Detecting mutations that confer oxazolidinone resistance in gram-positive bacteria. - Related Documents




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448701.0000Detecting mutations that confer oxazolidinone resistance in gram-positive bacteria. Resistance to oxazolidinone antibiotics, including linezolid, in Gram-positive bacteria is mediated by single-nucleotide polymorphisms (SNPs) in the 23S ribosomal RNA. A G2576U change (encoded by a G2576T mutation in the rRNA genes) is found in most resistant clinical isolates of enterococci and staphylococci; a variety of changes have been found in resistant mutants selected in vitro. Pyrosequencing can be used to detect SNPs known to confer oxazolidinone resistance, including the G2576T change. Most bacteria have more than one rRNA gene copy and Pyrosequencing can also be used for allele quantification, i.e., to estimate the proportions of mutant vs wild-type alleles. The number of mutated rRNA gene copies correlates roughly with the level of oxazolidinone resistance displayed by resistant isolates. This chapter summarizes the Pyrosequencing assays that have been developed in our laboratory for analyzing oxazolidinone-resistant enterococci and staphylococci.200717185761
449710.9998Detection 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
624720.9998Molecular 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
448830.9998The cfr and cfr-like multiple resistance genes. The Cfr methyl transferase causes an RNA methylation of the bacterial ribosomes impeding reduced or abolished binding of many antibiotics acting at the peptidyl transferase center. It provides multi-resistance to eight classes of antibiotics, most of which are in clinical and veterinary use. The cfr gene is found in various bacteria in many geographical locations and placed on plasmids or associated with transposons. Cfr-related genes providing similar resistance have been identified in Bacillales, and now also in the pathogens Clostridium difficile and Enterococcus faecium. In addition, the presence of the cfr gene has been detected in harbours and food markets.201829378339
449540.9998Mutations 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
449850.9998A 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
624860.9998Characterization 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
438070.9997Comparative 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
626680.9997Bacterial gene loss as a mechanism for gain of antimicrobial resistance. Acquisition of exogenous DNA by pathogenic bacteria represents the basis for much of the acquired antimicrobial resistance in pathogenic bacteria. A more extreme mechanism to avoid the effect of an antibiotic is to delete the drug target, although this would be predicted to be rare since drug targets are often essential genes. Here, we review and discuss the description of a novel mechanism of resistance to the cephalosporin drug ceftazidime caused by loss of a penicillin-binding protein (PBP) in a Gram-negative bacillus (Burkholderia pseudomallei). This organism causes melioidosis across south-east Asia and northern Australia, and is usually treated with two or more weeks of ceftazidime followed by oral antibiotics for three to six months. Comparison of clinical isolates from six patients with melioidosis found initial ceftazidime-susceptible isolates and subsequent ceftazidime-resistant variants. The latter failed to grow on commonly used culture media, rendering these isolates difficult to detect in the diagnostic laboratory. Genomic analysis using pulsed-field gel electrophoresis and array based genomic hybridisation revealed a large-scale genomic deletion comprising 49 genes in the ceftazidime-resistant strains. Mutational analysis of wild-type B. pseudomallei demonstrated that ceftazidime resistance was due to deletion of a gene encoding a PBP 3 present within the region of genomic loss. This provides one explanation for ceftazidime treatment failure, and may be a frequent but undetected event in patients with melioidosis.201223022568
449090.9997Mutation analysis of mycobacterial rpoB genes and rifampin resistance using recombinant Mycobacterium smegmatis. Rifampin is a major drug used to treat leprosy and tuberculosis. The rifampin resistance of Mycobacterium leprae and Mycobacterium tuberculosis results from a mutation in the rpoB gene, encoding the β subunit of RNA polymerase. A method for the molecular determination of rifampin resistance in these two mycobacteria would be clinically valuable, but the relationship between the mutations and susceptibility to rifampin must be clarified before its use. Analyses of mutations responsible for rifampin resistance using clinical isolates present some limitations. Each clinical isolate has its own genetic variations in some loci other than rpoB, which might affect rifampin susceptibility. For this study, we constructed recombinant strains of Mycobacterium smegmatis carrying the M. leprae or M. tuberculosis rpoB gene with or without mutation and disrupted their own rpoB genes on the chromosome. The rifampin and rifabutin susceptibilities of the recombinant bacteria were measured to examine the influence of the mutations. The results confirmed that several mutations detected in clinical isolates of these two pathogenic mycobacteria can confer rifampin resistance, but they also suggested that some mutations detected in M. leprae isolates or rifampin-resistant M. tuberculosis isolates are not involved in rifampin resistance.201222252831
5977100.9997Methods to determine antibiotic resistance gene silencing. The occurrence of antibiotic-resistant bacteria is an increasingly serious problem world-wide. In addition, to phenotypically resistant bacteria, a threat may also be posed by isolates with silent, but intact, antibiotic resistance genes. Such isolates, which have recently been described, possess wild-type genes that are not expressed, but may convert to resistance by activating expression of the silent genes. They may therefore compromise the efficacy of antimicrobial treatment, particularly if their presence has not been diagnosed. This chapter describes the detection of silent resistance genes by PCR and DNA sequencing. A method to detect five potentially silent acquired resistance genes; aadA, bla (OXA-2), strAB, sul1, and tet(A) is described. First, the susceptibility of the isolates to the relevant antibiotics is determined by an appropriate susceptibility testing method, such as E-test. Then the presence of the genes is investigated by PCR followed by agarose gel electrophoresis of the amplification products. If a resistance gene is detected in a susceptible isolate, the entire open-reading frame and promoter sequence of the gene is amplified by PCR and their DNA sequences obtained. The DNA sequences are then compared to those of known resistant isolates, to detect mutations that may account for susceptibility. If no mutations are detected the expression of the gene is investigated by RT-PCR following RNA extraction. The methods described here can be applied to all acquired resistance genes for which sequence and normal expression data are available.201020401584
4504110.9997Resistance of enterococci to aminoglycosides and glycopeptides. High-level resistance to aminoglycosides in enterococci often is mediated by aminoglycoside-modifying enzymes, and the corresponding genes generally are located on self-transferable plasmids. These enzymes are similar to those in staphylococci but differ from the modifying enzymes of gram-negative bacteria. Three classes of enzymes are distinguished, depending upon the reaction catalyzed. All but amikacin and netilmicin confer high-level resistance to the antibiotics that are modified in vitro. However, the synergistic activity of these last two antibiotics in combination with beta-lactam agents can be suppressed, as has always been found in relation to high-level resistance to the aminoglycosides. Acquisition of glycopeptide resistance by enterococci recently was reported. Strains of two phenotypes have been distinguished: those that are resistant to high levels of vancomycin and teicoplanin and those that are inducibly resistant to low levels of vancomycin and susceptible to teicoplanin. In strains of Enterococcus faecium highly resistant to glycopeptides, we have characterized plasmids ranging from 34 to 40 kilobases that are often self-transferable to other gram-positive organisms. The resistance gene vanA has been cloned, and its nucleotide sequence has been determined. Hybridization experiments showed that this resistance determinant is present in all of our enterococcal strains that are highly resistant to glycopeptides. The vanA gene is part of a cluster of plasmid genes responsible for synthesis of peptidoglycan precursors containing a depsipeptide instead of the usual D-alanyl-D-alanine terminus. Reduced affinity of glycopeptides to these precursors confers resistance to the antibiotics.19921520800
3600120.9997Uncultured 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
4414130.9997Macrolide resistance mechanisms in Gram-positive cocci. Two principal mechanisms of resistance to macrolides have been identified in Gram-positive bacteria. Erythromycin-resistant methylase is encoded by erm genes. Resultant structural changes to rRNA prevent macrolide binding and allow synthesis of bacterial proteins to continue. Presence of the erm gene results in high-level resistance. Modification of the mechanism whereby antibiotics are eliminated from the bacteria also brings about resistance. Bacteria carrying the gene encoding macrolide efflux (i.e. the mefE gene) display relatively low-level resistance. Azithromycin, because of its ability to achieve concentrations at sites of infections, is capable of eradicating mefE-carrying strains. Other resistance mechanisms, involving stimulation of enzymatic degradation, appear not to be clinically significant.200111574191
6258140.9997Alterations in GyrA and ParC associated with fluoroquinolone resistance in Enterococcus faecium. High-level quinolone resistance in Enterococcus faecium was associated with mutations in both gyrA and parC genes in 10 of 11 resistant strains. On low-level resistant strain without such mutations may instead possess an efflux mechanism or alterations in the other subunits of the gyrase or topoisomerase IV genes. These findings are similar to those for other gram-positive bacteria, such as Enterococcus faecalis.199910103206
4505150.9997Origin and evolution of genes specifying resistance to macrolide, lincosamide and streptogramin antibiotics: data and hypotheses. Resistance to macrolide, lincosamide and streptogramin antibiotics is due to alteration of the target site or detoxification of the antibiotic. Postranscriptional methylation of 23S ribosomal rRNA confers resistance to macrolide (M), lincosamide (L) and streptogramin (S) B-type antibiotics, the so-called MLSB phenotype. Several classes of rRNA methylases conferring resistance to MLSB antibiotics have been characterized in Gram-positive cocci, in Bacillus spp, and in strains of actinomycetes producing erythromycin. The enzymes catalyze N6-dimethylation of an adenine residue situated in a highly conserved region of prokaryotic 23S rRNA. In this review, we compare the amino acid sequences of the rRNA methylases and analyze the codon usage in the corresponding erm (erythromycin resistance methylase) genes. The homology detected at the protein level is consistent with the notion that an ancestor of the erm genes was implicated in erythromycin resistance in a producing strain. However, the rRNA methylases of producers and non-producers present substantial sequence diversity. In Gram-positive bacteria the preferential codon usage in the erm genes reflects the guanosine plus cytosine content of the chromosome of the host. These observations suggest that the presence of erm genes in these micro-organisms is ancient. By contrast, it would appear that enterobacteria have acquired only recently an rRNA methylase gene of the ermB class from a Gram-positive coccus since the genes isolated in Escherichia coli and in Gram-positive cocci are highly homologous (homology greater than 98%) and present a codon usage typical of the latter micro-organisms. As opposed to the MLSB phenotype which results from a single biochemical mechanism, inactivation of structurally related antibiotics of the MLS group involves synthesis of various other enzymes. In enterobacteria, resistance to erythromycin and oleandomycin is due to production of erythromycin esterases which hydrolyze the lactone ring of the 14-membered macrolides. We recently reported the nucleotide sequence of ereA and ereB (erythromycin resistance esterase) genes which encode erythromycin esterases type I and II, respectively. The amino acid sequences of the two isozymes do not exhibit statistically significant homology. Analysis of codon usage in both genes suggests that esterase type I is indigenous to E. coli, whereas the type II enzyme was acquired by E. coli from a phylogenetically remote micro-organism. Inactivation of lincosamides, first reported in staphylococci and lactobacilli of animal origin, was also recently detected in Gram-positive cocci isolated from humans.(ABSTRACT TRUNCATED AT 400 WORDS)19873326871
6265160.9997Fitness costs of fluoroquinolone resistance in Streptococcus pneumoniae. The fitness cost of the genes responsible for resistance to fluoroquinolones in clinical isolates of Streptococcus pneumoniae were estimated in vitro in a common genetic background. Naturally occurring parC, parE, and gyrA loci containing mutations in the quinolone-resistance-determining regions were introduced by transformation into S. pneumoniae strain R6 individually and in combinations. The fitness of these transformants was estimated by pairwise competition experiments with a common R6 strain. On average, single par and gyr mutants responsible for low-level MIC resistance (first-step resistance) impose a fitness burden of approximately 8%. Some of these mutants engender no measurable cost, while one, a parE mutant, reduces the fitness of these bacteria by more than 40%. Most interestingly, the addition of the second par or gyr mutations required for clinically significant, high-MIC fluoroquinolone resistance does not increase the fitness burden imposed by these single genes and can even reduce it. We discuss the implications of these results for the epidemiology of fluoroquinolone resistance and the evolution of acquired resistance in treated patients.200717116668
4526170.9997The tetracycline resistance gene tet(M) exhibits mosaic structure. Tetracycline resistance genes of the M class, tet(M), are typically found on mobile genetic elements as the conjugative transposons of gram-positive bacteria. By comparing the sequences of eight different tet(M) genes (from Enterococcus faecalis, Streptococcus pneumoniae, Staphylococcus aureus, Ureaplasma urealyticum, and Neisseria), a mosaic structure was detected which could be traced to two distinct alleles. The two alleles displayed a divergence of 8% and a different G/C content. The block structure of these genes provides evidence for the contribution of homologous recombination to the evolution and the heterogeneity of the tet(M) locus. Unlike described cases of chromosomally located mosaic loci, tet(M) is a relatively recently acquired determinant in the species examined and it would appear that mosaic structure within tet(M) has evolved after acquisition of the gene by the mobile genetic elements upon which it is located.19968812782
4471180.9997Update 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
5983190.9997Analysis of mutational patterns in quinolone resistance-determining regions of GyrA and ParC of clinical isolates. Fluoroquinolone (FQ)-resistant bacteria pose a major global health threat. Unanalysed genomic data from thousands of sequenced microbes likely contain important hints regarding the evolution of FQ resistance, yet this information lies fallow. Here we analysed the co-occurrence patterns of quinolone resistance mutations in genes encoding the FQ drug targets DNA gyrase (gyrase) and topoisomerase IV (topo-IV) from 36,402 bacterial genomes, representing 10 Gram-positive and 10 Gram-negative species. For 19 species, the likeliest routes toward resistance mutations in both targets were determined, and for 5 species those mutations necessary and sufficient to predict FQ resistance were also determined. Target mutation hierarchy was fixed in all examined Gram-negative species, with gyrase being the primary and topo-IV the secondary quinolone target, as well as in six of nine Gram-positive species, with topo-IV being the primary and gyrase the secondary target. By contrast, in three Gram-positive species (Staphylococcus haemolyticus, Streptococcus pneumoniae and Streptococcus suis), under some conditions gyrase became the primary and topo-IV the secondary target. The path through individual resistance mutations varied by species. Both linear and branched paths were identified in Gram-positive and Gram-negative organisms alike. Finally, FQ resistance could be predicted based solely on target gene quinolone resistance mutations for Acinetobacter baumannii, Escherichia coli and Staphylococcus aureus, but not Klebsiella pneumoniae or Pseudomonas aeruginosa. These findings have important implications both for sequence-based diagnostics and for understanding the emergence of FQ resistance.201930582984