could be an efficient way to find novel mobile resistance genes. DNA from polluted Indian river sediments were amplified using three sets of primers targeting class 1 integrons and sequenced by long- and short-read technologies to maintain both accuracy and context. RESULTS: Up to 89% of identified open reading frames encode known resistance genes, or variations thereof (> 1000). We identified putative novel ARGs to aminoglycosides, beta-lactams, trimethoprim, rifampicin, and chloramphenicol, including several novel OXA variants, providing reduced susceptibility to carbapenems. One dihydropteroate synthase gene, with less than 34% amino acid identity to the three known mobile sulfonamide resistance genes (sul1-3), provided complete resistance when expressed in Escherichia coli. The mobilized gene, here named sul4, is the first mobile sulfonamide resistance gene discovered since 2003. Analyses of adjacent DNA suggest that sul4 has been decontextualized from a set of chromosomal genes involved in folate synthesis in its original host, likely within the phylum Chloroflexi. The presence of an insertion sequence common region element could provide mobility to the entire integron. Screening of 6489 metagenomic datasets revealed that sul4 is already widespread in seven countries across Asia and Europe. CONCLUSIONS: Our findings show that exploring integrons from environmental communities with a history of antibiotic exposure can provide an efficient way to find novel, mobile resistance genes. The mobilization of a fourth sulfonamide resistance gene is likely to provide expanded opportunities for sulfonamide resistance to spread, with potential impacts on both human and animal health.201729246178
3712170.9995Enumeration 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
3872180.9995Functional metagenomic analysis reveals rivers are a reservoir for diverse antibiotic resistance genes. The environment harbours a significant diversity of uncultured bacteria and a potential source of novel and extant resistance genes which may recombine with clinically important bacteria disseminated into environmental reservoirs. There is evidence that pollution can select for resistance due to the aggregation of adaptive genes on mobile elements. The aim of this study was to establish the impact of waste water treatment plant (WWTP) effluent disposal to a river by using culture independent methods to study diversity of resistance genes downstream of the WWTP in comparison to upstream. Metagenomic libraries were constructed in Escherichia coli and screened for phenotypic resistance to amikacin, gentamicin, neomycin, ampicillin and ciprofloxacin. Resistance genes were identified by using transposon mutagenesis. A significant increase downstream of the WWTP was observed in the number of phenotypic resistant clones recovered in metagenomic libraries. Common β-lactamases such as blaTEM were recovered as well as a diverse range of acetyltransferases and unusual transporter genes, with evidence for newly emerging resistance mechanisms. The similarities of the predicted proteins to known sequences suggested origins of genes from a very diverse range of bacteria. The study suggests that waste water disposal increases the reservoir of resistance mechanisms in the environment either by addition of resistance genes or by input of agents selective for resistant phenotypes.201424636906
4669190.9995Functional Metagenome Mining of Soil for a Novel Gentamicin Resistance Gene. Extensive use of antibiotics over recent decades has led to bacterial resistance against antibiotics, including gentamicin, one of the most effective aminoglycosides. The emergence of resistance is problematic for hospitals, since gentamicin is an important broad-spectrum antibiotic for the control of bacterial pathogens in the clinic. Previous study to identify gentamicin resistance genes from environmental samples have been conducted using culture-dependent screening methods. To overcome these limitations, we employed a metagenome-based culture-independent protocol to identify gentamicin resistance genes. Through functional screening of metagenome libraries derived from soil samples, a fosmid clone was selected as it conferred strong gentamicin resistance. To identify a specific functioning gene conferring gentamicin resistance from a selected fosmid clone (35-40 kb), a shot-gun library was constructed and four shot-gun clones (2-3 kb) were selected. Further characterization of these clones revealed that they contained sequences similar to that of the RNA ligase, T4 rnlA that is known as a toxin gene. The overexpression of the rnlA-like gene in Escherichia coli increased gentamicin resistance, indicating that this toxin gene modulates this trait. The results of our metagenome library analysis suggest that the rnlA-like gene may represent a new class of gentamicin resistance genes in pathogenic bacteria. In addition, we demonstrate that the soil metagenome can provide an important resource for the identification of antibiotic resistance genes, which are valuable molecular targets in efforts to overcome antibiotic resistance.201626699755