Metabolic and Transcriptional Profiling of Fraxinus chinensis var. rhynchophylla Unravels Possible Constitutive Resistance against Agrilus planipennis

The emerald ash borer (EAB, Agrilus planipennis), an ash-tree wood-boring beetle, has caused widespread mortality of ash. Asian ash, which coevolved with EAB, is considered more resistant than its North American and European congeners. Although some compounds and proteins related to resistance to EAB have been identified, the underlying ash resistance mechanism to EAB still needs further study. The Asian ash species, Fraxinus chinensis var. rhynchophylla, is highly resistant to EAB. In this study, metabolic and transcriptional profiling of the phloem of this species was investigated, and differentially expressed metabolites and genes were analyzed by comparing them with those of the susceptible F. pennsylvanica. Four hundred and twenty-eight metabolites were detected in both species, and several coumarins and lignans, which were exclusive to F. chinensis var. rhynchophylla, were identified. Compared with susceptible F. pennsylvanica, genes related to phenylpropanoid biosynthesis, ethylene (ET), and jasmonic acid (JA) biosynthesis and signaling in F. chinensis var. rhynchophylla were found to be up-regulated. It was hypothesized that coumarins, lignans, and ET and JA signaling might contribute to greater resistance to EAB in F. chinensis var. rhynchophylla. This study suggests candidate metabolites and genes for biomarker development in future ash-breeding programs.

Detection of Inducible Resistance to Clindamycin among Methicillin Resistant and Sensitive strains of Staphylococcus aureus from India

The resistance to MLSB antibiotics, i.e. Macrolide-Lincosamide-Streptogramin B (MLSB), is an increasing problem among Methicillin-resistant Staphylococci. The resistance to macrolides can be by efflux mechanism or via inducible or constitutive resistance. Unfortunately, routine clindamycin susceptibility testing fails to detect the inducible resistance, which commonly results in treatment failure and necessitates incorporating a simple D-test to detect such resistance. A retrospective observational study was performed on S. aureus isolates from patients. The strains were subjected to antibiotic susceptibility testing followed by detection of mecA gene by a polymerase chain reaction and, the ‘D-test’ was performed to know the inducible resistance to clindamycin. A total of 235 isolates were identified as S. aureus. Antibiotic susceptibility test indicated 190 MRSA and 45 are sensitive to MLSB (MS). Inducible clindamycin resistance was found among 48 (20.4%) isolates and constitutive resistance in 104 (44.2%). MRSA strains had higher inducible and constitutive resistance than MSSA strains (22.1%, 51.6% and 13.3%, 13.3%, respectively). Clindamycin is a commonly used antibiotic in patients with MRSA infections to spare higher-end anti-MRSA antibiotics like linezolid and vancomycin. To detect inducible clindamycin to avoid treatment failures; the study showed the importance of incorporating the D-test in routine testing.

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

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.

INDUCTION OF CLINDAMYCIN RESISTANCE IN CLINICAL ISOLATES OF STAPHYLOCOCCUS AUREUS FROM A TERTIARY CARE HOSPITAL.

Nowadays in Staphylococcus aureus isolates resistant to lincosamide, macrolide and streptogramin B (MLSB) group of antibiotics are expanded. Therefore, clindamycin is preferred drug for the treatment of infections caused by S. aureus, but due to change in sensitivity patterns of clindamycin it is leading to treatment failure. The three resistance phenotypes of MLSB antibiotics are iMLSB (inducible resistance) and cMLSB (constitutive resistance) that are resistant to macrolides, lincosamides and streptogrammins B antibiotics, whereas MS resistance that is sole resistant to macrolides and streptogramins B antibiotics. Erythromycin ribosome methylase (erm) genes are responsible for expressing inducible clindamycin resistance among S. aureus. In the present investigation, a Double disc approximation/Disc induction test (D-test) and PCR were used. Out of 428 strains the prevalence of iMLSB, cMLSB and MS phenotypes were 36 (8.41%), 47 (10.98%) and 48(11.21%) respectively. It is concluded that D-test should be routinely done to avoid treatment failure due to clindamycin resistance. In addition, PCR is a simple, quick, reliable and sensitive method that could also be used in the detection of inducible clindamycin resistance. The reason for the lower prevalence of iMLSB phenotype in the present study could be due to the reason that samples included in this study were mostly from the rural areas as the exposure of antimicrobial agents is less. Keywords: Clindamycin resistance, D-test, ermA, ermC, iMLSB, S. aureus

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

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.

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

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.

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

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.

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

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.

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

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 subspecies 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.