The cme gene of Clostridium difficile confers multidrug resistance in Enterococcus faecalis. - Related Documents




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40201.0000The cme gene of Clostridium difficile confers multidrug resistance in Enterococcus faecalis. Antibiotic resistance in C. difficile by efflux has been previously suggested. The genome of C. difficile 630 was screened for sequences encoding putative proteins homologous to NorA from Staphylococcus aureus. Four ORFs homologous to efflux genes were cloned into the pAT79 shuttle vector under the control of transcription and translation signals of Gram-positive bacteria and expressed in Enterococcus faecalis JH2-2 and S. aureus RN4220. One of these sequences, designated cme conferred resistance to ethidium bromide, safranin O, and erythromycin in E. faecalis. The three other ORFs did not confer detectable resistance in both bacteria.200415336408
44310.9994Deletion mutant analysis of the Staphylococcus aureus plasmid pI258 mercury-resistance determinant. Deletion mutant analysis of the mercury-resistant determinant (mer operon) from the Staphylococcus aureus plasmid pI258 was used to verify the location of the merA and merB genes and to show the existence of mercuric ion transport gene(s). ORF5 was confirmed to be a transport gene and has an amino acid product sequence homologous to the merT gene products from several gram-negative bacteria and a Bacillus species. Deletion analysis established that inactivation of merA on a broad-spectrum mer resistance determinant resulted in a mercury-hypersensitive phenotype. Gene dosage had no apparent effect on the level of resistance conferred by the intact mer operon or on the expression of an inducible phenotype, except that when the intact pI258 mer operon was on a high copy number plasmid, uninduced cells possessed a volatilization rate that was at most only 3.5-fold less than that observed for induced cells. There was no need for mercury ion transport proteins for full resistance when the mer operon was expressed in a high copy number plasmid.19911954576
596320.9994Expression of the mphB gene for macrolide 2'-phosphotransferase II from Escherichia coli in Staphylococcus aureus. The genes mphA and mphB encode macrolide 2'-phosphotransferases I and II, respectively, and they confer resistance to macrolide antibiotics in Escherichia coli. To study the expression of these genes in Gram-positive bacteria, we constructed recombinant plasmids that consisted of an mph gene and the pUB110 vector in Bacillus subtilis. When these plasmids were introduced into Staphylococcus aureus, the mphB gene was active and macrolide 2'-phosphotransferase II was produced. The gene endowed S. aureus with high-level resistance to spiramycin, a macrolide antibiotic with a 16-membered ring. Moreover, transcription of the mphB gene in S. aureus began at the promoter that was active in E. coli.19989503630
596430.9993Heat shock treatment increases the frequency of loss of an erythromycin resistance-encoding transposable element from the chromosome of Lactobacillus crispatus CHCC3692. A 3,165-bp chromosomally integrated transposon, designatedTn3692, of the gram-positive strain Lactobacillus crispatus CHCC3692 contains an erm(B) gene conferring resistance to erythromycin at concentrations of up to 250 micrograms/ml. Loss of this resistance can occur spontaneously, but the rate is substantially increased by heat shock treatment. Heat shock treatment at 60 degrees C resulted in an almost 40-fold increase in the frequency of erythromycin-sensitive cells (erythromycin MIC, 0.047 micrograms/ml). The phenotypic change was followed by a dramatic increase in transcription of the transposase gene and the concomitant loss of an approximately 2-kb DNA fragment carrying the erm(B) gene from the 3,165-bp erm transposon. In cells that were not subjected to heat shock, transcription of the transposase gene was not detectable. The upstream sequence of the transposase gene did not show any homology to known heat shock promoters in the gene data bank. Significant homology (>99%) was observed between the erythromycin resistance-encoding gene from L. crispatus CHCC3692 and the erm(B) genes from other gram-positive bacteria, such as Streptococcus agalactiae, Streptococcus pyogenes, Enterococcus faecium, and Lactobacillus reuteri, which strongly indicates a common origin of the erm(B) gene for these species. The transposed DNA element was not translocated to other parts of the genome of CHCC3692, as determining by Southern blotting, PCR analysis, and DNA sequencing. No other major aberrations were observed, as judged by colony morphology, growth performance of the strain, and pulsed-field gel electrophoresis. These observations suggest that heat shock treatment could be used as a tool for the removal of unwanted antibiotic resistance genes harbored in transposons flanked by insertion sequence elements or transposases in lactic acid bacteria used for animal and human food production.200314660363
305240.9993Expression of antibiotic resistance genes from Escherichia coli in Bacillus subtilis. Bifunctional recombinant plasmids were constructed, comprised of the E. coli vectors pBR322, pBR325 and pACYC184 and different plasmids from Gram-positive bacteria, e.g. pBSU161-1 of B. subtilis and pUB110 and pC221 of S. aureus. The beta-lactamase (bla) gene and the chloramphenicol acetyltransferase (cat) gene from the E. coli plasmids were not transcribed and therefore not expressed in B. subtilis. However, tetracycline resistance from the E. coli plasmids was expressed in B. subtilis. Transcription of the tetracycline resistance gene(s) started in B. subtilis at or near the original E. coli promoter, the sequence of which is almost identical with the sequence recognized by sigma 55 of B. subtilis RNA polymerase.19836410152
450350.9993Evolution and transfer of aminoglycoside resistance genes under natural conditions. 3'-Aminoglycoside phosphotransferases [APH(3')] were chosen as a model to study the evolution and the transfer of aminoglycoside resistance genes under natural conditions. Comparison of the amino acid sequences of APH(3') enzymes from transposons Tn903 (type I) and Tn5 (type II) detected in Gram-negative bacteria, from the Gram-positive Staphylococcus and Streptococcus (type III), from the butirosin-producing Bacillus circulans (type IV) and from a neomycin-producing Streptomyces fradiae (type V) indicate that they have diverged from a common ancestor. These structural data support the hypothesis that the antibiotic-producing strains were the source of certain resistance determinants. We have shown that kanamycin resistance in Campylobacter coli BM2509 was due to the synthesis of an APH(3')-III, an enzyme not detected previously in a Gram-negative bacterium. The genes encoding APH(3')-III in Streptococcus and Campylobacter are identical. These findings constitute evidence for a recent in-vivo transfer of DNA between Gram-positive and Gram-negative bacteria.19863027020
42860.9993Identification and analysis of genes for tetracycline resistance and replication functions in the broad-host-range plasmid pLS1. The streptococcal plasmid pMV158 and its derivative pLS1 are able to replicate and confer tetracycline resistance in both Gram-positive and Gram-negative bacteria. Copy numbers of pLS1 were 24, 4 and 4 molecules per genome in Streptococcus pneumoniae, Bacillus subtilis and Escherichia coli, respectively. Replication of the streptococcal plasmids in E. coli required functional polA and recA genes. A copy-number mutation corresponding to a 332 base-pair deletion of pLS1 doubled the plasmid copy number in all three species. Determination of the complete DNA sequence of pLS1 revealed transcriptional and translational signals and four open reading frames. A putative inhibitory RNA was encoded in the region deleted by the copy-control mutation. Two putative mRNA transcripts encoded proteins for replication functions and tetracycline resistance, respectively. The repB gene encoded a trans-acting, 23,000 Mr protein necessary for replication, and the tet gene encoded a very hydrophobic, 50,000 Mr protein required for tetracycline resistance. The polypeptides corresponding to these proteins were identified by specific labeling of plasmid-encoded products. The tet gene of pLS1 was highly homologous to tet genes in two other plasmids of Gram-positive origin but different in both sequence and mode of regulation from tet genes of Gram-negative origin.19862438417
45770.9992Molecular characterization of the genes encoding DNA gyrase and topoisomerase IV of Listeria monocytogenes. The genes encoding subunits A and B of DNA gyrase and subunits C and E of topoisomerase IV of Listeria monocytogenes, gyrA, gyrB, parC and parE, respectively, were cloned and sequenced. Compared with the sequences of quinolone-susceptible bacteria, such as Escherichia coli and Bacillus subtilis, the quinolone resistance-determining region (QRDR) of DNA gyrase subunit A was altered; the deduced amino acid sequences revealed the substitutions Ser-84-->Thr and Asp/Glu-88-->Phe, two amino acid variations at hot spots, commonly associated with resistance to quinolones. No relevant divergences from QRDR consensus sequences were observed in GyrB or both topoisomerase IV subunits. Thus, it could be argued that the amino acid substitutions in GyrA would explain the intrinsic resistance of L. monocytogenes to nalidixic acid. In order to analyse the actual role of the GyrA alterations, a plasmid-encoded gyrA allele was mutated and transformed into L. monocytogenes. However, these heterodiploid strains were not affected in their resistance to nalidixic acid. The effects of the mutant plasmids on ciprofloxacin and sparfloxacin susceptibility were only modest.200212039883
625880.9992Alterations 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
45690.9992Cloning and nucleotide sequences of the topoisomerase IV parC and parE genes of Mycoplasma hominis. The topoisomerase IV parC and parE genes from the wall-less organism Mycoplasma hominis PG21 were cloned and sequenced. The coupled genes are located far from the DNA gyrase genes gyrA and gyrB. They encode proteins of 639 and 866 amino acids, respectively. As expected, the encoded ParE and ParC proteins exhibit higher homologies with the topoisomerase IV subunits of the gram-positive bacteria Staphylococcus aureus and Streptococcus pneumoniae than with their Escherichia coli counterparts. The conserved regions include the Tyr residue of the active site and the region involved in quinolone resistance (quinolone resistance-determining region [QRDR]) in ParC and the ATP-binding site and the QRDR in ParE.19989687401
441100.9992Preparation of a DNA gene probe for detection of mercury resistance genes in gram-negative bacterial communities. A DNA gene probe was prepared to study genetic change mechanisms responsible for adaptation to mercury in natural bacterial communities. The probe was constructed from a 2.6-kilobase NcoI-EcoRI DNA restriction fragment which spans the majority of the mercury resistance operon (mer) in the R-factor R100. The range of specificity of this gene probe was defined by hybridization to the DNA of a wide variety of mercury-resistant bacteria previously shown to possess the mercuric reductase enzyme. All of the tested gram-negative bacteria had DNA sequences homologous to the mer probe, whereas no such homologies were detected in DNA of the gram-positive strains. Thus, the mer probe can be utilized to study gene flow processes in gram-negative bacterial communities.19853994373
440110.9992Nucleotide sequence analysis reveals similarities between proteins determining methylenomycin A resistance in Streptomyces and tetracycline resistance in eubacteria. Previous studies had localised the gene (mmr) for resistance to methylenomycin A (Mm) to a 2.5-kb PstI fragment in the middle of a cluster of Mm biosynthetic genes from the Streptomyces coelicolor plasmid SCP1. In this paper, the gene has been more precisely located by sub-cloning, and the nucleotide sequence of the whole fragment has been determined. The predicted mmr-specified protein (Mr 49238) would be hydrophobic, with some homology at the amino acid level to tetracycline-resistance proteins from both Gram-positive and Gram-negative bacteria. Comparisons of hydropathy plots of the amino acid sequences reinforces the idea that the proteins are similar. It is suggested that Mm resistance may be conferred by a membrane protein, perhaps controlling efflux of the antibiotic. No significant homology was detected by hybridisation analysis between mmr and a cloned oxytetracycline (OTc)-resistance gene (tetB) of the OTc producer Streptomyces rimosus, and no cross-resistance was conferred by these genes. Sequences on both sides of mmr appear to encode proteins. The direction of translation in each case would be opposite to that of mmr translation. This suggests that mmr is transcribed as a monocistronic mRNA from a bidirectional promoter. An extensive inverted repeat sequence between the stop codons of mmr and the converging gene may function as a bidirectional transcription terminator.19872828187
4504120.9992Resistance 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
364130.9992Stenotrophomonas maltophilia D457R contains a cluster of genes from gram-positive bacteria involved in antibiotic and heavy metal resistance. A cluster of genes involved in antibiotic and heavy metal resistance has been characterized from a clinical isolate of the gram-negative bacterium Stenotrophomonas maltophilia. These genes include a macrolide phosphotransferase (mphBM) and a cadmium efflux determinant (cadA), together with the gene cadC coding for its transcriptional regulator. The cadC cadA region is flanked by a truncated IS257 sequence and a region coding for a bin3 invertase. Despite their presence in a gram-negative bacterium, these genetic elements share a common gram-positive origin. The possible origin of these determinants as a remnant composite transposon as well as the role of gene transfer between gram-positive and gram-negative bacteria for the acquisition of antibiotic resistance determinants in chronic, mixed infections is discussed.200010858330
4497140.9992Detection 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
3014150.9992Complete sequence of the multi-resistance plasmid pV7037 from a porcine methicillin-resistant Staphylococcus aureus. The aim of this study was to determine the complete sequence of the multi-resistance plasmid pV7037 to gain insight into the structure and organization of this plasmid. Of the four XbaI clones of pV7037, one clone of 17,577 bp has already been sequenced and shown to carry a multi-resistance gene cluster. The remaining three clones of approximately 12.5, 6.5 and 4.5 kb were sequenced, the entire plasmid sequence correctly assembled and investigated for reading frames. In addition, two reading frames one coding for an ABC transporter and the other coding for an rRNA methylase were cloned and expressed in a S. aureus host to see whether they confer antimicrobial resistance properties. Plasmid pV7037 proved to be 40,971 bp in size. Besides the previously determined resistance gene cluster, it carried a functionally active tet(L) gene for tetracycline resistance, a complete cadDX operon for cadmium resistance and also a variant of the β-lactamase transposon Tn552. Two single bp deletions, which resulted in frame shifts, functionally deleted the genes for the BlaZ β-lactamase and the signal transducer protein BlaR1 in this Tn552 variant of pV7037. Plasmid pV7037 seems to be composed of various parts previously known from plasmids and transposons of staphylococci and other Gram-positive bacteria. However, there are also parts of the plasmid which do not show any homology to so far known sequences deposited in the databases. The novel ABC transporter and rRNA methylase genes identified on pV7037 do not seem to play a role in antimicrobial resistance. The co-location of numerous antimicrobial resistance genes bears the risk of co-transfer and co-selection of resistance genes, but also persistence of resistance genes even if no direct selective pressure by the use of the respective antimicrobial agents is applied.201323953027
6324160.9992Genetic and biochemical basis of tetracycline resistance. Properties of several, well characterized, tetracycline resistance determinants were compared. The determinants in Tn1721 and Tn10 (both from Gram-negative bacteria) each contain two genes; one encodes a repressor that regulates both its own transcription and that of a membrane protein that confers resistance by promoting efflux of the drug. Determinants from Gram-positive bacteria also encode efflux proteins, but expression of resistance is probably regulated by translational attenuation. The likely tetracycline binding site (a common dipeptide) in each efflux protein was predicted. The presence of the common binding site is consistent with the ability of an efflux protein originating in Bacillus species to be expressed in Escherichia coli.19863542941
4505170.9992Origin 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
5981180.9992Alterations in the DNA topoisomerase IV grlA gene responsible for quinolone resistance in Staphylococcus aureus. A 4.2-kb DNA fragment conferring quinolone resistance was cloned from a quinolone-resistant clinical isolate of Staphylococcus aureus and was shown to possess a part of the grlB gene and a mutated grlA gene. S-80-->F and E-84-->K mutations in the grlA gene product were responsible for the quinolone resistance. The mutated grlA genes responsible for quinolone resistance were dominant over the wild-type allele, irrespective of gene dosage in a transformation experiment with the grlA gene alone. However, dominance by mutated grlA genes depended on gene dosage when bacteria were transformed with the grlA and grlB genes in combination. Quinolone-resistant gyrA mutants were easily isolated from a strain, S. aureus RN4220, carrying a plasmid with the mutated grlA gene, though this was not the case for other S. aureus strains lacking the plasmid. The elimination of this plasmid from such quinolone-resistant gyrA mutants resulted in marked increases in quinolone susceptibility. These results suggest that both DNA gyrase and DNA topoisomerase IV may be targets of quinolones and that the quinolone susceptibility of organisms may be determined by which of these enzymes is most quinolone sensitive.19968723458
3051190.9992Nucleotide sequence of the bacterial streptothricin resistance gene sat3. The nucleotide sequence of the sat3 gene which encodes resistance of enteric bacteria to the antibiotic streptothricin is reported. A protein with a molecular mass of about 23 kDa is expressed from this gene. The sat3 gene is not obviously related to any one of the streptothricin resistance determinants identified so far among Gram-negative or Gram-positive bacteria.19957640311