scholarly journals Genetic basis for clarithromycin resistance among isolates of Mycobacterium chelonae and Mycobacterium abscessus.

1996 ◽  
Vol 40 (7) ◽  
pp. 1676-1681 ◽  
Author(s):  
R J Wallace ◽  
A Meier ◽  
B A Brown ◽  
Y Zhang ◽  
P Sander ◽  
...  
Keyword(s):  
Point Mutation ◽  
Random Amplified Polymorphic Dna ◽  
Mycobacterium Abscessus ◽  
Single Step ◽  
23S Rrna ◽  
Mycobacterium Chelonae ◽  
Clarithromycin Resistance ◽  
Gene Coding ◽  
Disseminated Disease ◽  
Dna Pcr

Resistance to clarithromycin among isolates of Mycobacterium chelonae and M. abscessus was observed in 18 of 800 (2.3%) patients tested between 1990 and 1995. Patients whose isolates were resistant had either disseminated disease or chronic lung disease, and the resistant isolates were recovered after clarithromycin monotherapy. Sequencing of the gene coding for the 23S rRNA peptidyltransferase region revealed a point mutation involving adenine at position 2058 (38%) or adenine at position 2059 (62%) in 20 of 20 relapse isolates from the first 13 patients identified. By pulsed-field gel electrophoresis or random amplified polymorphic DNA PCR, initial and relapse isolates were shown to have identical DNA patterns. M. chelonae and M. abscessus isolates were found to have only a single chromosomal copy of the rRNA operon, thus making them susceptible to single-step mutations. Thus, clarithromycin resistance in these species of rapidly growing mycobacteria relates to a point mutation in the gene coding for 23S rRNA and occurs in limited clinical situations, but was identified in almost 5% of isolates tested in 1995.

scholarly journals Assessment of Clarithromycin Susceptibility in Strains Belonging to theMycobacterium abscessusGroup byerm(41) andrrlSequencing

2010 ◽  
Vol 55 (2) ◽  
pp. 775-781 ◽  
Author(s):  
Sylvaine Bastian ◽  
Nicolas Veziris ◽  
Anne-Laure Roux ◽  
Florence Brossier ◽  
Jean-Louis Gaillard ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Previous Treatment ◽  
Sensu Stricto ◽  
Mycobacterium Abscessus ◽  
23S Rrna ◽  
Rrna Gene ◽  
Drug Of Choice ◽  
Clarithromycin Resistance ◽  
Mycobacterium Bolletii ◽  
Species Specific

ABSTRACTClarithromycin was the drug of choice forMycobacterium abscessusinfections until inducible resistance due toerm(41) was described. BecauseM. abscessuswas split intoM. abscessussensu stricto,Mycobacterium massiliense, andMycobacterium bolletii, we looked forerm(41) in the three species and determined their clarithromycin susceptibility levels. Ninety strains were included: 87 clinical strains from cystic fibrosis patients (61%) and others (39%), representing 43M. abscessus, 30M. massiliense, and 14M. bolletiistrains identified on a molecular basis, and 3 reference strains. Clarithromycin and azithromycin MICs were determined by broth microdilution and Etest with a 14-day incubation period. Mutations inrrl(23S rRNA gene) known to confer acquired clarithromycin resistance were also sought.erm(41) was detected in all strains but with two deletions in allM. massiliensestrains. These strains were indeed susceptible to clarithromycin (MIC90of 1 μg/ml) except for four strains withrrlmutations.M. abscessusstrains harbored an intacterm(41) but had a T/C polymorphism at the 28th nucleotide: T28 strains (Trp10 codon) demonstrated inducible clarithromycin resistance (MIC90of >16 μg/ml), while C28 strains (Arg10) were susceptible (MIC90of 2 μg/ml) except for two strains withrrlmutations.M. bolletiistrains haderm(41) sequences similar to the sequence of the T28M. abscessusgroup, associated with inducible clarithromycin resistance (MIC90of >16 μg/ml).erm(41) sequences appeared species specific within theM. abscessusgroup and were fully concordant with clarithromycin susceptibility whenerm(41) sequencing was associated with detection ofrrlmutations. Clarithromycin-resistant strains, including the sixrrlmutants, were more often isolated in cystic fibrosis patients, but this was not significantly associated with a previous treatment.

scholarly journals A Real-Time PCR Assay for Rapid Identification of Inducible and Acquired Clarithromycin Resistance in Mycobacterium abscessus

2020 ◽  
Author(s):  
Meenu Kaushal Sharma ◽  
Yanni La ◽  
Debra Janella ◽  
Hafid Soualhine
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Acquired Resistance ◽  
Rapid Identification ◽  
Mycobacterium Abscessus ◽  
23S Rrna ◽  
Pcr Assay ◽  
Constitutive Resistance ◽  
Clarithromycin Resistance ◽  
Severe Infections

Abstract Background: Mycobacterium abscessus is a rapidly growing mycobacteria involved in severe infections of the lung, skin, or soft tissue. Macrolides such as clarithromycin are the recommended first line drugs for treatment of M. abscessus infections. However, M. abscessus has dual mechanisms of resistance to macrolides, making treatment by macrolides difficult. A functional erm(41) gene confers for inducible resistance while acquired mutations on the 23S rRNA rrl gene confer for constitutive resistance.Methods: We have developed a real-time PCR assay to detect both inducible and acquired resistance to clarithromycin, and compared the results to traditional erm(41) and rrl sequencing and phenotypic susceptibility testing using Sensititre™ plates. Results: Of the total 126 M. abscessus isolates tested, truncated erm(41) was found in 23/126 (18.3%) of the samples, 27/126 (21.4%) had a T28C mutation in erm(41), and 2/126 (1.6%) had an acquired A2058C mutation in rrl. The phenotypic results correlated with the expected sequencing results in 121/126 samples (96%). Phenotypic testing compared to real-time PCR resolved 2 of these discrepancies by showing the existence of both erm(41) alleles in the isolates that sequencing missed. One culture was found to be mixed with two M. abscessus subsp. as per hsp65 sequencing and 2 isolates had discordance between molecular and phenotypic results. It was presumed that 3 isolates showed discrepancy between sequencing and real-time PCR, but one culture was mixed and other 2 detected both alleles by real-time PCR leading to 100% concordance when compared to sequencing.Conclusion: In conclusion, real-time PCR is more accurate for detection of both acquired and induced clarithromycin resistance, specifically when mixed genic profiles are present in a sample.

scholarly journals A real-time PCR assay for rapid identification of inducible and acquired clarithromycin resistance in Mycobacterium abscessus

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Meenu Kaushal Sharma ◽  
Yanni La ◽  
Debra Janella ◽  
Hafid Soualhine
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Acquired Resistance ◽  
Rapid Identification ◽  
Mycobacterium Abscessus ◽  
23S Rrna ◽  
Pcr Assay ◽  
Constitutive Resistance ◽  
Clarithromycin Resistance ◽  
Severe Infections

Abstract Background Mycobacterium abscessus is a rapidly growing mycobacteria involved in severe infections of the lung, skin, or soft tissue. Macrolides such as clarithromycin are the recommended first line drugs for treatment of M. abscessus infections. However, M. abscessus has dual mechanisms of resistance to macrolides, making treatment by macrolides difficult. A functional erm(41) gene confers for inducible resistance while acquired mutations on the 23S rRNA rrl gene confer for constitutive resistance. Methods We have developed a real-time PCR assay to detect both inducible and acquired resistance to clarithromycin, and compared the results to traditional erm(41) and rrl sequencing and phenotypic susceptibility testing using Sensititre™ plates. Results Of the total 126 M. abscessus isolates tested, truncated erm(41) was found in 23/126 (18.3%) of the samples, 27/126 (21.4%) had a T28C mutation in erm(41), and 2/126 (1.6%) had an acquired A2058C mutation in rrl. The phenotypic results correlated with the expected sequencing results in 121/126 samples (96%). Phenotypic testing compared to real-time PCR resolved 2 of these discrepancies by showing the existence of both erm(41) alleles in the isolates that sequencing missed. One culture was found to be mixed with two M. abscessus subsp. as per hsp65 sequencing and 2 isolates had discordance between molecular and phenotypic results. It was presumed that 3 isolates showed discrepancy between sequencing and real-time PCR, but one culture was mixed and other 2 detected both alleles by real-time PCR leading to 100% concordance when compared to sequencing. Conclusion In conclusion, real-time PCR is more accurate for detection of both acquired and induced clarithromycin resistance, specifically when mixed genic profiles are present in a sample.

scholarly journals Helicobacter pylori eradication according to sequencing-based 23S ribosomal RNA point mutation associated with clarithromycin resistance

2019 ◽  
Author(s):  
Seung In Seo ◽  
Byoung Joo ◽  
Jin Gu Kang ◽  
Hyoung Su Kim ◽  
Myoung Kuk Jang ◽  
...  
Keyword(s):  
Point Mutation ◽  
Ribosomal Rna ◽  
Point Mutations ◽  
23S Rrna ◽  
Rrna Gene ◽  
Clarithromycin Resistance ◽  
Clinically Significant ◽  
H Pylori ◽  
23S Ribosomal Rna ◽  
Significant Point

Abstract Background Clarithromycin resistance in Helicobacter pylori (H. pylori) is associated with point mutations in the 23S ribosomal RNA (rRNA) gene. A sequencing-based method can detect more point mutations than a polymerase chain reaction (PCR)-based method. We investigated the point mutations in the 23S rRNA genes of patients infected with clarithromycin-resistant H. pylori and compared the H. pylori eradication rates based on the identified clinically significant point mutations. Methods A total of 431 adult patients with H. pylori infection were retrospectively recruited in Kangdong Sacred Heart Hospital in 2017 and 2018. Patients who did not have point mutations related to clarithromycin resistance and/or had clinically insignificant point mutations were treated with PAC (proton pump inhibitor, amoxicillin, clarithromycin) for 7 days, while patients with clinically significant point mutations were treated with PAM (proton pump inhibitor, amoxicillin, metronidazole) for 7 days. H. pylori eradication rates were compared between the two groups. Results Sequencing-based detection of point mutations identified four mutations that were considered clinically significant (A2142G, A2142C, A2143G, A2143C), while all the other mutations were considered clinically insignificant. The clarithromycin resistance rate was 21.3% in the overall group of patients. A2143G was the most clinically significant point mutation (84/431, 19.5%), while T2182C was the most clinically insignificant point mutation (283/431, 65.7%). The H. pylori eradication rate in the overall group of patients was 83.7%, and the 7-day PAM-treated clarithromycin-resistance group showed a significantly lower eradication rate than the 7-day PAC-treated nonresistance group [ITT; 55.4% (51/92) vs. 74.3% (252/339), P=0.001, PP; 66.2% (51/77) vs. 88.4% (252/285), P=0.0001]. Conclusions There were significantly lower eradication rates in the patients with clarithromycin-resistant H. pylori as identified by the sequencing of point mutations in the 23S rRNA gene when treated with PAM for 7 days. A future study comparing treatment regimens in clarithromycin-resistant H. pylori-infected patients may be necessary.

scholarly journals Multiplex Sequence Analysis Demonstrates the Competitive Growth Advantage of the A-to-G Mutants of Clarithromycin-Resistant Helicobacter pylori

1999 ◽  
Vol 43 (3) ◽  
pp. 683-685 ◽  
Author(s):  
Ge Wang ◽  
M. Sayeedur Rahman ◽  
M. Zafri Humayun ◽  
Diane E. Taylor
Keyword(s):  
Helicobacter Pylori ◽  
Sequence Analysis ◽  
Point Mutation ◽  
Growth Rates ◽  
23S Rrna ◽  
Competitive Growth ◽  
Clarithromycin Resistance ◽  
Rational Explanation ◽  
Different Types ◽  
Multiplex Sequencing

ABSTRACT Clarithromycin resistance in Helicobacter pylori is due to point mutation within the 23S rRNA. We examined the growth rates of different types of site-directed mutants and demonstrated quantitatively the competitive growth advantage of A-to-G mutants over other types of mutants by a multiplex sequencing assay. The results provide a rational explanation of why A-to-G mutants are predominantly observed among clarithromycin-resistant clinical isolates.

scholarly journals New Real-Time PCR Assays for Detection of Inducible and Acquired Clarithromycin Resistance in the Mycobacterium abscessus Group

2015 ◽  
Vol 53 (11) ◽  
pp. 3430-3437 ◽  
Author(s):  
Shamira J. Shallom ◽  
Natalia S. Moura ◽  
Kenneth N. Olivier ◽  
Elizabeth P. Sampaio ◽  
Steven M. Holland ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Melting Curve ◽  
Mycobacterium Abscessus ◽  
23S Rrna ◽  
Melting Curve Analysis ◽  
Rrna Gene ◽  
Nucleotide Position ◽  
Inducible Resistance ◽  
Clarithromycin Resistance

Members of theMycobacterium abscessusgroup (MAG) cause lung, soft tissue, and disseminated infections. The oral macrolides clarithromycin and azithromycin are commonly used for treatment. MAG can display clarithromycin resistance through the inducibleerm(41) gene or via acquired mutations in therrl(23S rRNA) gene. Strains harboring a truncation or a T28C substitution inerm(41) lose the inducible resistance trait. Phenotypic detection of clarithromycin resistance requires extended incubation (14 days), highlighting the need for faster methods to detect resistance. Two real-time PCR-based assays were developed to assess inducible and acquired clarithromycin resistance and tested on a total of 90 clinical and reference strains. A SYBR green assay was designed to distinguish between a full-length and truncatederm(41) gene by temperature shift in melting curve analysis. Single nucleotide polymorphism (SNP) allele discrimination assays were developed to distinguish T or C at position 28 oferm(41) and 23S rRNArrlgene mutations at position 2058 and/or 2059. Truncated and full-sizeerm(41) genes were detected in 21/90 and 69/90 strains, respectively, with 64/69 displaying T at nucleotide position 28 and 5/69 containing C at that position. Fifteen isolates showedrrlmutations conferring clarithromycin resistance, including A2058G (11 isolates), A2058C (3 isolates), and A2059G (1 isolate). Targeted sequencing and phenotypic assessment of resistance concurred with molecular assay results. Interestingly, we also noted cooccurring strains harboring an activeerm(41), inactiveerm(41), and/or acquired mutational resistance, as well as slowly growing MAG strains and also strains displaying an inducible resistance phenotype within 5 days, long before the recommended 14-day extended incubation.

scholarly journals A Real-Time PCR Assay for Rapid Identification of Inducible and Acquired Clarithromycin Resistance in Mycobacterium abscessus

2020 ◽  
Author(s):  
Meenu Kaushal Sharma ◽  
Yanni La ◽  
Debra Janella ◽  
Hafid Soualhine
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Acquired Resistance ◽  
Rapid Identification ◽  
Mycobacterium Abscessus ◽  
23S Rrna ◽  
Pcr Assay ◽  
Constitutive Resistance ◽  
Clarithromycin Resistance ◽  
Severe Infections

Abstract Background: Mycobacterium abscessus is a rapidly growing mycobacteria involved in severe infections of the lung, skin, or soft tissue. Macrolides such as clarithromycin are the recommended first line drugs for treatment of M. abscessus infections. However, M. abscessus has dual mechanisms of resistance to macrolides, making treatment by macrolides difficult. A functional erm(41) gene confers for inducible resistance while acquired mutations on the 23S rRNA rrl gene confer for constitutive resistance. Methods: We have developed a real-time PCR assay to detect both inducible and acquired resistance to clarithromycin, and compared the results to traditional erm(41) and rrl sequencing and phenotypic susceptibility testing using Sensititre™ plates. Results: Of the total 126 M. abscessus isolates tested, truncated erm(41) was found in 23/126 (18.3%) of the samples, 27/126 (21.4%) had a T28C mutation in erm(41), and 2/126 (1.6%) had an acquired A2058C mutation in rrl. The phenotypic results correlated with the expected sequencing results in 121/126 samples (96%). Phenotypic testing compared to real-time PCR resolved 2 of these discrepancies by showing the existence of both erm(41) alleles in the isolates that sequencing missed. One culture was found to be mixed with two M. abscessus subsp. as per hsp65 sequencing and 2 isolates had discordance between molecular and phenotypic results. It was presumed that 3 isolates showed discrepancy between sequencing and real-time PCR, but one culture was mixed and other 2 detected both alleles by real-time PCR leading to 100% concordance when compared to sequencing. Conclusion: In conclusion, real-time PCR is more accurate for detection of both acquired and induced clarithromycin resistance, specifically when mixed genic profiles are present in a sample.

scholarly journals A Real-Time PCR Assay for Rapid Identification of Inducible and Acquired Clarithromycin Resistance in Mycobacterium abscessus

2020 ◽  
Author(s):  
Meenu Kaushal Sharma ◽  
Yanni La ◽  
Debra Janella ◽  
Hafid Soualhine
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Acquired Resistance ◽  
Rapid Identification ◽  
Mycobacterium Abscessus ◽  
23S Rrna ◽  
Pcr Assay ◽  
Constitutive Resistance ◽  
Clarithromycin Resistance ◽  
Severe Infections

Abstract Abstract Background: Mycobacterium abscessus is a rapidly growing mycobacteria involved in severe infections of the lung, skin, or soft tissue. Macrolides such as clarithromycin are the recommended first line drugs for treatment of M. abscessus infections. However, M. abscessus has dual mechanisms of resistance to macrolides, making treatment by macrolides difficult. A functional erm(41) gene confers for inducible resistance while acquired mutations on the 23S rRNA rrl gene confer for constitutive resistance. Methods: We have developed a real-time PCR assay to detect both inducible and acquired resistance to clarithromycin, and compared the results to traditional erm(41) and rrl sequencing and phenotypic susceptibility testing using Sensititre™ plates. Results: Of the total 126 M. abscessus isolates tested, truncated erm(41) was found in 23/126 (18.3%) of the samples, 27/126 (21.4%) had a T28C mutation in erm(41), and 2/126 (1.6%) had an acquired A2058C mutation in rrl. The phenotypic results correlated with the expected sequencing results in 121/126 samples (96%). Phenotypic testing compared to real-time PCR resolved 2 of these discrepancies by showing the existence of both erm(41) alleles in the isolates that sequencing missed. One culture was found to be mixed with two M. abscessus subsp. as per hsp65 sequencing and 2 isolates had discordance between molecular and phenotypic results. It was presumed that 3 isolates showed discrepancy between sequencing and real-time PCR, but one culture was mixed and other 2 detected both alleles by real-time PCR leading to 100% concordance when compared to sequencing. Conclusion: In conclusion, real-time PCR is more accurate for detection of both acquired and induced clarithromycin resistance, specifically when mixed genic profiles are present in a sample.

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