Genetic Manipulation of Mycobacterium abscessus

Medjahed, Halima; Singh, Anil Kumar

doi:10.1002/9780471729259.mc10d02s18

Cited by 27 publications

(25 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…RNA samples were extracted from mid-log-phase bacterial cultures according to the protocols recommended by Medjahed and Singh ( 34 ). cDNA was synthesized using the RT reagent kit with gDNA Eraser (TaKaRa, Shiga, Japan).…”

Section: Methodsmentioning

confidence: 99%

Determination of MIC Distribution and Mechanisms of Decreased Susceptibility to Bedaquiline among Clinical Isolates of Mycobacterium abscessus

Guo

et al. 2018

Antimicrob Agents Chemother

View full text Add to dashboard Cite

Chemotherapeutic options against Mycobacterium abscessus infections are very limited. Bedaquiline, a new antituberculosis (anti-TB) drug, is effective for the treatment of multidrug-resistant TB. However, few data are available on bedaquiline for treatment of M. abscessus infections. In this study, we determined the profile for in vitro susceptibility of M. abscessus clinical isolates to bedaquiline and investigated the potential molecular mechanisms of decreased susceptibility. A total of 197 M. abscessus clinical isolates were collected from sputum and bronchoalveolar fluid of patients with lung infections. Standard broth microdilution test revealed that bedaquiline exhibited high in vitro killing activity against M. abscessus isolates, with a MIC50 of 0.062 and a MIC90 of 0.125 mg/liter. Whole-genome sequencing data showed that no nonsynonymous mutation occurred in atpE, the gene encoding the bedaquiline-targeted protein. However, of 6 strains with decreased susceptibility of bedaquiline (MIC = 0.5 to 1 mg/liter), 3 strains had nonsynonymous mutations in mab_4384, the gene encoding the repressor of efflux pump MmpS5/MmpL5. Quantitative reverse transcription-PCR (qRT-PCR) analysis showed that the expression of MmpS5/MmpL5 in the group with decreased susceptibility to bedaquiline was significantly higher than in those with medium MICs (MIC = 0.125 to 0.5 mg/liter) or in the low-MIC group (MIC ≤ 0.062 mg/liter). Two isolates with increased MICs did not show overexpression of MmpS5/MmpL5, which could not be explained by known molecular mechanisms. This is the first report showing the association of MmpS5/MmpL5 with decreased bedaquiline susceptibility in M. abscessus clinical isolates and suggesting the presence of other, yet-to-be identified mechanisms for decreased bedaquiline susceptibility in M. abscessus.

show abstract

Section: Methodsmentioning

confidence: 99%

Determination of MIC Distribution and Mechanisms of Decreased Susceptibility to Bedaquiline among Clinical Isolates of Mycobacterium abscessus

Guo

et al. 2018

Antimicrob Agents Chemother

View full text Add to dashboard Cite

show abstract

“…Subcloning and Sequencing of Tn-Adjacent Genomic Regions. Genomic DNA was extracted from each mutant, ClaI restricted, and subcloned in pUC19 (Invitrogen, Thermo Fisher Scientific) (61). Genomic DNA was sequenced using a 5′ primer in the Tn (TTGACGAGTTCTTCTGA).…”

Section: Cell Infectionmentioning

confidence: 99%

“…ΔEccB 4 Mutant Construction and Complementation. Disruption of the eccB 4 gene was performed using the recombineering system as described previously (10,61). Complementation was performed after amplifying and cloning eccB 4 into the integrative plasmid pMVH361 as described (SI Appendix, Fig.…”

Section: Cell Infectionmentioning

confidence: 99%

Identification of genes required for Mycobacterium abscessus growth in vivo with a prominent role of the ESX-4 locus

Laencina

Dubois

Moigne

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

101

146

View full text Add to dashboard Cite

, a rapidly growing mycobacterium (RGM) and an opportunistic human pathogen, is responsible for a wide spectrum of clinical manifestations ranging from pulmonary to skin and soft tissue infections. This intracellular organism can resist the bactericidal defense mechanisms of amoebae and macrophages, an ability that has not been observed in other RGM. can up-regulate several virulence factors during transient infection of amoebae, thereby becoming more virulent in subsequent respiratory infections in mice. Here, we sought to identify the genes required for replication within amoebae. To this end, we constructed and screened a transposon () insertion library of an subspcies clinical isolate for attenuated clones. This approach identified five genes within the ESX-4 locus, which in encodes an ESX-4 type VII secretion system that exceptionally also includes the ESX conserved EccE component. To confirm the screening results and to get further insight into the contribution of ESX-4 to growth and survival in amoebae and macrophages, we generated a deletion mutant of that encodes a core structural element of ESX-4. This mutant was less efficient at blocking phagosomal acidification than its parental strain. Importantly, and in contrast to the wild-type strain, it also failed to damage phagosomes and showed reduced signs of phagosome-to-cytosol contact, as demonstrated by a combination of cellular and immunological assays. This study attributes an unexpected and genuine biological role to the underexplored mycobacterial ESX-4 system and its substrates.

show abstract

“…Relative expression of the genes was assessed by comparing the quantity of mRNA expressed by the organism cultured in the presence and absence of CLA using the same technical approach reported previously. 28 A culture incubated in the presence of half its MIC of CLA was shaken at 37°C for 3 h; the RNA was then extracted according to the protocols described by Medjahed et al 29 cDNA was synthesized using the HiScript III RT SuperMix with gDNA wiper (Vazyme Biotech Co., Ltd). qRT-PCR was performed using ChamQ Universal SYBR Master Mix (Vazyme Biotech Co., Ltd) on a QS6 Real-Time PCR System (Applied Biosystems, Carlsbad, CA).…”

Section: Rna Extraction and Quantitative Reverse Transcription Pcr (Qmentioning

confidence: 99%

Efflux Pumps Contribute to Intrinsic Clarithromycin Resistance in Clinical, Mycobacterium abscessus Isolates

Guo

Chen

Zhang

et al. 2020

IDR

View full text Add to dashboard Cite

The emergence of clarithromycin resistance is a challenge in treating Mycobacterium abscessus infections. Known mechanisms that contribute to intrinsic clarithromycin resistance focus on rrl gene-related mutations, but resistant clinical isolates often exhibit an inconsistent rrl genotype. Patients and Methods: In this study, 194 clinical Mycobacterium abscessus isolates were collected from patients with lung infections and the whole genome of each isolate was sequenced. A comprehensive examination of the molecular mechanisms underlying intrinsic clarithromycin resistance was performed, combining MIC determination, comparative genome sequence analysis and qRT-PCR. Results: Of the 194 isolates, 13 (6.7%) were clarithromycin resistant; only seven of these harbored a rrl 2270/2271 mutation. The remaining six resistant isolates did not exhibit a specific resistance-associated mutation in the clarithromycin target-site genes, rrl, rplC, rplD and rplV, or in the rrl modification gene erm(41). qRT-PCR analysis showed that the increased expression of the efflux pump genes, MAB_2355c, MAB_1409c and MAB_1846, as well as their positive regulatory gene whiB7, consistently correlated with increased clarithromycin resistance. The presence of efflux pump inhibitors significantly decreased the MIC of clarithromycin for nonsusceptible isolates, especially the intrinsic resistant isolates that exhibited no rrl 2270/2271 mutation. Conclusion: These findings indicate that efflux pumps play a prominent role in the intrinsic resistance of M. abscessus to clarithromycin, complementing other known resistance mechanisms.

show abstract

Genetic Manipulation of Mycobacterium abscessus

Cited by 27 publications

References 31 publications

Determination of MIC Distribution and Mechanisms of Decreased Susceptibility to Bedaquiline among Clinical Isolates of Mycobacterium abscessus

Determination of MIC Distribution and Mechanisms of Decreased Susceptibility to Bedaquiline among Clinical Isolates of Mycobacterium abscessus

Identification of genes required for Mycobacterium abscessus growth in vivo with a prominent role of the ESX-4 locus

<p>Efflux Pumps Contribute to Intrinsic Clarithromycin Resistance in Clinical, <em>Mycobacterium abscessus</em> Isolates</p>

Contact Info

Product

Resources

About