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305300.9907Expression in Escherichia coli of cryptic tetracycline resistance genes from bacteroides R plasmids. The putative clindamycin resistance region of the Bacteroides fragilis R plasmid pBF4 was cloned in the vector R300B in Escherichia coli. This 3.8-kb EcoRI D fragment from pBF4 expressed noninducible tetracycline resistance in E. coli under aerobic but not anaerobic growth conditions. The fragment does not express tetracycline resistance in Bacteroides, a strict anaerobe. The separate tetracycline resistance transfer system in the Bacteroides host strain V479-1 has no homology to the cryptic determinant on pBF4. In addition, this aerobic tetracycline resistance determinant is not homologous to the three major plasmid mediated tetracycline resistance regions found in facultative gram-negative bacteria, represented by R100, RK2, and pBR322. A similar cryptic tetracycline resistance fragment was cloned from pCP1, a separate clindamycin resistance plasmid from Bacteroides that shares homology with the EcoRI D fragment of pBF4. This study identifies cryptic drug resistance determinants in Bacteroides that are expressed when inserted into an aerobically growing organism.19846379711
544010.9896Molecular structure and evolution of the conjugative multiresistance plasmid pRE25 of Enterococcus faecalis isolated from a raw-fermented sausage. Plasmid pRE25 from Enterococcus faecalis transfers resistances against kanamycin, neomycin, streptomycin, clindamycin, lincomycin, azithromycin, clarithromycin, erythromycin, roxithromycin, tylosin, chloramphenicol, and nourseothricin sulfate by conjugation in vitro to E. faecalis JH2-2, Lactococcus lactis Bu2, and Listeria innocua L19. Its nucleotide sequence of 50237 base pairs represents the largest, fully sequenced conjugative multiresistance plasmid of enterococci (Plasmid 46 (2001) 170). The gene for chloramphenicol resistance (cat) was identified as an acetyltransferase identical to the one of plasmid pIP501 of Streptococcus agalactiae. Erythromycin resistance is due to a 23S ribosomal RNA methyl transferase, again as found in pIP501 (ermB). The aminoglycoside resistance genes are packed in tandem as in transposon Tn5405 of Staphylococcus aureus: an aminoglycoside 6-adenyltransferase, a streptothricin acetyl transferase, and an aminoglycoside phosphotransferase.). Identical resistance genes are known from pathogens like Streptococcus pyogenes, S. agalactiae, S. aureus, Campylobacter coli, Clostridium perfringens, and Clostridium difficile. pRE25 is composed of a 30.5-kbp segment almost identical to pIP501. Of the 15 genes involved in conjugative transfer, 10 codes for putative transmembrane proteins (e.g. trsB, traC, trsF, trsJ, and trsL). The enterococcal part is joined into the pIP501 part by insertion elements IS1216V of E. faecium Tn1545 (three copies), and homologs of IS1062 (E. faecalis) and IS1485 (E. faecium). pRE25 demonstrates that enterococci from fermented food do participate in the molecular communication between Gram-positive and Gram-negative bacteria of the human and animal microflora.200314597005
35820.9895Resistance factors in anaerobic bacteria. Resistance transfer factors have been described in both Bacteroides and clostridia. The clindamycin (Cln) resistance transfer factors from the Bacteroides fragilis group of organisms have been best studied, including our own plasmid pBFTM10. The clindamycin resistance determinant (Cln X) of pBFTM10 can be detected in 90% of Cln resistant Bacteroides isolated from dispersed geographical areas. This determinant can be located in the chromosome and on plasmids. Recent studies from our laboratory have shown that the Cln X genes of pBFTM 10 are carried on a compound transposon, Tn4400. Bacteroides plasmids have been cloned in Escherichia coli and shuttle vectors have been developed that allow transfers of DNA from E. coli back to B. fragilis, using the broad host range plasmid RK2 to supply essential conjugation functions. We have shown that shuttle vectors containing pBFTM 10 can be retransferred from B. fragilis back to E. coli. In addition, a tetracycline transfer element from B. fragilis strain TM230 is able to promote high frequency conjugation between B. fragilis and E. coli. The results of these investigations indicate that Bacteroides has efficient mechanisms to exchange genetic material and that genetic exchange can occur between Bacteroides and E. coli, which exist in intimate contact in the human colon.19863029859
35930.9894Construction of shuttle cloning vectors for Bacteroides fragilis and use in assaying foreign tetracycline resistance gene expression. Shuttle vectors capable of replication in both Escherichia coli and Bacteroides fragilis have been developed. Conjugal transfer of these plasmids from E. coli to B. fragilis is facilitated by inclusion of the origin of transfer of the IncP plasmid RK2. The vectors pDK1 and pDK2 provide unique sites for cloning selectable markers in Bacteroides. pOA10 is a cosmid vector containing the replication region of pCP1 necessary for maintenance in Bacteroides. pDK3, pDK4.1, and pDK4.2 contain the Bacteroides clindamycin resistance gene allowing selection and maintenance in B. fragilis of plasmids containing inserted DNA fragments. pDK3 was used to test the expression in B. fragilis of five foreign tetracycline resistance (TcR) genes. The tetA, -B, and -C markers from facultative gram-negative bacteria, as well as a TcR determinant from Clostridium perfringens, did not express TcR in B. fragilis. The tetM gene, originally described in streptococci, encoded a small but reproducible increase of TcR in Bacteroides. These studies demonstrate the utility of shuttle vectors for introducing cloned genes into Bacteroides and underscore the differences in gene expression in these anaerobes.19883071818
596740.9893Metronidazole- and carbapenem-resistant bacteroides thetaiotaomicron isolated in Rochester, Minnesota, in 2014. Emerging antimicrobial resistance in members of the Bacteroides fragilis group is a concern in clinical medicine. Although metronidazole and carbapenem resistance have been reported in Bacteroides thetaiotaomicron, a member of the B. fragilis group, they have not, to the best of our knowledge, been reported together in the same B. thetaiotaomicron isolate. Herein, we report isolation of piperacillin-tazobactam-, metronidazole-, clindamycin-, ertapenem-, and meropenem-resistant B. thetaiotaomicron from a patient with postoperative intra-abdominal abscess and empyema. Whole-genome sequencing demonstrated the presence of nimD with at least a portion of IS1169 upstream, a second putative nim gene, two β-lactamase genes (one of which has not been previously reported), two tetX genes, tetQ, ermF, two cat genes, and a number of efflux pumps. This report highlights emerging antimicrobial resistance in B. thetaiotaomicron and the importance of identification and antimicrobial susceptibility testing of selected anaerobic bacteria.201525941219
542750.9892PCR-based detection of resistance genes in anaerobic bacteria isolated from intra-abdominal infections. Little information is available on the distribution of antimicrobial resistance genes in anaerobes in Japan. To understand the background of antimicrobial resistance in anaerobes involved in intra-abdominal infections, we investigated the distribution of eight antimicrobial resistance genes (cepA, cfiA, cfxA, ermF, ermB, mefA, tetQ, and nim) and a mutation in the gyrA gene in a total of 152 organisms (Bacteroides spp., Prevotella spp., Fusobacterium spp., Porphyromonas spp., Bilophila wadsworthia, Desulfovibrio desulfuricans, Veillonella spp., gram-positive cocci, and non-spore-forming gram-positive bacilli) isolated between 2003 and 2004 in Japan. The cepA gene was distributed primarily in Bacteroides fragilis. Gene cfxA was detected in about 9 % of the Bacteroides isolates and 75 % of the Prevotella spp. isolates and did not appear to contribute to cephamycin resistance. Two strains of B. fragilis contained the metallo-β-lactamase gene cfiA, but they did not produce the protein product. Gene tetQ was detected in about 81, 44, and 63 % of B. fragilis isolates, other Bacteroides spp., and Prevotella spp. isolates, respectively. The ermF gene was detected in 25, 13, 56, 64, and 16 % of Bacteroides spp., Prevotella spp., Fusobacterium spp., B. wadsworthia, and anaerobic cocci, respectively. Gene mefA was found in only 10 % of the B. fragilis strains and 3 % of the non-B. fragilis strains. Genes nim and ermB were not detected in any isolate. Substitution at position 82 (Ser to Phe) in gyrA was detected in B. fragilis isolates that were less susceptible or resistant to moxifloxacin. This study is the first report on the distribution of resistance genes in anaerobes isolated from intra-abdominal infections in Japan. We expect that the results might help in understanding the resistance mechanisms of specific anaerobes.201323338012
301360.9892Nucleotide sequence and organization of the multiresistance plasmid pSCFS1 from Staphylococcus sciuri. OBJECTIVES: The multiresistance plasmid pSCFS1 from Staphylococcus sciuri was sequenced completely and analysed with regard to its gene organization and the putative role of a novel ABC transporter in antimicrobial resistance. METHODS: Plasmid pSCFS1 was transformed into Staphylococcus aureus RN4220, overlapping restriction fragments were cloned into Escherichia coli plasmid vectors and sequenced. For further analysis of the ABC transporter, a approximately 3 kb EcoRV-HpaI fragment was cloned into the staphylococcal plasmid pT181MCS and the respective S. aureus RN4220 transformants were subjected to MIC determination. RESULTS: A total of 14 ORFs coding for proteins of >100 amino acids were detected within the 17 108 bp sequence of pSCFS1. Five of them showed similarity to recombination/mobilization genes while another two were similar to plasmid replication genes. In addition to the previously described genes cfr for chloramphenicol/florfenicol resistance and erm(33) for inducible resistance to macrolide-lincosamide-streptogramin B resistance, a Tn554-like spectinomycin resistance gene and Tn554-related transposase genes were identified. Moreover, a novel ABC transporter was detected and shown to mediate low-level lincosamide resistance. CONCLUSION: Plasmid pSCFS1 is composed of various parts which show similarity to sequences known to occur on plasmids or transposons of Gram-positive, but also Gram-negative bacteria. It is likely that pSCFS1 represents the result of inter-plasmid recombination events also involving the truncation of a Tn554-like transposon.200415471995
542670.9891First Report of Antibiotic Resistance Markers cfiA and nim Among Bacteroides fragilis Group Strains in Ecuadorian Patients. In recent years, increasing resistance of Bacteroides fragilis to several antibiotics has been reported in different countries. The aim of this study was to evaluate the antibiotic resistance profiles of Bacteroides spp. isolated from clinical samples by phenotypic and molecular methods. A total of 40 nonrepetitive isolates of the B. fragilis group were studied from 2018 to 2019. The species was identified by API 20A system. The minimum inhibitory concentrations (MICs) were determined by Sensititre anaerobe MIC plate. The presence of the nim and cfiA genes was checked by conventional PCR. The association between genes and insertion sequence (IS) was performed by whole genome sequencing. Eleven isolates were categorized as metronidazole-resistant and only 2 isolates harbored the nim gene. Five isolates were imipenem-resistant, but cfiA gene was detected in two isolates. cfiA gene was closely related to the cfiA-4 allele and associated with IS614B. The nim gene was not related to any nim gene type and was considered a new variant named nimL. IS612 was found upstream of nimL gene. In view of the scarcity of data on B. fragilis, there is a need to surveil antibiotic resistance levels and molecular mechanisms to implement better antimicrobial therapies against this important group of bacteria.202337733248
49080.9891Mercuric resistance genes in gram-positive oral bacteria. Mercury-resistant bacteria isolated from the oral cavities of children carried one of two types of merA gene that appear to have evolved from a common ancestor. Streptococcus oralis, Streptococcus mitis and a few other species had merA genes that were very similar to merA of Bacillus cereus strain RC607. Unlike the B. cereus RC607 merA gene, however, the streptococcal merA genes were not carried on Tn5084-like transposons. Instead, comparisons with microbial genomic sequences suggest the merA gene is located on a novel type II transposon. Coagulase-negative staphylococci and Streptococcus parasanguis had identical merA genes that represent a new merA variant.200415251199
36190.9890Plasmid transfer from Escherichia coli to Bacteroides fragilis: differential expression of antibiotic resistance phenotypes. A unique shuttle plasmid, pDP1, has been constructed to mediate gene transfer between Escherichia coli and the Gram-negative anaerobe Bacteroides fragilis. pDP1 contains the pBR322 replicon and the Bacteroides clindamycin resistance plasmid pCP1 linked to the transfer origin of the broad host range plasmid RK2. pDP1 can be transferred from E. coli to B. fragilis by the RK2 conjugation system even though RK2 itself is not maintained in the Bacteroides recipients. The antibiotic resistance and replication functions of pDP1 have been mapped by deletion analysis, and a 5-kilobase portion of the plasmid has been identified as the essential region for maintenance in Bacteroides. Comparison of the resistance conferred by pDP1 on E. coli and B. fragilis shows that antibiotic resistance genes are expressed differently in aerobic and anaerobic bacteria. These results document the feasibility of gene transfer from E. coli to B. fragilis and demonstrate the usefulness of this conjugation system to study genetic structure and expression in Bacteroides.19846095273
407100.9889Molecular cloning and characterization of two lincomycin-resistance genes, lmrA and lmrB, from Streptomyces lincolnensis 78-11. Two different lincomycin-resistance determinants (lmrA and lmrB) from Streptomyces lincolnensis 78-11 were cloned in Streptomyces lividans 66 TK23. The gene lmrA was localized on a 2.16 kb fragment, the determined nucleotide sequence of which encoded a single open reading frame 1446 bp long. Analysis of the deduced amino acid sequence suggested the presence of 12 membrane-spanning domains and showed significant similarities to the methylenomycin-resistance protein (Mmr) from Streptomyces coelicolor, the QacA protein from Staphylococcus aureus, and several tetracycline-resistance proteins from both Gram-positive and Gram-negative bacteria, as well as to some sugar-transport proteins from Escherichia coli. The lmrB gene was actively expressed from a 2.7 kb fragment. An open reading frame of 837 bp could be localized which encoded a protein that was significantly similar to 23S rRNA adenine(2058)-N-methyltransferases conferring macrolide-lincosamide-streptogramin resistance. LmrB also had putative rRNA methyltransferase activity since lincomycin resistance of ribosomes was induced in lmrB-containing strains. Surprisingly, both enzymes, LmrA and LmrB, had a substrate specificity restricted to lincomycin and did not cause resistance to other lincosamides such as celesticetin and clindamycin, or to macrolides.19921328813
4475110.9889Clindamycin resistance in anaerobic bacteria. Knowledge of the mechanisms of antimicrobial resistance and resistance transfer in anaerobic bacteria has been gained over the past several years. There is widespread resistance to the beta-lactam antibiotics in the B. fragilis group of organisms and there is emerging penicillin resistance in other Bacteroides species. These resistances are usually mediated by chromosomal beta-lactamases. There have been two new beta-lactamases described in Bacteroides; a penicillinase which inactivates ureidopenicillins and another that inactivates cefoxitin. The transfer of the common beta-lactamase, penicillinase, and cefoxitin resistance has been documented in B. fragilis. The mechanism of tetracycline resistance in B. fragilis is the lack of accumulation of intracellular drug; the resistance is widespread in anaerobic bacteria and is seen in two-thirds of the B. fragilis strains. The transfer of tetracycline resistance is common, however, no transfer factor has yet been isolated. Clindamycin-erythromycin resistance in Bacteroides was first recognized in the mid-1970s and transferable resistance was described in 1979. The mechanism of resistance is probably similar to macrolide-lincosamide-streptinogramin-resistance seen in aerobic bacteria. Two clindamycin resistance transfer factors, pBFTM10 and pIP410 (pBF4) have been described. A common resistance determinant found both on plasmids and chromosomes is widely distributed in nature and it probably resides on a transposon. DNA homology studies indicate that there is more than one type of clindamycin resistance in Bacteroides; a newly recognized clindamycin resistance determinant is transferable. Local outbreaks of clindamycin resistance have been noted in the United States and in Europe. The susceptibility of Bacteroides in the United States in 1983 from a multi-center study reveals a 5% incidence of resistance in B. fragilis and 1% in Bacteroides species. The rate of clindamycin resistance has remained steady over the past three years in the Bacteroides fragilis group.19846598519
5227120.9887Mutation at the position 2058 of the 23S rRNA as a cause of macrolide resistance in Streptococcus pyogenes. BACKGROUND: In streptococci, three macrolide resistance determinants (erm(B), erm(TR) and mef(A)) have been found. In addition, certain mutations at the ribosomal 23S RNA can cause resistance to macrolides. Mutation at the position 2058 of the 23S rRNA of the Streptococcus pyogenes as a cause of macrolide resistance has not been described before. METHODS: Antibiotic resistance determinations for the clinical S. pyogenes strain ni4277 were done using the agar dilution technique. Macrolide resistance mechanisms were studied by PCR and sequencing. All six rRNA operons were amplified using operon-specific PCR. The PCR products were partially sequenced in order to resolve the sequences of different 23S rRNA genes. RESULTS: One clinical isolate of S. pyogenes carrying an adenine to guanine mutation at the position 2058 of the 23S rRNA in five of the six possible rRNA genes but having no other known macrolide resistance determinants is described. The strain was highly resistant to macrolides and azalides, having erythromycin and azithromycin MICs > 256 microgram/ml. It was resistant to lincosamides (clindamycin MIC 16 microgram/ml) and also MIC values for ketolides were clearly elevated. The MIC for telithromycin was 16 microgram/ml. CONCLUSION: In this clinical S. pyogenes strain, a mutation at the position 2058 was detected. No other macrolide resistance-causing determinants were detected. This mutation is known to cause macrolide resistance in other bacteria. We can conclude that this mutation was the most probable cause of macrolide, lincosamide and ketolide resistance in this strain.200415128458
6181130.9887Two distinct major facilitator superfamily drug efflux pumps mediate chloramphenicol resistance in Streptomyces coelicolor. Chloramphenicol, florfenicol, and thiamphenicol are used as antibacterial drugs in clinical and veterinary medicine. Two efflux pumps of the major facilitator superfamily encoded by the cmlR1 and cmlR2 genes mediate resistance to these antibiotics in Streptomyces coelicolor, a close relative of Mycobacterium tuberculosis. The transcription of both genes was observed by reverse transcription-PCR. Disruption of cmlR1 decreased the chloramphenicol MIC 1.6-fold, while disruption of cmlR2 lowered the MIC 16-fold. The chloramphenicol MIC of wild-type S. coelicolor decreased fourfold and eightfold in the presence of reserpine and Phe-Arg-beta-naphthylamide, respectively. These compounds are known to potentiate the activity of some antibacterial drugs via efflux pump inhibition. While reserpine is known to potentiate drug activity against gram-positive bacteria, this is the first time that Phe-Arg-beta-naphthylamide has been shown to potentiate drug activity against a gram-positive bacterium.200919687245
3567140.9887Cloning and sequence analysis of ermQ, the predominant macrolide-lincosamide-streptogramin B resistance gene in Clostridium perfringens. The erythromycin resistance determinant from Clostridium perfringens JIR100 has been cloned, sequenced, and shown to be expressed in Escherichia coli. An open reading frame with sequence similarity to erm genes from other bacteria was identified and designated the ermQ gene. On the basis of comparative sequence analysis, it was concluded that the ermQ gene represented a new Erm hybridization class, designated ErmQ. Genes belonging to the ErmQ class were found to be widespread in C. perfringens, since 30 of 38 macrolide-lincosamide-streptogramin B-resistant C. perfringens strains, from diverse sources, hybridized to an ermQ-specific gene probe. The ermQ gene therefore represents the most common erythromycin resistance determinant in C. perfringens.19948067735
491150.9886Class II broad-spectrum mercury resistance transposons in Gram-positive bacteria from natural environments. We have studied the mechanisms of the horizontal dissemination of a broad-spectrum mercury resistance determinant among Bacillus and related species. This mer determinant was first described in Bacillus cereus RC607 from Boston Harbor, USA, and was then found in various Bacillus and related species in Japan, Russia and England. We have shown that the mer determinant can either be located at the chromosome, or on a plasmid in the Bacillus species, and is carried by class II mercury resistance transposons: Tn5084 from B. cereus RC607 and B. cereus VKM684 (ATCC10702) and Tn5085 from Exiguobacterium sp. TC38-2b. Tn5085 is identical in nucleotide sequence to TnMERI1, the only other known mer transposon from Bacillus species, but it does not contain an intron like TnMERI1. Tn5085 is functionally active in Escherichia coli. Tn5083, which we have isolated from B. megaterium MK64-1, contains an RC607-like mer determinant, that has lost some mercury resistance genes and possesses a merA gene which is a novel sequence variant that has not been previously described. Tn5083 and Tn5084 are recombinants, and are comprised of fragments from several transposons including Tn5085, and a relative of a putative transposon from B. firmus (which contains similar genes to the cadmium resistance operon of Staphylococcus aureus), as well as others. The sequence data showed evidence for recombination both between transposition genes and between mer determinants.200111446519
360160.9886Broad host range cloning vectors for gram-negative bacteria. A series of cloning vectors has been constructed based on the broad-host-range plasmid R300B. One of these vectors, pGSS33, has a size of 13.4 kb and carries four antibiotic resistance genes [ampicillin (Apr), chloramphenicol (Cmr), streptomycin (Smr) and tetracycline (Tcr)], all of which have restriction sites for insertional inactivation. The derivation, structure and uses of the plasmids are described.19846092235
3049170.9886Characterisation of plasmids purified from Acetobacter pasteurianus 2374. Four cryptic plasmids pAP1, pAP2, pAP3, and pAP4 with their replication regions AP were isolated from Gram-negative bacteria Acetobacter pasteurianus 2374 and characterised by sequence analyses. All plasmids were carrying the kanamycin resistance gene. Three of four plasmids pAP2, pAP3, and pAP4 encode an enzyme that confers ampicillin resistance to host cells. Moreover, the tetracycline resistance gene was identified only in pAP2 plasmid. All plasmids are capable to coexist with each other in Acetobacter cells. On the other hand, the coexistence of more than one plasmid is excluded in Escherichia coli. The nucleotide sequence of replication regions showed significant homology. The nucleotide and protein sequence analyses of resistance genes of all plasmids were compared with transposons Tn3, Tn10, and Tn903 which revealed significant differences in the primary structure, however no functional changes of gene were obtained.200314511653
456180.9885Cloning 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
418190.9885Plasmid-mediated mechanisms of resistance to aminoglycoside-aminocyclitol antibiotics and to chloramphenicol in group D streptococci. Genes conferring resistance to aminoglycoside-aminocyclitol antibiotics in three group D streptococcal strains, Streptococcus faecalis JH1 and JH6 and S. faecium JH7, and to chloramphenicol in JH6 are carried by plasmids that can transfer to other S. faecalis cells. The aminoglycoside resistance is mediated by constitutively synthesized phosphotransferase enzymes that have substrate profiles very similar to those of aminoglycoside phosphotransferases found in gram-negative bacteria. Phosphorylation probably occurs at the aminoglycoside 3'-hydroxyl group. Plasmid-borne streptomycin resistance is due to production of the enzyme streptomycin adenylyltransferase, which, as in staphylococci and in contrast to that detected in gram-negative bacteria, is less effective against spectinomycin as substrate. Resistance to chloramphenicol is by enzymatic acetylation. The chloramphenicol acetyltransferase is inducible and bears a close resemblance to the type D chloramphenicol acetyltransferase variant from staphylococci.197896732