Resident microbiota of the gypsy moth midgut harbors antibiotic resistance determinants. - Related Documents




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451201.0000Resident microbiota of the gypsy moth midgut harbors antibiotic resistance determinants. Little is known about the significance of insects as environmental reservoirs of antibiotic-resistant bacteria. We characterized the antibiotic resistome of the microbial community in gypsy moth larval midguts by applying functional metagenomics to cultured isolates. The minimum inhibitory concentrations of 12 antibiotics were determined for 44 cultured isolates, and antibiotic resistance genes were selected from metagenomic libraries derived from DNA extracted from a pool of the isolates. Six unique clones were identified. Two were highly resistant to penicillin-type beta-lactams, two were moderately resistant to erythromycin, and two were moderately resistant to a range of antibiotics, including erythromycin, carbenicillin, and chloramphenicol. Sequence analysis predicted that the active genes encoded efflux pumps, a transcriptional activator of efflux pump protein expression, and an extended-spectrum class A beta-lactamase. Insect guts are a reservoir of antibiotic resistance genes with the potential for dissemination.200919206998
360010.9997Uncultured 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
335820.9997Novel class 1 integron harboring antibiotic resistance genes in wastewater-derived bacteria as revealed by functional metagenomics. Combatting antibiotic resistance is critical to our ability to treat infectious diseases. Here, we identified and characterized diverse antimicrobial resistance genes, including potentially mobile elements, from synthetic wastewater treatment microcosms exposed to the antibacterial agent triclosan. After seven weeks of exposure, the microcosms were subjected to functional metagenomic selection across 13 antimicrobials. This was achieved by cloning the combined genetic material from the microcosms, introducing this genetic library into E. coli, and selecting for clones that grew on media supplemented with one of the 13 antimicrobials. We recovered resistant clones capable of growth on media supplemented with a single antimicrobial, yielding 13 clones conferring resistance to at least one antimicrobial agent. Antibiotic susceptibility analysis revealed resistance ranging from 4 to >50 fold more resistant, while one clone showed resistance to multiple antibiotics. Using both Sanger and SMRT sequencing, we identified the predicted active gene(s) on each clone. One clone that conferred resistance to tetracycline contained a gene encoding a novel tetA-type efflux pump that was named TetA(62). Three clones contained predicted active genes on class 1 integrons. One integron had a previously unreported genetic arrangement and was named In1875. This study demonstrated the diversity and potential for spread of resistance genes present in human-impacted environments.202133515651
596130.9997Characterization 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
360140.9997R 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
340750.9997The culturable soil antibiotic resistome: a community of multi-drug resistant bacteria. Understanding the soil bacterial resistome is essential to understanding the evolution and development of antibiotic resistance, and its spread between species and biomes. We have identified and characterized multi-drug resistance (MDR) mechanisms in the culturable soil antibiotic resistome and linked the resistance profiles to bacterial species. We isolated 412 antibiotic resistant bacteria from agricultural, urban and pristine soils. All isolates were multi-drug resistant, of which greater than 80% were resistant to 16-23 antibiotics, comprising almost all classes of antibiotic. The mobile resistance genes investigated, (ESBL, bla NDM-1, and plasmid mediated quinolone resistance (PMQR) resistance genes) were not responsible for the respective resistance phenotypes nor were they present in the extracted soil DNA. Efflux was demonstrated to play an important role in MDR and many resistance phenotypes. Clinically relevant Burkholderia species are intrinsically resistant to ciprofloxacin but the soil Burkholderia species were not intrinsically resistant to ciprofloxacin. Using a phenotypic enzyme assay we identified the antibiotic specific inactivation of trimethoprim in 21 bacteria from different soils. The results of this study identified the importance of the efflux mechanism in the soil resistome and variations between the intrinsic resistance profiles of clinical and soil bacteria of the same family.201323776501
467160.9997Detection by metagenomic functional analysis and improvement by experimental evolution of β-lactams resistance genes present in oil contaminated soils. The spread of antibiotic resistance genes has become a global health concern identified by the World Health Organization as one of the greatest threats to health. Many of antimicrobial resistance determinants found in bacterial pathogens originate from environmental bacteria, so identifying the genes that confer resistance to antibiotics in different habitats is mandatory to better understand resistance mechanisms. Soil is one of the most diverse environments considered reservoir of antimicrobial resistance genes. The aim of this work is to study the presence of genes that provide resistance to antibiotics used in clinical settings in two oil contaminated soils by metagenomic functional analysis. Using fosmid vectors that efficiently transcribe metagenomic DNA, we have selected 12 fosmids coding for two class A β-lactamases, two subclass B1 and two subclass B3 metallo-β-lactamases, one class D β-lactamase and three efflux pumps that confer resistance to cefexime, ceftriaxone, meropenem and/or imipenem. In some of them, detection of the resistance required heterologous expression from the fosmid promoter. Although initially, these environmental genes only provide resistance to low concentrations of antibiotics, we have obtained, by experimental evolution, fosmid derivatives containing β-lactamase ORFs with a single base substitution, which substantially increase their β-lactamase activity and resistance level. None of the mutations affect β-lactamase coding sequences and are all located upstream of them. These results demonstrate the presence of enzymes that confer resistance to relevant β-lactams in these soils and their capacity to rapidly adapt to provide higher resistance levels.202235768448
451170.9997Whole genome sequences to assess the link between antibiotic and metal resistance in three coastal marine bacteria isolated from the mummichog gastrointestinal tract. Antibiotic resistance is a global public health issue and metal exposure can co-select for antibiotic resistance. We examined genome sequences of three multi-drug and metal resistant bacteria: one Shewanella sp., and two Vibrio spp., isolated from the gut of the mummichog fish (Fundulus heteroclitus). Our primary goal was to understand the mechanisms of co-selection. Phenotypically, the strains showed elevated resistance to arsenate, mercury, and various types of β-lactams. The genomes contained genes of public health concern including one carbapenemase (bla(OXA-48)). Our analyses indicate that the co-selection phenotype is mediated by chromosomal resistance genes and cross-resistance. No evidence of co-resistance was found; most resistance genes were chromosomally located. Moreover, the identification of many efflux pump gene homologs indicates that cross-resistance and/or co-regulation may further contribute to resistance. We suggest that the mummichog gut microbiota may be a source of clinically relevant antibiotic resistance genes.201830301067
370180.9997Genetic 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
449790.9997Detection and expression analysis of tet(B) in Streptococcus oralis. Tetracycline resistance can be achieved through tet genes, which code for efflux pumps, ribosomal protection proteins and inactivation enzymes. Some of these genes have only been described in either Gram-positive or Gram-negative bacteria. This is the case of tet(B), which codes for an efflux pump and, so far, had only been found in Gram-negative bacteria. In this study, tet(B) was detected in two clinical Streptococcus oralis strains isolated from the gingival sulci of two subjects. In both cases, the gene was completely sequenced, yielding 100% shared identity and coverage with other previously published sequences of tet(B). Moreover, we studied the expression of tet(B) using RT-qPCR in the isolates grown with and without tetracycline, detecting constitutive expression in only one of the isolates, with no signs of expression in the other one. This is the first time that the presence and expression of the tet(B) gene has been confirmed in Gram-positive bacteria, which highlights the potential of the genus Streptococcus to become a reservoir and a disseminator of antibiotic resistance genes in an environment so prone to horizontal gene transfer as is the oral biofilm.201931448060
3871100.9997Functional characterization of bacteria isolated from ancient arctic soil exposes diverse resistance mechanisms to modern antibiotics. Using functional metagenomics to study the resistomes of bacterial communities isolated from different layers of the Canadian high Arctic permafrost, we show that microbial communities harbored diverse resistance mechanisms at least 5,000 years ago. Among bacteria sampled from the ancient layers of a permafrost core, we isolated eight genes conferring clinical levels of resistance against aminoglycoside, β-lactam and tetracycline antibiotics that are naturally produced by microorganisms. Among these resistance genes, four also conferred resistance against amikacin, a modern semi-synthetic antibiotic that does not naturally occur in microorganisms. In bacteria sampled from the overlaying active layer, we isolated ten different genes conferring resistance to all six antibiotics tested in this study, including aminoglycoside, β-lactam and tetracycline variants that are naturally produced by microorganisms as well as semi-synthetic variants produced in the laboratory. On average, we found that resistance genes found in permafrost bacteria conferred lower levels of resistance against clinically relevant antibiotics than resistance genes sampled from the active layer. Our results demonstrate that antibiotic resistance genes were functionally diverse prior to the anthropogenic use of antibiotics, contributing to the evolution of natural reservoirs of resistance genes.201525807523
4508110.9997Tetracycline Resistance Genes Identified from Distinct Soil Environments in China by Functional Metagenomics. Soil microbiota represents one of the ancient evolutionary origins of antibiotic resistance and has been increasingly recognized as a potentially vast unstudied reservoir of resistance genes with possibilities to exchange with pathogens. Tetracycline resistance is one of the most abundant antibiotic resistances that may transfer among clinical and commensal microorganisms. To investigate tetracycline resistance genes from soil bacteria in different habitats, we performed functional analysis of three metagenomic libraries derived from soil samples collected from Yunnan, Sichuan, and Tibet, respectively, in China. We found efflux transporter genes form all the libraries, including 21 major facilitator superfamily efflux pump genes and one multidrug and toxic compound extrusion (MATE) transporter gene. Interestingly, we also identified two tetracycline destructase genes, belonging to a newly described family of tetracycline-inactivating enzymes that scarcely observed in clinical pathogens, from the Tibet library. The inactivation activity of the putative enzyme was confirmed in vitro by biochemical analysis. Our results indicated that efflux pumps distributed predominantly across habitats. Meanwhile, the mechanism of enzymatic inactivation for tetracycline resistance should not be neglected and merits further investigation.201728790997
4506120.9996Molecular profiling of multidrug-resistant river water isolates: insights into resistance mechanism and potential inhibitors. Polluted waters are an important reservoir for antibiotic resistance genes and multidrug-resistant bacteria. This report describes the microbial community, antibiotic resistance genes, and the genetic profile of extended spectrum β-lactamase strains isolated from rivers at, Pune, India. ESBL-producing bacteria isolated from diverse river water catchments running through Pune City were characterized for their antibiotic resistance. The microbial community and types of genes which confer antibiotic resistance were identified followed by the isolation of antibiotic-resistant bacteria on selective media and their genome analysis. Four representative isolates were sequenced using next generation sequencing for genomic analysis. They were identified as Pseudomonas aeruginosa, Escherichia coli, and two isolates were Enterobacter cloacae. The genes associated with the multidrug efflux pumps, such as tolC, macA, macB, adeL, and rosB, were detected in the isolates. As MacAB-TolC is an ABC type efflux pump responsible for conferring resistance in bacteria to several antibiotics, potential efflux pump inhibitors were identified by molecular docking. The homology model of their MacB protein with that from Escherichia coli K12 demonstrated structural changes in different motifs of MacB. Molecular docking of reported efflux pump inhibitors revealed the highest binding affinity of compound MC207-110 against MacB. It also details the potential efflux pump inhibitors that can serve as possible drug targets in drug development and discovery.202031236860
3394130.9996Antibiotic resistance patterns of Pseudomonas spp. isolated from faecal wastes in the environment and contaminated surface water. The Pseudomonas genus, which includes environmental and pathogenic species, is known to present antibiotic resistances, and can receive resistance genes from multi-resistant enteric bacteria released into the environment via faecal rejects. This study was aimed to investigate the resistome of Pseudomonas populations that have been in contact with these faecal bacteria. Thus, faecal discharges originating from human or cattle were sampled (from 12 points and two sampling campaigns) and 41 Pseudomonas species identified (316 isolates studied). The resistance phenotype to 25 antibiotics was determined in all isolates, and we propose a specific antibiotic resistance pattern for 14 species (from 2 to 9 resistances). None showed resistance to aminoglycosides, tetracycline, or polymyxins. Four species carried a very low number of resistances, with none to β-lactams. Interestingly, we observed the absence of the transcriptional activator soxR gene in these four species. No plasmid transfer was highlighted by conjugation assays, and a few class 1 but no class 2 integrons were detected in strains that may have received resistance genes from Enterobacteria. These results imply that the contribution of the Pseudomonas genus to the resistome of an ecosystem first depends on the structure of the Pseudomonas populations, as they may have very different resistance profiles.202031930390
3595140.9996Antibiotic Susceptibility, Resistance Gene Determinants and Corresponding Genomic Regions in Lactobacillus amylovorus Isolates Derived from Wild Boars and Domestic Pigs. Restrictions on the use of antibiotics in pigs lead to the continuous search for new probiotics serving as an alternative to antibiotics. One of the key parameters for probiotic bacteria selection is the absence of horizontally transmissible resistance genes. The aim of our study was to determine antibiotic susceptibility profiles in 28 Lactobacillus amylovorus isolates derived from the digestive tract of wild boars and farm pigs by means of the broth microdilution method and whole genome sequencing (WGS). We revealed genetic resistance determinants and examined sequences flanking resistance genes in these strains. Our findings indicate that L. amylovorus strains from domestic pigs are predominantly resistant to tetracycline, erythromycin and ampicillin. WGS analysis of horizontally transmissible genes revealed only three genetic determinants (tetW, ermB and aadE) of which all tetW and ermB genes were present only in strains derived from domestic pigs. Sequence analysis of coding sequences (CDS) in the neighborhood of the tetW gene revealed the presence of site-specific recombinase (xerC/D), site-specific DNA recombinase (spoIVCA) or DNA-binding transcriptional regulator (xre), usually directly downstream of the tetW gene. In the case of ermB, CDS for omega transcriptional repressor or mobilization protein were detected upstream of the ermB gene.202236677394
4669150.9996Functional 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
6259160.9996Evidence of an efflux pump in Serratia marcescens. Spontaneous mutants resistant to fluoroquinolones were obtained by exposing Serratia marcescens NIMA (wild-type strain) to increasing concentrations of ciprofloxacin both in liquid and on solid media. Frequencies of mutation ranged from 10(-7) to 10(-9). Active expulsion of antibiotic was explored as a possible mechanism of resistance in mutants as well as changes in topoisomerase target genes. The role of extrusion mechanisms in determining the emergence of multidrug-resistant bacteria was also examined. Mutants resistant to high concentrations of fluoroquinolones had a single mutation in their gyrA QRDR sequences, whereas the moderate resistance in the rest of mutants was due to extrusion of the drug.200010990265
3872170.9996Functional 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
3395180.9996Presence of multidrug-resistant enteric bacteria in dairy farm topsoil. In addition to human and veterinary medicine, antibiotics are extensively used in agricultural settings, such as for treatment of infections, growth enhancement, and prophylaxis in food animals, leading to selection of drug and multidrug-resistant bacteria. To help circumvent the problem of bacterial antibiotic resistance, it is first necessary to understand the scope of the problem. However, it is not fully understood how widespread antibiotic-resistant bacteria are in agricultural settings. The lack of such surveillance data is especially evident in dairy farm environments, such as soil. It is also unknown to what extent various physiological modulators, such as salicylate, a component of aspirin and known model modulator of multiple antibiotic resistance (mar) genes, influence bacterial multi-drug resistance. We isolated and identified enteric soil bacteria from local dairy farms within Roosevelt County, NM, determined the resistance profiles to antibiotics associated with mar, such as chloramphenicol, nalidixic acid, penicillin G, and tetracycline. We then purified and characterized plasmid DNA and detected mar phenotypic activity. The minimal inhibitory concentrations (MIC) of antibiotics for the isolates ranged from 6 to >50 microg/mL for chloramphenicol, 2 to 8 microg/mL for nalidixic acid, 25 to >300 microg/mL for penicillin G, and 1 to >80 microg/mL for tetracycline. On the other hand, many of the isolates had significantly enhanced MIC for the same antibiotics in the presence of 5 mM salicylate. Plasmid DNA extracted from 12 randomly chosen isolates ranged in size from 6 to 12.5 kb and, in several cases, conferred resistance to chloramphenicol and penicillin G. It is concluded that enteric bacteria from dairy farm topsoil are multidrug resistant and harbor antibiotic-resistance plasmids. A role for dairy topsoil in zoonoses is suggested, implicating this environment as a reservoir for development of bacterial resistance against clinically relevant antibiotics.200515778307
3357190.9996Detection of 140 clinically relevant antibiotic-resistance genes in the plasmid metagenome of wastewater treatment plant bacteria showing reduced susceptibility to selected antibiotics. To detect plasmid-borne antibiotic-resistance genes in wastewater treatment plant (WWTP) bacteria, 192 resistance-gene-specific PCR primer pairs were designed and synthesized. Subsequent PCR analyses on total plasmid DNA preparations obtained from bacteria of activated sludge or the WWTP's final effluents led to the identification of, respectively, 140 and 123 different resistance-gene-specific amplicons. The genes detected included aminoglycoside, beta-lactam, chloramphenicol, fluoroquinolone, macrolide, rifampicin, tetracycline, trimethoprim and sulfonamide resistance genes as well as multidrug efflux and small multidrug resistance genes. Some of these genes were only recently described from clinical isolates, demonstrating genetic exchange between clinical and WWTP bacteria. Sequencing of selected resistance-gene-specific amplicons confirmed their identity or revealed that the amplicon nucleotide sequence is very similar to a gene closely related to the reference gene used for primer design. These results demonstrate that WWTP bacteria are a reservoir for various resistance genes. Moreover, detection of about 64 % of the 192 reference resistance genes in bacteria obtained from the WWTP's final effluents indicates that these resistance determinants might be further disseminated in habitats downstream of the sewage plant.200919389756