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Jin Wang - One of the best experts on this subject based on the ideXlab platform.
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Deciphering the late steps of Rifamycin biosynthesis.
Nature Communications, 2018Co-Authors: Chao Lei, Jin Wang, Xingwang Zhang, Wei Zhang, Zhen Fan, Gong-li Tang, Youli XiaoAbstract:Rifamycin-derived drugs, including rifampin, rifabutin, rifapentine, and rifaximin, have long been used as first-line therapies for the treatment of tuberculosis and other deadly infections. However, the late steps leading to the biosynthesis of the industrially important Rifamycin SV and B remain largely unknown. Here, we characterize a network of reactions underlying the biosynthesis of Rifamycin SV, S, L, O, and B. The two-subunit transketolase Rif15 and the cytochrome P450 enzyme Rif16 are found to mediate, respectively, a unique C–O bond formation in Rifamycin L and an atypical P450 ester-to-ether transformation from Rifamycin L to B. Both reactions showcase interesting chemistries for these two widespread and well-studied enzyme families. The enzymes Rif15 and Rif16 are involved in the late steps of the biosynthesis of Rifamycins, a group of antibiotics. Here, the authors characterized these two proteins and found that they catalyse unusual biochemical reactions.
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a feedback regulatory model for rifq mediated repression of Rifamycin export in amycolatopsis mediterranei
Microbial Cell Factories, 2018Co-Authors: Jingzhi Wang, Guoping Zhao, Jin WangAbstract:Due to the important role of Rifamycin in curing tuberculosis infection, the study on Rifamycin has never been stopped. Although RifZ, which locates within the Rifamycin biosynthetic cluster, has recently been characterized as a pathway-specific regulator for Rifamycin biosynthesis, little is known about the regulation of Rifamycin export. In this work, we proved that the expression of the Rifamycin efflux pump (RifP) was regulated by RifQ, a TetR-family transcriptional regulator. Deletion of rifQ had little impact on bacterial growth, but resulted in improved Rifamycin production, which was consistent with the reverse transcription PCR results that RifQ negatively regulated rifP’s transcription. With electrophoretic mobility shift assay and DNase I Footprinting assay, RifQ was found to directly bind to the promoter region of rifP, and a typical inverted repeat was identified within the RifQ-protected sequences. The transcription initiation site of rifP was further characterized and found to be upstream of the RifQ binding sites, well explaining the RifQ-mediated repression of rifP’s transcription in vivo. Moreover, Rifamycin B (the end product of Rifamycin biosynthesis) remarkably decreased the DNA binding affinity of RifQ, which led to derepression of Rifamycin export, reducing the intracellular concentration of Rifamycin B as well as its toxicity against the host. Here, we proved that the export of Rifamycin B was repressed by RifQ in Amycolatopsis mediterranei, and the RifQ-mediated repression could be specifically relieved by Rifamycin B, the end product of Rifamycin biosynthesis, based on which a feedback model was proposed for regulation of Rifamycin export. With the findings here, one could improve the antibiotic yield by simply inactivating the negative regulator of the antibiotic transporter.
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MOESM8 of A feedback regulatory model for RifQ-mediated repression of Rifamycin export in Amycolatopsis mediterranei
2018Co-Authors: Chao Lei, Jingzhi Wang, Guoping Zhao, Yuanyuan Liu, Xinqiang Liu, Jin WangAbstract:Additional file 8: Figure S8. Effect of Rifamycins to the transcription of rifP. Rifamycin SV and Rifamycin B were added into the LYZL11 culture medium, respectively, and the rifP transcriptional level was measured at 24Â h after the addition of Rifamycins. DMSO was used as a blank control and rpoB was used as an internal control
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A feedback regulatory model for RifQ-mediated repression of Rifamycin export in Amycolatopsis mediterranei
BMC, 2018Co-Authors: Chao Lei, Jingzhi Wang, Guoping Zhao, Yuanyuan Liu, Xinqiang Liu, Jin WangAbstract:Abstract Background Due to the important role of Rifamycin in curing tuberculosis infection, the study on Rifamycin has never been stopped. Although RifZ, which locates within the Rifamycin biosynthetic cluster, has recently been characterized as a pathway-specific regulator for Rifamycin biosynthesis, little is known about the regulation of Rifamycin export. Results In this work, we proved that the expression of the Rifamycin efflux pump (RifP) was regulated by RifQ, a TetR-family transcriptional regulator. Deletion of rifQ had little impact on bacterial growth, but resulted in improved Rifamycin production, which was consistent with the reverse transcription PCR results that RifQ negatively regulated rifP’s transcription. With electrophoretic mobility shift assay and DNase I Footprinting assay, RifQ was found to directly bind to the promoter region of rifP, and a typical inverted repeat was identified within the RifQ-protected sequences. The transcription initiation site of rifP was further characterized and found to be upstream of the RifQ binding sites, well explaining the RifQ-mediated repression of rifP’s transcription in vivo. Moreover, Rifamycin B (the end product of Rifamycin biosynthesis) remarkably decreased the DNA binding affinity of RifQ, which led to derepression of Rifamycin export, reducing the intracellular concentration of Rifamycin B as well as its toxicity against the host. Conclusions Here, we proved that the export of Rifamycin B was repressed by RifQ in Amycolatopsis mediterranei, and the RifQ-mediated repression could be specifically relieved by Rifamycin B, the end product of Rifamycin biosynthesis, based on which a feedback model was proposed for regulation of Rifamycin export. With the findings here, one could improve the antibiotic yield by simply inactivating the negative regulator of the antibiotic transporter
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a preliminary study of the mechanism of nitrate stimulated remarkable increase of Rifamycin production in amycolatopsis mediterranei u32 by rna seq
Microbial Cell Factories, 2015Co-Authors: Zhi Hui Shao, Jian Xu, Guoping Zhao, Jin WangAbstract:Background Rifamycin is an important antibiotic for the treatment of infectious disease caused by Mycobacteria tuberculosis. It was found that in Amycolatopsis mediterranei U32, an industrial producer for Rifamycin SV, supplementation of nitrate into the medium remarkably stimulated the yield of Rifamycin SV. However, the molecular mechanism of this nitrate-mediated stimulation remains unknown.
Guoping Zhao - One of the best experts on this subject based on the ideXlab platform.
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a feedback regulatory model for rifq mediated repression of Rifamycin export in amycolatopsis mediterranei
Microbial Cell Factories, 2018Co-Authors: Jingzhi Wang, Guoping Zhao, Jin WangAbstract:Due to the important role of Rifamycin in curing tuberculosis infection, the study on Rifamycin has never been stopped. Although RifZ, which locates within the Rifamycin biosynthetic cluster, has recently been characterized as a pathway-specific regulator for Rifamycin biosynthesis, little is known about the regulation of Rifamycin export. In this work, we proved that the expression of the Rifamycin efflux pump (RifP) was regulated by RifQ, a TetR-family transcriptional regulator. Deletion of rifQ had little impact on bacterial growth, but resulted in improved Rifamycin production, which was consistent with the reverse transcription PCR results that RifQ negatively regulated rifP’s transcription. With electrophoretic mobility shift assay and DNase I Footprinting assay, RifQ was found to directly bind to the promoter region of rifP, and a typical inverted repeat was identified within the RifQ-protected sequences. The transcription initiation site of rifP was further characterized and found to be upstream of the RifQ binding sites, well explaining the RifQ-mediated repression of rifP’s transcription in vivo. Moreover, Rifamycin B (the end product of Rifamycin biosynthesis) remarkably decreased the DNA binding affinity of RifQ, which led to derepression of Rifamycin export, reducing the intracellular concentration of Rifamycin B as well as its toxicity against the host. Here, we proved that the export of Rifamycin B was repressed by RifQ in Amycolatopsis mediterranei, and the RifQ-mediated repression could be specifically relieved by Rifamycin B, the end product of Rifamycin biosynthesis, based on which a feedback model was proposed for regulation of Rifamycin export. With the findings here, one could improve the antibiotic yield by simply inactivating the negative regulator of the antibiotic transporter.
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MOESM8 of A feedback regulatory model for RifQ-mediated repression of Rifamycin export in Amycolatopsis mediterranei
2018Co-Authors: Chao Lei, Jingzhi Wang, Guoping Zhao, Yuanyuan Liu, Xinqiang Liu, Jin WangAbstract:Additional file 8: Figure S8. Effect of Rifamycins to the transcription of rifP. Rifamycin SV and Rifamycin B were added into the LYZL11 culture medium, respectively, and the rifP transcriptional level was measured at 24Â h after the addition of Rifamycins. DMSO was used as a blank control and rpoB was used as an internal control
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A feedback regulatory model for RifQ-mediated repression of Rifamycin export in Amycolatopsis mediterranei
BMC, 2018Co-Authors: Chao Lei, Jingzhi Wang, Guoping Zhao, Yuanyuan Liu, Xinqiang Liu, Jin WangAbstract:Abstract Background Due to the important role of Rifamycin in curing tuberculosis infection, the study on Rifamycin has never been stopped. Although RifZ, which locates within the Rifamycin biosynthetic cluster, has recently been characterized as a pathway-specific regulator for Rifamycin biosynthesis, little is known about the regulation of Rifamycin export. Results In this work, we proved that the expression of the Rifamycin efflux pump (RifP) was regulated by RifQ, a TetR-family transcriptional regulator. Deletion of rifQ had little impact on bacterial growth, but resulted in improved Rifamycin production, which was consistent with the reverse transcription PCR results that RifQ negatively regulated rifP’s transcription. With electrophoretic mobility shift assay and DNase I Footprinting assay, RifQ was found to directly bind to the promoter region of rifP, and a typical inverted repeat was identified within the RifQ-protected sequences. The transcription initiation site of rifP was further characterized and found to be upstream of the RifQ binding sites, well explaining the RifQ-mediated repression of rifP’s transcription in vivo. Moreover, Rifamycin B (the end product of Rifamycin biosynthesis) remarkably decreased the DNA binding affinity of RifQ, which led to derepression of Rifamycin export, reducing the intracellular concentration of Rifamycin B as well as its toxicity against the host. Conclusions Here, we proved that the export of Rifamycin B was repressed by RifQ in Amycolatopsis mediterranei, and the RifQ-mediated repression could be specifically relieved by Rifamycin B, the end product of Rifamycin biosynthesis, based on which a feedback model was proposed for regulation of Rifamycin export. With the findings here, one could improve the antibiotic yield by simply inactivating the negative regulator of the antibiotic transporter
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a preliminary study of the mechanism of nitrate stimulated remarkable increase of Rifamycin production in amycolatopsis mediterranei u32 by rna seq
Microbial Cell Factories, 2015Co-Authors: Zhi Hui Shao, Jian Xu, Guoping Zhao, Jin WangAbstract:Background Rifamycin is an important antibiotic for the treatment of infectious disease caused by Mycobacteria tuberculosis. It was found that in Amycolatopsis mediterranei U32, an industrial producer for Rifamycin SV, supplementation of nitrate into the medium remarkably stimulated the yield of Rifamycin SV. However, the molecular mechanism of this nitrate-mediated stimulation remains unknown.
John A Fuerst - One of the best experts on this subject based on the ideXlab platform.
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lc ms based metabolomics study of marine bacterial secondary metabolite and antibiotic production in salinispora arenicola
Marine Drugs, 2015Co-Authors: Utpal Bose, Amitha K Hewavitharana, John A Fuerst, Yi Kai Ng, P N Shaw, Mark P HodsonAbstract:An LC-MS-based metabolomics approach was used to characterise the variation in secondary metabolite production due to changes in the salt content of the growth media as well as across different growth periods (incubation times). We used metabolomics as a tool to investigate the production of Rifamycins (antibiotics) and other secondary metabolites in the obligate marine actinobacterial species Salinispora arenicola, isolated from Great Barrier Reef (GBR) sponges, at two defined salt concentrations and over three different incubation periods. The results indicated that a 14 day incubation period is optimal for the maximum production of Rifamycin B, whereas Rifamycin S and W achieve their maximum concentration at 29 days. A “chemical profile” link between the days of incubation and the salt concentration of the growth medium was shown to exist and reliably represents a critical point for selection of growth medium and harvest time.
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diversity of mycobacterium species from marine sponges and their sensitivity to antagonism by sponge derived Rifamycin synthesizing actinobacterium in the genus salinispora
Fems Microbiology Letters, 2010Co-Authors: Hiroshi Izumi, Amitha K Hewavitharana, Nicholas P Shaw, Marie Gauthier, Bernard M Degnan, John A FuerstAbstract:Eleven isolates of Mycobacterium species as well as an antimycobacterial Salinispora arenicola strain were cultured from the sponge Amphimedon queenslandica. The 16S rRNA, rpoB, and hsp65 genes from these Mycobacterium isolates were sequenced, and phylogenetic analysis of a concatenated alignment showed the formation of a large clade with Mycobacterium poriferae isolated previously from another sponge species. The separation of these Mycobacterium isolates into three species-level groups was evident from sequence similarity and phylogenetic analyses. In addition, an isolate that is phylogenetically related to Mycobacterium tuberculosis was recovered from the sponge Fascaplysinopsis sp. Several different mycobacteria thus appear to co-occur in the same sponge. An actinobacterium closely related to S. arenicola, a known producer of the antimycobacterial Rifamycins, was coisolated from the same A. queenslandica specimen from which mycobacteria had been isolated. This Salinispora isolate was confirmed to synthesize Rifamycin and displayed inhibitory effects against representatives from two of three Mycobacterium phylotype groups. Evidence for antagonism of sponge-derived Salinispora against sponge-derived Mycobacterium strains from the same sponge specimen and the production of antimycobacterial antibiotics by this Salinispora strain suggest that the synthesis of such antibiotics may have functions in competition between sponge microbial community members.
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Screening of Rifamycin producing marine sponge bacteria by LC-MS-MS.
Journal of Chromatography B, 2007Co-Authors: Amitha K Hewavitharana, P. Nicholas Shaw, Tae Kyung Kim, John A FuerstAbstract:HPLC-MS-MS has been used for the identification and characterisation of Rifamycin B and Rifamycin SV in various strains of the marine sponge-derived bacterium Salinispora. Gradient elution using acetonitrile/water/ammonium acetate was used to separate the Rifamycins from the matrix and negative ion-electrospray mass spectrometry was used for detection and confirmation. The presence of Rifamycin in bacterial extracts was confirmed by matching retention times, parent ion spectra and the fragmentated parent ion spectra of the standard compounds and the bacterial extracts. All strains of the marine sponge bacterium Salinispora tested were found to contain Rifamycin thus an alternate actinobacterial source of Rifamycin was established.
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discovery of a new source of Rifamycin antibiotics in marine sponge actinobacteria by phylogenetic prediction
Applied and Environmental Microbiology, 2006Co-Authors: Amitha K Hewavitharana, Nicholas P Shaw, John A FuerstAbstract:Phylogenetic analysis of the ketosynthase (KS) gene sequences of marine sponge-derived Salinispora strains of actinobacteria indicated that the polyketide synthase (PKS) gene sequence most closely related to that of Salinispora was the Rifamycin B synthase of Amycolatopsis mediterranei. This result was not expected from taxonomic species tree phylogenetics using 16S rRNA sequences. From the PKS sequence data generated from our sponge-derived Salinispora strains, we predicted that such strains might synthesize Rifamycin-like compounds. Liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis was applied to one sponge-derived Salinispora strain to test the hypothesis of Rifamycin synthesis. The analysis reported here demonstrates that this Salinispora isolate does produce compounds of the Rifamycin class, including Rifamycin B and Rifamycin SV. A Rifamycin-specific KS primer set was designed, and that primer set increased the number of Rifamycin-positive strains detected by PCR screening relative to the number detectable using a conserved KS-specific set. Thus, the Salinispora group of actinobacteria represents a potential new source of Rifamycins outside the genus Amycolatopsis and the first recorded source of Rifamycins from marine bacteria.
Christopher K Murphy - One of the best experts on this subject based on the ideXlab platform.
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efficacy of a novel Rifamycin derivative abi 0043 against staphylococcus aureus in an experimental model of foreign body infection
Antimicrobial Agents and Chemotherapy, 2007Co-Authors: Andrej Trampuz, David M. Rothstein, Christopher K Murphy, Andreas F Widmer, Regine Landmann, Werner ZimmerliAbstract:We compared the efficacy of a novel Rifamycin derivative, ABI-0043, with that of rifampin, alone and in combination with levofloxacin, against methicillin-susceptible Staphylococcus aureus ATCC 29213 in a guinea pig tissue-cage infection model. The MIC, logarithmic-growth-phase minimal bactericidal concentration, and stationary-growth-phase minimal bactericidal concentration of ABI-0043 were 0.001, 0.008, and 0.25 μg/ml, respectively; the corresponding concentrations of rifampin were 0.016, 0.8, and 3.6 μg/ml, respectively. After a single intraperitoneal dose of 12.5 mg/kg of body weight, the peak concentration in cage fluid was 1.13 μg/ml of ABI-0043 and 0.98 μg/ml of rifampin. Five days after completion of treatment, levofloxacin administered alone (5 mg/kg/12 h) resulted in bacterial counts in cage fluid that were similar to those for untreated controls (>8.0 log10 CFU/ml), whereas rifampin and ABI-0043 administered alone (12.5 mg/kg/12 h) decreased the mean titers of bacteria ± standard deviations to 1.43 ± 0.28 log10 and 1.57 ± 0.53 log10 CFU/ml, respectively, in cage fluid. In combination with levofloxacin, both Rifamycins cleared bacteria from the cage fluid. The cure rates of cage-associated infections with rifampin and ABI-0043 administered alone were 46% and 58%, respectively, and increased to 88% and 92% in combination with levofloxacin. Emergence of Rifamycin resistance was observed in 42% of cages after ABI-0043 therapy and in 38% of cages after rifampin therapy; no emergence of resistance occurred with combination treatment with levofloxacin. In conclusion, ABI-0043 had cure rates comparable to that of rifampin. ABI-0043 in combination with a quinolone has the potential for treatment of implant-associated infections caused by susceptible strains of S. aureus, potentially without drug-drug interactions.
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In Vitro Time-kill Activities of Rifalazil, Alone and in Combination with Vancomycin, against Logarithmic and Stationary Cultures of Staphylococcus aureus
The Journal of Antibiotics, 2006Co-Authors: Marcia S. Osburne, David M. Rothstein, Ronnie Farquhar, Christopher K MurphyAbstract:Rifalazil is a novel Rifamycin that, like other members of this class, inhibits bacterial transcription by targeting the β subunit of prokaryotic DNA-dependent RNA polymerase. To address the high-frequency resistance seen with Rifamycins, we assessed the ability of rifalazil, alone and in combination with vancomycin, to both kill cells and to suppress the appearance of resistant mutants in log and stationary phase Staphylococcus aureus cultures, using high cell densities in an in vitro kill curve model. We found that 1) rifalazil alone killed log-phase cultures more rapidly than rifampicin, but both drugs quickly selected for resistant mutants, 2) co-treatment of log phase cultures with rifalazil and vancomycin increased bacterial killing by about 3-Log_10 over either drug used alone and delayed the appearance of Rifamycin-resistant mutants, 3) rifalazil and vancomycin in combination killed stationary phase cultures by 3∼4 Log_10 by 48 hours.
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In vitro time-kill activities of rifalazil, alone and in combination with vancomycin, against logarithmic and stationary cultures of Staphylococcus aureus.
The Journal of Antibiotics, 2006Co-Authors: Marcia S. Osburne, David M. Rothstein, Ronnie Farquhar, Christopher K MurphyAbstract:Rifalazil is a novel Rifamycin that, like other members of this class, inhibits bacterial transcription by targeting the beta subunit of prokaryotic DNA-dependent RNA polymerase. To address the high-frequency resistance seen with Rifamycins, we assessed the ability of rifalazil, alone and in combination with vancomycin, to both kill cells and to suppress the appearance of resistant mutants in log and stationary phase Staphylococcus aureus cultures, using high cell densities in an in vitro kill curve model. We found that (1) rifalazil alone killed log-phase cultures more rapidly than rifampicin, but both drugs quickly selected for resistant mutants, (2) co-treatment of log phase cultures with rifalazil and vancomycin increased bacterial killing by about 3-Log10 over either drug used alone and delayed the appearance of Rifamycin-resistant mutants, (3) rifalazil and vancomycin in combination killed stationary phase cultures
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In Vitro Activity of Novel Rifamycins against Rifamycin-Resistant Staphylococcus aureus
Antimicrobial Agents and Chemotherapy, 2006Co-Authors: Christopher K Murphy, Michael H. Cynamon, Marcia S. Osburne, John Van Duzer, Steve Mullin, Jim Siedlecki, Kathy Kerstein, David M. RothsteinAbstract:We describe novel Rifamycin derivatives (new chemical entities [NCEs]) that retain significant activity against a comprehensive collection of Staphylococcus aureus strains that are resistant to Rifamycins. This collection of resistant strains contains 21 of the 26 known single-amino-acid alterations in RpoB, the target of Rifamycins. Some NCEs also demonstrated a lower frequency of resistance development than rifampin and rifalazil in S. aureus as measured in a resistance emergence test. When assayed for activity against the strongest Rifamycin-resistant mutants, several NCEs had MICs of 2 μg/ml, in contrast to MICs of rifampin and rifalazil, which were 512 μg/ml for the same strains. The properties of these NCEs therefore demonstrate a significant improvement over those of earlier Rifamycins, which have been limited primarily to combination therapy due to resistance development, and suggest a potential use of these NCEs for monotherapy in several clinical indications.
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Activity of Novel BenzoxazinoRifamycins against Rifamycin-Resistant Streptococcus pyogenes
Antimicrobial Agents and Chemotherapy, 2006Co-Authors: Steve Mullin, David M. Rothstein, Christopher K MurphyAbstract:Serious gram-positive infections are frequently caused by Staphylococcus aureus or Streptococcus pyogenes. The Rifamycin rifampin (RIF) is often used as part of the treatment for such infections, because it has potent bactericidal activity against both free-living and biofilm-associated gram-positive bacteria (13) and has favorable pharmacokinetics (2). Despite this, RIF's use as monotherapy has been limited because of treatment failure due to the emergence of Rifamycin-resistant organisms (3, 4, 7, 8). Resistance results from amino acid substitutions within the conserved Rifamycin resistance-determining region (RRDR) of the RpoB subunit of RNA polymerase, the target of Rifamycins. Because of resistance emergence, RIF is used mainly as part of a multidrug treatment regimen in which the partner drug(s) is able to prevent outgrowth of resistant subpopulations. A previous study described the activity of the benzoxazinoRifamycins rifalazil (RFZ) and related new chemical entities (NCEs) against RIF-resistant S. aureus (10). The maximum MICs of the most potent NCEs were 2 to 4 μg/ml against the most Rifamycin-resistant S. aureus strains. RIF and RFZ have MICs of >512 μg/ml against these same strains (10). Considering that a large proportion of serious gram-positive infections are caused by streptococci, we sought to determine in the present study whether NCEs had improved activity against Rifamycin-resistant strains of S. pyogenes. The two mutations conferring RIF resistance in clinical isolates of S. pyogenes (4, 7) and several mutations in other streptococci (1, 5, 6, 9, 12) all reside in the RRDR of RpoB in these organisms. S. pyogenes ATCC 19615 mutants with decreased susceptibility to RIF were selected for and characterized as described previously (10). The resistance frequencies of S. pyogenes for RIF were 7.0 × 10−8 (at 0.24 μg/ml), 6.0 × 10−8 (at 0.12 μg/ml), and 2.9 × 10−8 (at 0.06 μg/ml). Thirty-three colonies with decreased susceptibility to RIF were isolated, and oligonucleotide primers SPY-F and SPY-R (5′-AACCGTCGTATCCGTGCCGTTGGT-3′ and 5′-TGCCGTCGCAACAGCAACTACCTG-3′, respectively) were used to amplify and DNA sequence their RRDR regions, encoded by base pairs 1228 to 1884 (S. pyogenes MGAS315 coordinates; GenBank accession number NC 004070). Seventeen discrete amino acid changes at 10 different positions within the S. pyogenes RRDR were identified (Table (Table1).1). Changes at H486, R489, and S491 correspond to positions that, when altered, confer the high levels of resistance to Rifamycins in other bacteria (10). The H486N substitution was previously identified in a RIF-resistant clinical strain of S. pyogenes (7). TABLE 1. Susceptibility of S. pyogenes RIF-resistant mutants to RIF, RFZ, and select NCEs The MICs of RIF, RFZ, and NCEs for S. pyogenes ATCC 19615 and its Rifamycin-resistant derivatives were determined using standard methods (11). S. pyogenes ATCC 19615 was 16- to 32-fold more susceptible to RFZ and NCEs than was S. aureus ATCC 29213 (Table (Table1)1) (10). MICs of the most potent NCEs (e.g., ABI-0043) against Rifamycin-resistant S. pyogenes strains ranged from 0.00025 μg/ml to 0.25 μg/ml for the most resistant strain (Table (Table1)1) and 0.016 μg/ml to 4 μg/ml for the most Rifamycin-resistant S. aureus strains (10). In fact, the MICs of NCEs against S. pyogenes mutants (Table (Table1)1) were consistently ≥8-fold lower than those for S. aureus mutants containing mutations in the corresponding codon of the rpoB gene (10). It is anticipated that NCEs having in vivo efficacy against Rifamycin-resistant S. aureus strains would be effective against the most resistant S. pyogenes mutants. Experimental infection models using resistant strains from both of these species should verify this assumption.
Megan Murray - One of the best experts on this subject based on the ideXlab platform.
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rifampicin and rifabutin resistance in 1003 mycobacterium tuberculosis clinical isolates
Journal of Antimicrobial Chemotherapy, 2019Co-Authors: Maha R Farhat, Jaimie D Sixsmith, Roger Calderon, Nathan D Hicks, Sarah M Fortune, Megan MurrayAbstract:OBJECTIVES Drug-resistant TB remains a public health challenge. Rifamycins are among the most potent anti-TB drugs. They are known to target the RpoB subunit of RNA polymerase; however, our understanding of how Rifamycin resistance is genetically encoded remains incomplete. Here we investigated rpoB genetic diversity and cross-resistance between the two Rifamycin drugs rifampicin and rifabutin. METHODS We performed WGS of 1003 Mycobacterium tuberculosis clinical isolates and determined MICs of both Rifamycin agents on 7H10 agar using the indirect proportion method. We generated rpoB mutants in a laboratory strain and measured their antibiotic susceptibility using the alamarBlue reduction assay. RESULTS Of the 1003 isolates, 766 were rifampicin resistant and 210 (27%) of these were rifabutin susceptible; 102/210 isolates had the rpoB mutation D435V (Escherichia coli D516V). Isolates with discordant resistance were 17.2 times more likely to harbour a D435V mutation than those resistant to both agents (OR 17.2, 95% CI 10.5-27.9, P value <10-40). Compared with WT, the D435V in vitro mutant had an increased IC50 of both Rifamycins; however, in both cases to a lesser degree than the S450L (E. coli S531L) mutation. CONCLUSIONS The observation that the rpoB D435V mutation produces an increase in the IC50 of both drugs contrasts with findings from previous smaller studies that suggested that isolates with the D435V mutation remain rifabutin susceptible despite being rifampicin resistant. Our finding thus suggests that the recommended critical testing concentration for rifabutin should be revised.
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rifampicin and rifabutin resistance in 1000 mycobacterium tuberculosis clinical isolates
bioRxiv, 2018Co-Authors: Maha R Farhat, Jaimie D Sixsmith, Roger Calderon, Nathan D Hicks, Sarah M Fortune, Megan MurrayAbstract:Drug resistant tuberculosis (TB) remains a public health challenge with limited treatment options and high associated mortality. Rifamycins are among the most potent anti-TB drugs, and the loss of susceptibility to these agents, a hallmark of MDR TB, is considered a substantial therapeutic challenge. Rifamycins are known to target the RpoB subunit of RNA polymerase; however, our understanding of how Rifamycin resistance is genetically encoded remains incomplete. Here we investigated rpoB genetic diversity and cross resistance between the two Rifamycin drugs rifampicin (RIF) and rifabutin (RFB). We performed whole genome sequencing of 1005 MTB clinical isolates and measured minimum inhibitory concentration (MIC) to both agents on 7H10 agar using the indirect proportion method. Of the 1005 isolates, 767 were RIF resistant, and of these, 211 (27%) were sensitive to RFB at the critical concentration of 0.5ug/ml; 101/211 isolates had the rpoB mutation D435V (E.coli D516V). Isolates with discrepant resistance (RIF R and RFB S) 16.9 times more likely to harbor a D435V mutation as those resistant to both agents (OR 95% CI 10.5-27.9, P-value <10-40). To further understand this discrepancy, we generated both D435V and S450L (E.coli S531L) rpoB mutants in a laboratory strain and measured their antibiotic susceptibility using the alamar blue reduction assay. Compared with wildtype, D435V increased the 50% inhibitory concentration (IC50) to both RIF and RFB, however in both cases to a lesser degree than the S450L mutation. The observation that the rpoB D435V mutation produces an increase in the IC50 for both drugs contrasts with findings from previous smaller studies that suggested that isolates with D435V mutation remain RFB susceptible despite being RIF resistant. Our finding thus suggests that the recommended critical testing concentration for RFB should be revised.
