Genome-wide identification of fitness-genes in aminoglycoside-resistant Escherichia coli during antibiotic stress. - Related Documents




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595801.0000Genome-wide identification of fitness-genes in aminoglycoside-resistant Escherichia coli during antibiotic stress. Resistance against aminoglycosides is widespread in bacteria. This study aimed to identify genes that are important for growth of E. coli during aminoglycoside exposure, since such genes may be targeted to re-sensitize resistant E. coli to treatment. We constructed three transposon mutant libraries each containing > 230.000 mutants in E. coli MG1655 strains harboring streptomycin (aph(3″)-Ib/aph(6)-Id), gentamicin (aac(3)-IV), or neomycin (aph(3″)-Ia) resistance gene(s). Transposon Directed Insertion-site Sequencing (TraDIS), a combination of transposon mutagenesis and high-throughput sequencing, identified 56 genes which were deemed important for growth during streptomycin, 39 during gentamicin and 32 during neomycin exposure. Most of these fitness-genes were membrane-located (n = 55) and involved in either cell division, ATP-synthesis or stress response in the streptomycin and gentamicin exposed libraries, and enterobacterial common antigen biosynthesis or magnesium sensing/transport in the neomycin exposed library. For validation, eight selected fitness-genes/gene-clusters were deleted (minCDE, hflCK, clsA and cpxR associated with streptomycin and gentamicin resistance, and phoPQ, wecA, lpp and pal associated with neomycin resistance), and all mutants were shown to be growth attenuated upon exposure to the corresponding antibiotics. In summary, we identified genes that are advantageous in aminoglycoside-resistant E. coli during antibiotic stress. In addition, we increased the understanding of how aminoglycoside-resistant E. coli respond to antibiotic exposure.202438378700
380510.9997De Novo Characterization of Genes That Contribute to High-Level Ciprofloxacin Resistance in Escherichia coli. Sensitization of resistant bacteria to existing antibiotics depends on the identification of candidate targets whose activities contribute to resistance. Using a transposon insertion library in an Escherichia coli mutant that was 2,000 times less susceptible to ciprofloxacin than its parent and the relative fitness scores, we identified 19 genes that contributed to the acquired ciprofloxacin resistance and mapped the shortest genetic path that increased the antibiotic susceptibility of the resistant bacteria back to a near wild-type level.201627431218
596120.9997Characterization 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
626230.9997Potential of Tetracycline Resistance Proteins To Evolve Tigecycline Resistance. Tigecycline is a glycylcycline antibiotic active against multidrug-resistant bacterial pathogens. The objectives of our study were to examine the potential of the Tet(A), Tet(K), Tet(M), and Tet(X) tetracycline resistance proteins to acquire mutations causing tigecycline resistance and to determine how this affects resistance to earlier classes of tetracyclines. Mutations in all four tet genes caused a significant increase in the tigecycline MIC in Escherichia coli, and strains expressing mutant Tet(A) and Tet(X) variants reached clinically relevant MICs (2 mg/liter and 3 mg/liter, respectively). Mutations predominantly accumulated in transmembrane domains of the efflux pumps, most likely increasing the accommodation of tigecycline as a substrate. All selected Tet(M) mutants contained at least one mutation in the functionally most important loop III of domain IV. Deletion of leucine 505 of this loop led to the highest increase of the tigecycline MIC (0.5 mg/liter) among Tet(M) mutants. It also caused collateral sensitivity to earlier classes of tetracyclines. A majority of the Tet(X) mutants showed increased activity against all three classes of tetracylines. All tested Tet proteins have the potential to acquire mutations leading to increased MICs of tigecycline. As tet genes are widely found in pathogenic bacteria and spread easily by horizontal gene transfer, resistance development by alteration of existing Tet proteins might compromise the future medical use of tigecycline. We predict that Tet(X) might become the most problematic future Tet determinant, since its weak intrinsic tigecycline activity can be mutationally improved to reach clinically relevant levels without collateral loss in activity to other tetracyclines.201626596936
449840.9997A naturally occurring gene amplification leading to sulfonamide and trimethoprim resistance in Streptococcus agalactiae. Gene amplifications have been detected as a transitory phenomenon in bacterial cultures. They are predicted to contribute to rapid adaptation by simultaneously increasing the expression of genes clustered on the chromosome. However, genome amplifications have rarely been described in natural isolates. Through DNA array analysis, we have identified two Streptococcus agalactiae strains carrying tandem genome amplifications: a fourfold amplification of 13.5 kb and a duplication of 92 kb. Both amplifications were located close to the terminus of replication and originated independently from any long repeated sequence. They probably arose in the human host and showed different stabilities, the 13.5-kb amplification being lost at a frequency of 0.003 per generation and the 92-kb tandem duplication at a frequency of 0.035 per generation. The 13.5-kb tandem amplification carried the five genes required for dihydrofolate biosynthesis and led to both trimethoprim (TMP) and sulfonamide (SU) resistance. Resistance to SU probably resulted from the increased synthesis of dihydropteroate synthase, the target of this antibiotic, whereas the amplification of the whole pathway was responsible for TMP resistance. This revealed a new mechanism of resistance to TMP involving an increased dihydrofolate biosynthesis. This is, to our knowledge, the first reported case of naturally occurring antibiotic resistance resulting from genome amplification in bacteria. The low stability of DNA segment amplifications suggests that their role in antibiotic resistance might have been underestimated.200818024520
360150.9997R 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
625960.9997Evidence 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
449670.9997Phenotypic and genetic barriers to establishment of horizontally transferred genes encoding ribosomal protection proteins. BACKGROUND: Ribosomal protection proteins (RPPs) interact with bacterial ribosomes to prevent inhibition of protein synthesis by tetracycline. RPP genes have evolved from a common ancestor into at least 12 distinct classes and spread by horizontal genetic transfer into a wide range of bacteria. Many bacterial genera host RPP genes from multiple classes but tet(M) is the predominant RPP gene found in Escherichia coli. OBJECTIVES: We asked whether phenotypic barriers (low-level resistance, high fitness cost) might constrain the fixation of other RPP genes in E. coli. METHODS: We expressed a diverse set of six different RPP genes in E. coli, including tet(M), and quantified tetracycline susceptibility and growth phenotypes as a function of expression level, and evolvability to overcome identified phenotypic barriers. RESULTS: The genes tet(M) and tet(Q) conferred high-level tetracycline resistance without reducing fitness; tet(O) and tet(W) conferred high-level resistance but significantly reduced growth fitness; tetB(P) conferred low-level resistance and while mutants conferring high-level resistance were selectable these had reduced growth fitness; otr(A) did not confer resistance and resistant mutants could not be selected. Evolution experiments suggested that codon usage patterns in tet(O) and tet(W), and transcriptional silencing associated with nucleotide composition in tetB(P), accounted for the observed phenotypic barriers. CONCLUSIONS: With the exception of tet(Q), the data reveal significant phenotypic and genetic barriers to the fixation of additional RPP genes in E. coli.202133655294
470280.9996Increased antimicrobial resistance of acid-adapted pathogenic Escherichia coli, and transcriptomic analysis of polymyxin-resistant strain. This study investigated the acid adaptation and antimicrobial resistance of seven pathogenic Escherichia coli strains and one commensal strain under nutrient-rich acidic conditions. After acid adaptation, three pathogenic E. coli survived during 100 h incubation in tryptic soy broth at pH 3.25. Acid-adapted (AA) strains showed increased resistance to antimicrobials including ampicillin, ciprofloxacin and especially polymyxins (colistin and polymyxin B), the last resort antimicrobial for multidrug-resistant Gram-negative bacteria. Enterotoxigenic E. coli strain (NCCP 13717) showed significantly increased resistance to acids and polymyxins. Transcriptome analysis of the AA NCCP 13717 revealed upregulation of genes related to the acid fitness island and the arn operon, which reduces lipopolysaccharide binding affinity at the polymyxin site of action. Genes such as eptA, tolC, and ompCF were also upregulated to alter the structure of the cell membrane, reducing the outer membrane permeability compared to the control, which is likely to be another mechanism for polymyxin resistance. This study highlights the emergence of antimicrobial resistance in AA pathogenic E. coli strains, particularly polymyxin resistance, and the mechanisms behind the increased antimicrobial resistance, providing important insights for the development of risk management strategies to effectively control the antimicrobial resistant foodborne pathogens.202439307200
359990.9996Distribution 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
5836100.9996Identification of Pseudomonas aeruginosa genes associated with antibiotic susceptibility. Pseudomonas aeruginosa causes acute and chronic infections in humans and these infections are difficult to treat due to the bacteria's high-level of intrinsic and acquired resistance to antibiotics. To address this problem, it is crucial to investigate the molecular mechanisms of antibiotic resistance in this organism. In this study, a P. aeruginosa transposon insertion library of 17000 clones was constructed and screened for altered susceptibility to seven antibiotics. Colonies grown on agar plates containing antibiotics at minimum inhibitory concentrations (MICs) and those unable to grow at 1/2 MIC were collected. The transposon-disrupted genes in 43 confirmed mutants that showed at least a three-fold increase or a two-fold decrease in susceptibility to at least one antibiotic were determined by semi-random PCR and subsequent sequencing analysis. In addition to nine genes known to be associated with antibiotic resistance, including mexI, mexB and mexR, 24 new antibiotic resistance-associated genes were identified, including a fimbrial biogenesis gene pilY1 whose disruption resulted in a 128-fold increase in the MIC of carbenicillin. Twelve of the 43 genes identified were of unknown function. These genes could serve as targets to control or reverse antibiotic resistance in this important human pathogen.201020953948
6264110.9996Multi-drug resistance pattern and genome-wide SNP detection in levofloxacin-resistant uropathogenic Escherichia coli strains. OBJECTIVES: Antibiotic treatment is extremely stressful for bacteria and has profound effects on their viability. Such administration induces physiological changes in bacterial cells, with considerable impact on their genome structure that induces mutations throughout the entire genome. This study investigated drug resistance profiles and structural changes in the entire genome of uropathogenic Escherichia coli (UPEC) strains isolated from six adapted clones that had evolved under laboratory conditions. METHODS: Eight UPEC strains, including two parental strains and six adapted clones, with different fluoroquinolone resistance levels originally isolated from two patients were used. The minimum inhibitory concentration (MIC) of 28 different antibiotics including levofloxacin was determined for each of the eight strains. In addition, the effects of mutations acquired with increased drug resistance in the levofloxacin-resistant strains on expression of genes implicated to be involved in drug resistance were examined. RESULTS: Of the eight UPEC strains used to test the MIC of 28 different antibiotics, two highly fluoroquinolone-resistant strains showed increased MIC in association with many of the antibiotics. As drug resistance increased, some genes acquired mutations, including the transcriptional regulator acrR and DNA-binding transcriptional repressor marR. Two strain groups with genetically different backgrounds (GUC9 and GFCS1) commonly acquired mutations in acrR and marR. Notably, acquired mutations related to efflux pump upregulation also contributed to increases in MIC for various antibiotics other than fluoroquinolone. CONCLUSIONS: The present results obtained using strains with artificially acquired drug resistance clarify the underlying mechanism of resistance to fluoroquinolones and other types of antibiotics.202438041251
5837120.9996The secondary resistome of multidrug-resistant Klebsiella pneumoniae. Klebsiella pneumoniae causes severe lung and bloodstream infections that are difficult to treat due to multidrug resistance. We hypothesized that antimicrobial resistance can be reversed by targeting chromosomal non-essential genes that are not responsible for acquired resistance but essential for resistant bacteria under therapeutic concentrations of antimicrobials. Conditional essentiality of individual genes to antimicrobial resistance was evaluated in an epidemic multidrug-resistant clone of K. pneumoniae (ST258). We constructed a high-density transposon mutant library of >430,000 unique Tn5 insertions and measured mutant depletion upon exposure to three clinically relevant antimicrobials (colistin, imipenem or ciprofloxacin) by Transposon Directed Insertion-site Sequencing (TraDIS). Using this high-throughput approach, we defined three sets of chromosomal non-essential genes essential for growth during exposure to colistin (n = 35), imipenem (n = 1) or ciprofloxacin (n = 1) in addition to known resistance determinants, collectively termed the "secondary resistome". As proof of principle, we demonstrated that inactivation of a non-essential gene not previously found linked to colistin resistance (dedA) restored colistin susceptibility by reducing the minimum inhibitory concentration from 8 to 0.5 μg/ml, 4-fold below the susceptibility breakpoint (S ≤ 2 μg/ml). This finding suggests that the secondary resistome is a potential target for developing antimicrobial "helper" drugs that restore the efficacy of existing antimicrobials.201728198411
6247130.9996Molecular basis and evolutionary cost of a novel macrolides/lincosamides resistance phenotype in Staphylococcus haemolyticus. Staphylococcus haemolyticus (S. haemolyticus) is a coagulase-negative Staphylococcus that has become one of the primary causes of nosocomial infection. After a long period of antibiotic use, S. haemolyticus has developed multiple resistance phenotypes for macrolides and lincosamides. Herein, we evaluated four S. haemolyticus clinical isolates, of which three had antibiotic resistance patterns reported previously. The fourth isolate was resistant to both erythromycin and clindamycin in the absence of erythromycin induction. This novel phenotype, known as constitutive macrolides-lincosamides-streptogramins resistance, has been reported in other bacteria but has not been previously reported in S. haemolyticus. Investigation of the isolate demonstrated a deletion in the methyltransferase gene ermC, upstream leader peptide. This deletion resulted in constitutive MLS resistance based on whole-genome sequencing and experimental verification. Continuous expression of ermC was shown to inhibit the growth of S. haemolyticus, which turned out to be the fitness cost with no MLS pressure. In summary, this study is the first to report constitutive MLS resistance in S. haemolyticus, which provides a better understanding of MLS resistance in clinical medicine. IMPORTANCE This study identified a novel phenotype of macrolides/lincosamides resistance in Staphylococcus haemolyticus which improved a better guidance for clinical treatment. It also clarified the mechanistic basis for this form of antibiotic resistance that supplemented the drug resistance mechanism of Staphylococcus. In addition, this study elaborated on a possibility that continuous expression of some resistance genes was shown to inhibit the growth of bacteria themselves, which turned out to be the fitness cost in the absence of antibiotic pressure.202337724875
5757140.9996The expression regulation of recA gene and bacterial class 2 integron-associated genes induced by antibiotics. OBJECTIVE: To investigate the effects and mechanisms of common antibiotics induction on the expression of class 2 integron integrase and variable region resistance genes in bacteria, as well as potential structural mutations. METHODS: Clinical isolates containing non-functional class 2 integrons and functional class 2 integrons were selected. Strains containing non-functional class 2 integrons or functional class 2 integrons were constructed using isolated DNA templates. These strains were subjected to continuous induction with drug concentrations of 1/2 MIC and 1/4 MIC (ciprofloxacin, ampicillin, and kanamycin) and a concentration of 0.2 μg/ml (mitomycin C) over 8 days. The relative expression levels of relevant genes were measured on days 1, 3, and 8. Drug resistance in the experimental strains was assessed before and after induction to identify any differences. Finally, the sequence of the non-functional class 2 integron integrase gene was analyzed for structural changes that occurred as a result of induction. RESULTS: All drugs selected in this study increased the relative expression levels of recA, intI2, dfrA1, sat2, and aadA1. Significant differences in inductive abilities were observed among the drugs. The 1/2 MIC concentrations were more effective than 1/4 MIC concentrations in increasing the relative expression levels of target genes and enhancing the resistance of the experimental strains. The relative expression levels of recA, intI2, and dfrA1 rose on day 1, peaked on day 3, and slightly declined by day 8. Induced strains exhibited increased resistance to the drugs, with the most significant changes observed in the clinical isolates, particularly concerning CIP resistance. Notably, clinical isolate 7b induced with 1/2 MIC KAN exhibited the loss of one base at position 12bp in the integrase sequence. However, none of the four drugs induced mutations at the 444 bp position of class 2 integrons. CONCLUSION: Sub-MIC concentrations of drugs have been shown to induce an increase in the relative expression level of the SOS response-related gene recA, as well as the integrase and resistance genes of class 2 integrons. Continuous induction leads to sustained upregulation of these genes, which stabilizes or slightly decreases upon reaching a plateau. However, the capacity of different drugs to induce expression varies significantly. Short-term antibiotic exposure did not result in critical mutations that convert class 2 integrons into functional forms.202540950603
4380150.9996Comparative 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
6248160.9996Characterization of a stable, metronidazole-resistant Clostridium difficile clinical isolate. BACKGROUND: Clostridium difficile are gram-positive, spore forming anaerobic bacteria that are the leading cause of healthcare-associated diarrhea, usually associated with antibiotic usage. Metronidazole is currently the first-line treatment for mild to moderate C. difficile diarrhea however recurrence occurs at rates of 15-35%. There are few reports of C. difficile metronidazole resistance in the literature, and when observed, the phenotype has been transient and lost after storage or exposure of the bacteria to freeze/thaw cycles. Owing to the unstable nature of the resistance phenotype in the laboratory, clinical significance and understanding of the resistance mechanisms is lacking. METHODOLOGY/PRINCIPAL FINDINGS: Genotypic and phenotypic characterization was performed on a metronidazole resistant clinical isolate of C. difficile. Whole-genome sequencing was used to identify potential genetic contributions to the phenotypic variation observed with molecular and bacteriological techniques. Phenotypic observations of the metronidazole resistant strain revealed aberrant growth in broth and elongated cell morphology relative to a metronidazole-susceptible, wild type NAP1 strain. Comparative genomic analysis revealed single nucleotide polymorphism (SNP) level variation within genes affecting core metabolic pathways such as electron transport, iron utilization and energy production. CONCLUSIONS/SIGNIFICANCE: This is the first characterization of stable, metronidazole resistance in a C. difficile isolate. The study provides an in-depth genomic and phenotypic analysis of this strain and provides a foundation for future studies to elucidate mechanisms conferring metronidazole resistance in C. difficile that have not been previously described.201323349739
5651170.9996Class 1 integron causes vulnerability to formaldehyde in Escherichia coli. In this study, the relationships of integron 1 element, formaldehyde dehydrogenase, and orfF genes with the level of formaldehyde resistance of isolated E. coli were investigated. E. coli bacteria were isolated from apparently healthy and colibacillosis-affected broilers of Fars Province, Iran. Formaldehyde resistance level and the presence of genetic markers were measured using MIC, and PCR tests, respectively. The prevalence of integron 1 element, orfF, and formaldehyde dehydrogenase genes in E. coli isolates were 61%, 8%, and 94%, respectively. In addition, according to our cut off definition, 15% and 85% of isolates were resistant and sensitive to formaldehyde, respectively. None of the genes had a statistically significant relationship with the formaldehyde resistance; however, the isolates containing integron 1 were significantly more sensitive to formaldehyde in the MIC test than those without integron 1. Integron 1 gene cassette could carry some bacterial surface proteins and porins with different roles in bacterial cells. Formaldehyde could also interfere with the protein functions by alkylating and cross-linking, and this compound would affect bacterial cell surface proteins in advance. Through an increase in the cell surface proteins, the presence of integron 1 gene cassette might make E. coli more sensitive to formaldehyde. As integron 1 was always involved in increasing bacterial resistance to antibiotics and disinfectants such as QACs, this is the first report of bacterial induction of sensitivity to a disinfectant through integron 1. Finally, integron 1 does not always add an advantage to E. coli bacteria, and it could be assumed as a cause of vulnerability to formaldehyde.202134148112
4669180.9996Functional 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
4495190.9996Mutations in the bacterial ribosomal protein l3 and their association with antibiotic resistance. Different groups of antibiotics bind to the peptidyl transferase center (PTC) in the large subunit of the bacterial ribosome. Resistance to these groups of antibiotics has often been linked with mutations or methylations of the 23S rRNA. In recent years, there has been a rise in the number of studies where mutations have been found in the ribosomal protein L3 in bacterial strains resistant to PTC-targeting antibiotics but there is often no evidence that these mutations actually confer antibiotic resistance. In this study, a plasmid exchange system was used to replace plasmid-carried wild-type genes with mutated L3 genes in a chromosomal L3 deletion strain. In this way, the essential L3 gene is available for the bacteria while allowing replacement of the wild type with mutated L3 genes. This enables investigation of the effect of single mutations in Escherichia coli without a wild-type L3 background. Ten plasmid-carried mutated L3 genes were constructed, and their effect on growth and antibiotic susceptibility was investigated. Additionally, computational modeling of the impact of L3 mutations in E. coli was used to assess changes in 50S structure and antibiotic binding. All mutations are placed in the loops of L3 near the PTC. Growth data show that 9 of the 10 mutations were well accepted in E. coli, although some of them came with a fitness cost. Only one of the mutants exhibited reduced susceptibility to linezolid, while five exhibited reduced susceptibility to tiamulin.201525845869