m abscessus  (ATCC)

Journal: mLife

Article Title: CRISPR‐based genome editing reveals the roles of efflux pumps in Mycobacterium abscessus

doi: 10.1002/mlf2.70048

Figure Lengend Snippet: ETH improves the transformation efficiency of Mycobacterium abscessus . (A, B) Susceptibility of the indicated M. abscessus to the drug ETH (A) and EMB (B). The MIC 50 was defined as the concentration of the compound that inhibited bacterial growth by 50% and was analyzed by a nonlinear fit model in GraphPad Prism 8.0. Data are expressed as the mean ± SD of triplicate samples and are representative of two independent experiments. (C) Transformation efficiency of M. abscessus when different concentrations of ETH or EMB were present in the culture medium used for competent cell preparation. ETH (10, 20, and 40 μg/ml) or EMB (200, 400, and 800 μg/ml) was added to the M. abscessus culture. The transformation efficiency was defined as the total number of CFU generated per transformation. Statistically significant differences were determined using multiple paired t ‐tests. EMB, ethambutol; ETH, ethionamide. * p < 0.05; ** p < 0.01; **** p < 0.0001.

Article Snippet: Initially, M. abscessus ATCC 19977 was recovered on 7H10 solid medium.

Techniques: Transformation Assay, Concentration Assay, Generated

Journal: mLife

Article Title: CRISPR‐based genome editing reveals the roles of efflux pumps in Mycobacterium abscessus

doi: 10.1002/mlf2.70048

Figure Lengend Snippet: Genome editing using CRISPR‐Cas9‐assisted recombineering in M. abscessus. (A) Schematic showing the lacZ ‐targeting sgRNA and oligonucleotides used for recombineering. The oligonucleotides lacZ .lag (79 nt) and lacZ .lead (79 nt, sequences reverse and complementary to lacZ .lag) targeting the lagging strand and the leading strand of DNA replication were designed to introduce a continuous stop codon into the lacZ open reading frame, respectively. (B) Number of CFU per transformation and percentage of white transformant colonies after electroporation of the pSG‐ lacZ ‐1 plasmids and oligonucleotides into M. abscessus ATCC 19977 harboring the recombinase‐expression plasmid. lacZ‐1 and lacZ‐2 denote sgRNAs targeting two different sites in the lacZ gene. The transformants were plated on X‐gal plates for blue‐white screening. The black bar corresponds to the left Y axis, indicating the number of transformants, and the gray bar corresponds to the right Y axis, indicating the percentage of white colonies. Results represent the average of two independent experiments. (C) Transformation and recombination efficiency of the dual‐reporter detection system resulting from electroporation of the shear plasmid expressing gfp and the oligonucleotides targeting lacZ . Recombination efficiency was defined as the percentage of white colonies on the X‐gal plate. GFP positivity was calculated based on the proportion of GFP‐positive colonies. lacZ‐1 and lacZ‐2 denote sgRNAs targeting two different sites in the lacZ gene. (D) Transformation efficiency and recombination efficiency of gene deletion using CRISPR‐Cas9‐assisted dsDNA recombineering. Deletions of 620 bp were introduced into M. abscessus chromosomal DNA using approximately 1 kb dsDNA fragments. X‐gal, 5‐bromo‐4‐chloro‐3‐indolyl β‐ d ‐galactopyranoside.

Article Snippet: Initially, M. abscessus ATCC 19977 was recovered on 7H10 solid medium.

Techniques: CRISPR, Introduce, Transformation Assay, Electroporation, Expressing, Plasmid Preparation, Shear

Journal: mLife

Article Title: CRISPR‐based genome editing reveals the roles of efflux pumps in Mycobacterium abscessus

doi: 10.1002/mlf2.70048

Figure Lengend Snippet: MAB_2806–2807 is required for antibiotic resistance of Mycobacterium abscessus. (A) Disk diffusion assay of the MAB_2806‐2807 operon to different antibiotics. The zone of inhibition for FOX, LZD, and CLR was measured for Δ MAB_2806 , Δ MAB_2807 , and Δ MAB_2806–2807 , as well as their single‐ or double‐gene complementary strains. Statistically significant differences were determined using a paired two‐tailed t ‐test. All p values were calculated by comparison with the wild type. ** p < 0.01; *** p < 0.001; and **** p < 0.0001. (B) Uptake of EtBr by wild type, Δ MAB_2806‐2807 , and the complementary strain. CLR, clarithromycin; EtBr, ethidium bromide; FOX, cefoxitin; LZD, linezolid.

Article Snippet: Initially, M. abscessus ATCC 19977 was recovered on 7H10 solid medium.

Techniques: Diffusion-based Assay, Inhibition, Two Tailed Test, Comparison

Journal: mLife

Article Title: CRISPR‐based genome editing reveals the roles of efflux pumps in Mycobacterium abscessus

doi: 10.1002/mlf2.70048

Figure Lengend Snippet: Screening of efflux pump genes affecting the survival of M. abscessus in Galleria mellonella larvae. (A) Screening of an M.abscessus efflux pump mutant library in G. mellonella larvae. Efflux pump mutants and the wild‐type ATCC 19977 strain of M. abscessus harboring barcodes were mixed and used to infect G. mellonella larvae. At 3 and 5 days postinfection, the larvae were homogenized and plated. The plated bacteria were collected, and the barcode sequences were amplified for next‐generation sequencing analysis. Created in BioRender. Duan, D. (2025) https://BioRender.com/bi8qu91 . (B) Volcano plots depict the log 2 FC values and p values of each mutant from the library at 3 days (left) and 5 (right) days postinjection of G. mellonella larvae. Blue dots represent mutants depleted from G. mellonella larvae, while red dots represent those that are enriched. Gray dots indicate mutants showing no significant difference. The horizontal dashed line indicates a p ‐value of 0.05, while the left and right vertical dashed lines indicate log 2 FC of −1 and 1, respectively.

Article Snippet: Initially, M. abscessus ATCC 19977 was recovered on 7H10 solid medium.

Techniques: Mutagenesis, Bacteria, Amplification, Next-Generation Sequencing

Journal: mLife

Article Title: CRISPR‐based genome editing reveals the roles of efflux pumps in Mycobacterium abscessus

doi: 10.1002/mlf2.70048

Figure Lengend Snippet: ETH improves the transformation efficiency of Mycobacterium abscessus . (A, B) Susceptibility of the indicated M. abscessus to the drug ETH (A) and EMB (B). The MIC 50 was defined as the concentration of the compound that inhibited bacterial growth by 50% and was analyzed by a nonlinear fit model in GraphPad Prism 8.0. Data are expressed as the mean ± SD of triplicate samples and are representative of two independent experiments. (C) Transformation efficiency of M. abscessus when different concentrations of ETH or EMB were present in the culture medium used for competent cell preparation. ETH (10, 20, and 40 μg/ml) or EMB (200, 400, and 800 μg/ml) was added to the M. abscessus culture. The transformation efficiency was defined as the total number of CFU generated per transformation. Statistically significant differences were determined using multiple paired t ‐tests. EMB, ethambutol; ETH, ethionamide. * p < 0.05; ** p < 0.01; **** p < 0.0001.

Article Snippet: The M. abscessus ATCC 19977 WT and efflux pump mutant strains were cultured in 7H9 without OADC until the OD 600 reached ~1.0, followed by dilution to an OD 600 of ~0.7.

Techniques: Transformation Assay, Concentration Assay, Generated

Journal: mLife

Article Title: CRISPR‐based genome editing reveals the roles of efflux pumps in Mycobacterium abscessus

doi: 10.1002/mlf2.70048

Figure Lengend Snippet: Genome editing using CRISPR‐Cas9‐assisted recombineering in M. abscessus. (A) Schematic showing the lacZ ‐targeting sgRNA and oligonucleotides used for recombineering. The oligonucleotides lacZ .lag (79 nt) and lacZ .lead (79 nt, sequences reverse and complementary to lacZ .lag) targeting the lagging strand and the leading strand of DNA replication were designed to introduce a continuous stop codon into the lacZ open reading frame, respectively. (B) Number of CFU per transformation and percentage of white transformant colonies after electroporation of the pSG‐ lacZ ‐1 plasmids and oligonucleotides into M. abscessus ATCC 19977 harboring the recombinase‐expression plasmid. lacZ‐1 and lacZ‐2 denote sgRNAs targeting two different sites in the lacZ gene. The transformants were plated on X‐gal plates for blue‐white screening. The black bar corresponds to the left Y axis, indicating the number of transformants, and the gray bar corresponds to the right Y axis, indicating the percentage of white colonies. Results represent the average of two independent experiments. (C) Transformation and recombination efficiency of the dual‐reporter detection system resulting from electroporation of the shear plasmid expressing gfp and the oligonucleotides targeting lacZ . Recombination efficiency was defined as the percentage of white colonies on the X‐gal plate. GFP positivity was calculated based on the proportion of GFP‐positive colonies. lacZ‐1 and lacZ‐2 denote sgRNAs targeting two different sites in the lacZ gene. (D) Transformation efficiency and recombination efficiency of gene deletion using CRISPR‐Cas9‐assisted dsDNA recombineering. Deletions of 620 bp were introduced into M. abscessus chromosomal DNA using approximately 1 kb dsDNA fragments. X‐gal, 5‐bromo‐4‐chloro‐3‐indolyl β‐ d ‐galactopyranoside.

Article Snippet: The M. abscessus ATCC 19977 WT and efflux pump mutant strains were cultured in 7H9 without OADC until the OD 600 reached ~1.0, followed by dilution to an OD 600 of ~0.7.

Techniques: CRISPR, Introduce, Transformation Assay, Electroporation, Expressing, Plasmid Preparation, Shear

Journal: mLife

Article Title: CRISPR‐based genome editing reveals the roles of efflux pumps in Mycobacterium abscessus

doi: 10.1002/mlf2.70048

Figure Lengend Snippet: MAB_2806–2807 is required for antibiotic resistance of Mycobacterium abscessus. (A) Disk diffusion assay of the MAB_2806‐2807 operon to different antibiotics. The zone of inhibition for FOX, LZD, and CLR was measured for Δ MAB_2806 , Δ MAB_2807 , and Δ MAB_2806–2807 , as well as their single‐ or double‐gene complementary strains. Statistically significant differences were determined using a paired two‐tailed t ‐test. All p values were calculated by comparison with the wild type. ** p < 0.01; *** p < 0.001; and **** p < 0.0001. (B) Uptake of EtBr by wild type, Δ MAB_2806‐2807 , and the complementary strain. CLR, clarithromycin; EtBr, ethidium bromide; FOX, cefoxitin; LZD, linezolid.

Article Snippet: The M. abscessus ATCC 19977 WT and efflux pump mutant strains were cultured in 7H9 without OADC until the OD 600 reached ~1.0, followed by dilution to an OD 600 of ~0.7.

Techniques: Diffusion-based Assay, Inhibition, Two Tailed Test, Comparison

Journal: mLife

Article Title: CRISPR‐based genome editing reveals the roles of efflux pumps in Mycobacterium abscessus

doi: 10.1002/mlf2.70048

Figure Lengend Snippet: Screening of efflux pump genes affecting the survival of M. abscessus in Galleria mellonella larvae. (A) Screening of an M.abscessus efflux pump mutant library in G. mellonella larvae. Efflux pump mutants and the wild‐type ATCC 19977 strain of M. abscessus harboring barcodes were mixed and used to infect G. mellonella larvae. At 3 and 5 days postinfection, the larvae were homogenized and plated. The plated bacteria were collected, and the barcode sequences were amplified for next‐generation sequencing analysis. Created in BioRender. Duan, D. (2025) https://BioRender.com/bi8qu91 . (B) Volcano plots depict the log 2 FC values and p values of each mutant from the library at 3 days (left) and 5 (right) days postinjection of G. mellonella larvae. Blue dots represent mutants depleted from G. mellonella larvae, while red dots represent those that are enriched. Gray dots indicate mutants showing no significant difference. The horizontal dashed line indicates a p ‐value of 0.05, while the left and right vertical dashed lines indicate log 2 FC of −1 and 1, respectively.

Article Snippet: The M. abscessus ATCC 19977 WT and efflux pump mutant strains were cultured in 7H9 without OADC until the OD 600 reached ~1.0, followed by dilution to an OD 600 of ~0.7.

Techniques: Mutagenesis, Bacteria, Amplification, Next-Generation Sequencing