m abscessus atcc 19977  (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

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Protective mechanism of action of the antifungal drug naftifine against Mycobacterium abscessus infection

doi: 10.1128/aac.01105-25

Figure Lengend Snippet: Naftifine inhibits intracellular M. abscessus growth in a concentration- and time-dependent manner. THP-1 macrophages were infected with ( A ) ATCC 19977 T smooth variant, ( B ) ATCC 19977 T rough variant, and clinical isolates ( C ) 23-S-10, ( D ) 23-S-12, and ( E ) 23-S-13. The ATCC 19977 T strains are sensitive to both clarithromycin and amikacin, while the clinical isolates are resistant to both antibiotics. Following infection, cells were treated with increasing concentrations of naftifine (3–90 μM) or 10 μM clarithromycin as a control. Intracellular bacterial burden was quantified by CFU enumeration at 24, 48, and 72 hours post-treatment. Data represent mean ± SD from three independent experiments. Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons test for comparisons between each naftifine concentration and the DMSO control at corresponding time points. ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: THP-1 cells were infected with M. abscessus ATCC 19977 T smooth or rough variants, or with clinical isolates, at an MOI of 5 in the presence of 40 ng/mL PMA.

Techniques: Concentration Assay, Infection, Variant Assay, Control

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Protective mechanism of action of the antifungal drug naftifine against Mycobacterium abscessus infection

doi: 10.1128/aac.01105-25

Figure Lengend Snippet: Dose-dependent cytotoxicity analysis in uninfected and M. abscessus -infected cells. ( A ) Representative flow cytometry dot plots show Annexin V and 7-AAD staining patterns in uninfected (UI) and infected (IN) cells treated with increasing concentrations (0, 10, 30, and 90 μM) of the test compound. THP-1 cells were infected with M. abscessus ATCC 19977 T smooth variants and treated with naftifine for 3 days. Gated regions indicate dead cells (Annexin V + , including early and late apoptotic and necrotic populations). ( B and C ) Quantification of cell death shows the percentage of Annexin V + population (both 7-AAD + and 7-AAD⁻) in uninfected ( B ) and infected ( C ) cells. Data represent mean ± SEM from at least three independent experiments. Statistical significance was determined by one-way ANOVA followed by Dunnett’s post hoc test for multiple comparisons against the control. ** P < 0.01 and **** P < 0.0001 compared to untreated control (0 μM).

Article Snippet: THP-1 cells were infected with M. abscessus ATCC 19977 T smooth or rough variants, or with clinical isolates, at an MOI of 5 in the presence of 40 ng/mL PMA.

Techniques: Infection, Flow Cytometry, Staining, Control

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Protective mechanism of action of the antifungal drug naftifine against Mycobacterium abscessus infection

doi: 10.1128/aac.01105-25

Figure Lengend Snippet: Naftifine exhibits bacteriostatic activity against M. abscessus in axenic culture. Time-kill assays were performed to evaluate the antimicrobial activity of naftifine against different M. abscessus strains and morphological variants. ( A ) ATCC 19977 T smooth variant, ( B ) ATCC 19977 T rough variant, and clinical isolates ( C ) 23-S-10, ( D ) 23-S-12, and ( E ) 23-S-13. The ATCC 19977 T strains are sensitive to both clarithromycin and amikacin, while the clinical isolates are resistant to both antibiotics. Bacterial cultures in logarithmic growth phase were treated with naftifine at concentrations ranging from 2 to 64 µg/mL (equivalent to 6–198 μM) or DMSO vehicle control. Samples were collected at 24-hour intervals over a 96-hour incubation period, and bacterial viability was assessed by CFU enumeration following serial dilution plating. Data represent mean ± SEM from two independent experiments performed in triplicate. Statistical comparisons between naftifine-treated and DMSO control at each time point were performed using two-way ANOVA with Tukey’s multiple comparisons test. Statistical significance is indicated as * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: THP-1 cells were infected with M. abscessus ATCC 19977 T smooth or rough variants, or with clinical isolates, at an MOI of 5 in the presence of 40 ng/mL PMA.

Techniques: Activity Assay, Variant Assay, Control, Incubation, Serial Dilution

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Protective mechanism of action of the antifungal drug naftifine against Mycobacterium abscessus infection

doi: 10.1128/aac.01105-25

Figure Lengend Snippet: Naftifine reduces M. abscessus burden in the lungs and spleens of immunosuppressed mice. BALB/c mice were immunosuppressed with dexamethasone and subsequently infected intranasally with 1,000 CFU of M. abscessus ATCC 19977 T rough variant. Treatment began 3 days post-infection and continued once daily for 14 days. Mice received either naftifine HCl (50 mg/kg of body weight, intraperitoneal), cefoxitin (200 mg/kg of body weight, subcutaneous, positive control), or PBS vehicle control (intraperitoneal). After the treatment period, mice were euthanized, and bacterial loads in the ( A ) lungs and ( B ) spleens were quantified by CFU enumeration and expressed as log 10 CFU per organ. Data represent individual mice with mean ± SEM. ( C ) Representative hematoxylin and eosin-stained lung sections from infected mice treated with PBS vehicle control (top panels) or naftifine (bottom panels) are shown at 4×, 10×, and 40× magnifications to illustrate histopathological changes. Statistical comparisons between treatment groups were performed with the Kruskal–Wallis test followed by Dunn’s multiple comparisons test. * P < 0.05; ** P < 0.01; *** P < 0.001 and ns, not significant.

Article Snippet: THP-1 cells were infected with M. abscessus ATCC 19977 T smooth or rough variants, or with clinical isolates, at an MOI of 5 in the presence of 40 ng/mL PMA.

Techniques: Infection, Variant Assay, Positive Control, Control, Staining

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Protective mechanism of action of the antifungal drug naftifine against Mycobacterium abscessus infection

doi: 10.1128/aac.01105-25

Figure Lengend Snippet: Autophagy contributes to the intracellular efficacy of naftifine against M. abscessus . Intracellular bacterial survival (log 10 CFU) of ( A ) naftifine-sensitive M. abscessus ATCC 19977 T S (Mab S, blue) strain and naftifine-resistant M. abscessus strain (Mab-Naft R1, red) in wild-type RAW 264.7 macrophages (filled circles) or ( B ) ATG16L1 knockdown macrophages (open circles) treated with naftifine at 0, 5, 10, and 20 µM. Bacterial loads were quantified on days 1 (D1) and 2 (D2) post-infection. ( C and D ) Representative immunoblots showing LC3-I/LC3-II expression and β-actin (loading control) in macrophage lysates at day 1 post-infection with ( C ) Mab-Naft S or ( D ) Mab-Naft R1, treated with indicated naftifine concentrations (μM). Data represent mean ± SEM. Statistical significance was determined by two-way ANOVA. ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: THP-1 cells were infected with M. abscessus ATCC 19977 T smooth or rough variants, or with clinical isolates, at an MOI of 5 in the presence of 40 ng/mL PMA.

Techniques: Knockdown, Infection, Western Blot, Expressing, Control

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Protective mechanism of action of the antifungal drug naftifine against Mycobacterium abscessus infection

doi: 10.1128/aac.01105-25

Figure Lengend Snippet: Synergistic interactions between naftifine and β-lactam antibiotics against M. abscessus . Checkerboard assays were performed to evaluate drug interactions between naftifine ( y -axis) and various antibiotics ( x -axis) against M. abscessus ATCC 19977 T smooth variant. Heat maps display the percentage of bacterial growth, with darker blue indicating greater growth. Twofold serial dilutions of each drug were tested in combination, and growth inhibition was assessed after incubation. Naftifine showed indifferent interactions with clarithromycin ( A ) and tigecycline ( E ), additive/partial synergistic effects with amikacin ( B ), but demonstrated synergistic effects when combined with the β-lactam antibiotics cefoxitin ( C ) and imipenem ( D ), particularly at sub-inhibitory concentrations. Synergy was defined as FICI ≤ 0.5, where the combined effect significantly exceeded the sum of individual drug activities. Data were analyzed and plotted using GraphPad Prism version 10.

Article Snippet: THP-1 cells were infected with M. abscessus ATCC 19977 T smooth or rough variants, or with clinical isolates, at an MOI of 5 in the presence of 40 ng/mL PMA.

Techniques: Variant Assay, Inhibition, Incubation

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Protective mechanism of action of the antifungal drug naftifine against Mycobacterium abscessus infection

doi: 10.1128/aac.01105-25

Figure Lengend Snippet: Autophagy contributes to the intracellular efficacy of naftifine against M. abscessus . Intracellular bacterial survival (log 10 CFU) of ( A ) naftifine-sensitive M. abscessus ATCC 19977 T S (Mab S, blue) strain and naftifine-resistant M. abscessus strain (Mab-Naft R1, red) in wild-type RAW 264.7 macrophages (filled circles) or ( B ) ATG16L1 knockdown macrophages (open circles) treated with naftifine at 0, 5, 10, and 20 µM. Bacterial loads were quantified on days 1 (D1) and 2 (D2) post-infection. ( C and D ) Representative immunoblots showing LC3-I/LC3-II expression and β-actin (loading control) in macrophage lysates at day 1 post-infection with ( C ) Mab-Naft S or ( D ) Mab-Naft R1, treated with indicated naftifine concentrations (μM). Data represent mean ± SEM. Statistical significance was determined by two-way ANOVA. ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: The full-length MAB_4508 gene from M. abscessus ATCC 19977 T S (wild type) as well as from the naftifine-resistant Naft R1 and Naft R2 mutants was amplified by PCR and cloned in-frame to the mycobacterial expression vector pMV261.

Techniques: Knockdown, Infection, Western Blot, Expressing, Control

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Protective mechanism of action of the antifungal drug naftifine against Mycobacterium abscessus infection

doi: 10.1128/aac.01105-25

Figure Lengend Snippet: Naftifine inhibits intracellular M. abscessus growth in a concentration- and time-dependent manner. THP-1 macrophages were infected with ( A ) ATCC 19977 T smooth variant, ( B ) ATCC 19977 T rough variant, and clinical isolates ( C ) 23-S-10, ( D ) 23-S-12, and ( E ) 23-S-13. The ATCC 19977 T strains are sensitive to both clarithromycin and amikacin, while the clinical isolates are resistant to both antibiotics. Following infection, cells were treated with increasing concentrations of naftifine (3–90 μM) or 10 μM clarithromycin as a control. Intracellular bacterial burden was quantified by CFU enumeration at 24, 48, and 72 hours post-treatment. Data represent mean ± SD from three independent experiments. Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons test for comparisons between each naftifine concentration and the DMSO control at corresponding time points. ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: For this purpose, we engineered the M. abscessus ATCC 19977 type strain (ATCC 19977 T ) to express the red fluorescent protein tdTomato, enabling quantification of intracellular bacterial burden via fluorescence intensity.

Techniques: Concentration Assay, Infection, Variant Assay, Control

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Protective mechanism of action of the antifungal drug naftifine against Mycobacterium abscessus infection

doi: 10.1128/aac.01105-25

Figure Lengend Snippet: Dose-dependent cytotoxicity analysis in uninfected and M. abscessus -infected cells. ( A ) Representative flow cytometry dot plots show Annexin V and 7-AAD staining patterns in uninfected (UI) and infected (IN) cells treated with increasing concentrations (0, 10, 30, and 90 μM) of the test compound. THP-1 cells were infected with M. abscessus ATCC 19977 T smooth variants and treated with naftifine for 3 days. Gated regions indicate dead cells (Annexin V + , including early and late apoptotic and necrotic populations). ( B and C ) Quantification of cell death shows the percentage of Annexin V + population (both 7-AAD + and 7-AAD⁻) in uninfected ( B ) and infected ( C ) cells. Data represent mean ± SEM from at least three independent experiments. Statistical significance was determined by one-way ANOVA followed by Dunnett’s post hoc test for multiple comparisons against the control. ** P < 0.01 and **** P < 0.0001 compared to untreated control (0 μM).

Article Snippet: For this purpose, we engineered the M. abscessus ATCC 19977 type strain (ATCC 19977 T ) to express the red fluorescent protein tdTomato, enabling quantification of intracellular bacterial burden via fluorescence intensity.

Techniques: Infection, Flow Cytometry, Staining, Control

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Protective mechanism of action of the antifungal drug naftifine against Mycobacterium abscessus infection

doi: 10.1128/aac.01105-25

Figure Lengend Snippet: Naftifine exhibits bacteriostatic activity against M. abscessus in axenic culture. Time-kill assays were performed to evaluate the antimicrobial activity of naftifine against different M. abscessus strains and morphological variants. ( A ) ATCC 19977 T smooth variant, ( B ) ATCC 19977 T rough variant, and clinical isolates ( C ) 23-S-10, ( D ) 23-S-12, and ( E ) 23-S-13. The ATCC 19977 T strains are sensitive to both clarithromycin and amikacin, while the clinical isolates are resistant to both antibiotics. Bacterial cultures in logarithmic growth phase were treated with naftifine at concentrations ranging from 2 to 64 µg/mL (equivalent to 6–198 μM) or DMSO vehicle control. Samples were collected at 24-hour intervals over a 96-hour incubation period, and bacterial viability was assessed by CFU enumeration following serial dilution plating. Data represent mean ± SEM from two independent experiments performed in triplicate. Statistical comparisons between naftifine-treated and DMSO control at each time point were performed using two-way ANOVA with Tukey’s multiple comparisons test. Statistical significance is indicated as * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: For this purpose, we engineered the M. abscessus ATCC 19977 type strain (ATCC 19977 T ) to express the red fluorescent protein tdTomato, enabling quantification of intracellular bacterial burden via fluorescence intensity.

Techniques: Activity Assay, Variant Assay, Control, Incubation, Serial Dilution

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Protective mechanism of action of the antifungal drug naftifine against Mycobacterium abscessus infection

doi: 10.1128/aac.01105-25

Figure Lengend Snippet: Naftifine reduces M. abscessus burden in the lungs and spleens of immunosuppressed mice. BALB/c mice were immunosuppressed with dexamethasone and subsequently infected intranasally with 1,000 CFU of M. abscessus ATCC 19977 T rough variant. Treatment began 3 days post-infection and continued once daily for 14 days. Mice received either naftifine HCl (50 mg/kg of body weight, intraperitoneal), cefoxitin (200 mg/kg of body weight, subcutaneous, positive control), or PBS vehicle control (intraperitoneal). After the treatment period, mice were euthanized, and bacterial loads in the ( A ) lungs and ( B ) spleens were quantified by CFU enumeration and expressed as log 10 CFU per organ. Data represent individual mice with mean ± SEM. ( C ) Representative hematoxylin and eosin-stained lung sections from infected mice treated with PBS vehicle control (top panels) or naftifine (bottom panels) are shown at 4×, 10×, and 40× magnifications to illustrate histopathological changes. Statistical comparisons between treatment groups were performed with the Kruskal–Wallis test followed by Dunn’s multiple comparisons test. * P < 0.05; ** P < 0.01; *** P < 0.001 and ns, not significant.

Article Snippet: For this purpose, we engineered the M. abscessus ATCC 19977 type strain (ATCC 19977 T ) to express the red fluorescent protein tdTomato, enabling quantification of intracellular bacterial burden via fluorescence intensity.

Techniques: Infection, Variant Assay, Positive Control, Control, Staining

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Protective mechanism of action of the antifungal drug naftifine against Mycobacterium abscessus infection

doi: 10.1128/aac.01105-25

Figure Lengend Snippet: MmpL3 mutations confer resistance to naftifine. Spot dilution assays demonstrate naftifine susceptibility across different M. abscessus strains and complementation constructs. Serial 10-fold dilutions (10 -5 to 10 0 ) of bacterial cultures were spotted onto 7H10-oleic albumin dextrose catalase agar plates containing no drug (0 μg/mL, left), 16 μg/mL naftifine (middle), or 32 μg/mL naftifine (right). ( A ) Wild-type M. abscessus ATCC 19977 (Mab) and strains transformed with vector control, wild-type MmpL3, or mutant MmpL3 alleles (S302T or V299G) show that expression of mutant MmpL3 variants confers naftifine resistance compared to wild-type controls. ( B ) Naftifine-resistant strain Naft R1 (harboring MmpL3 S302T mutation) and its derivatives transformed with vector control or wild-type MmpL3 clones demonstrate that complementation with wild-type MmpL3 does not restore naftifine sensitivity. ( C ) Naftifine-resistant strain Naft R2 (harboring MmpL3 V299G mutation) and its complemented derivatives show that wild-type MmpL3 expression cannot overcome the resistance conferred by the V299G mutation. Colony growth was assessed after 4 days of incubation, indicating that these MmpL3 mutations act dominantly to confer naftifine resistance.

Article Snippet: For this purpose, we engineered the M. abscessus ATCC 19977 type strain (ATCC 19977 T ) to express the red fluorescent protein tdTomato, enabling quantification of intracellular bacterial burden via fluorescence intensity.

Techniques: Construct, Transformation Assay, Plasmid Preparation, Control, Mutagenesis, Expressing, Clone Assay, Incubation

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Protective mechanism of action of the antifungal drug naftifine against Mycobacterium abscessus infection

doi: 10.1128/aac.01105-25

Figure Lengend Snippet: Autophagy contributes to the intracellular efficacy of naftifine against M. abscessus . Intracellular bacterial survival (log 10 CFU) of ( A ) naftifine-sensitive M. abscessus ATCC 19977 T S (Mab S, blue) strain and naftifine-resistant M. abscessus strain (Mab-Naft R1, red) in wild-type RAW 264.7 macrophages (filled circles) or ( B ) ATG16L1 knockdown macrophages (open circles) treated with naftifine at 0, 5, 10, and 20 µM. Bacterial loads were quantified on days 1 (D1) and 2 (D2) post-infection. ( C and D ) Representative immunoblots showing LC3-I/LC3-II expression and β-actin (loading control) in macrophage lysates at day 1 post-infection with ( C ) Mab-Naft S or ( D ) Mab-Naft R1, treated with indicated naftifine concentrations (μM). Data represent mean ± SEM. Statistical significance was determined by two-way ANOVA. ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: For this purpose, we engineered the M. abscessus ATCC 19977 type strain (ATCC 19977 T ) to express the red fluorescent protein tdTomato, enabling quantification of intracellular bacterial burden via fluorescence intensity.

Techniques: Knockdown, Infection, Western Blot, Expressing, Control

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Protective mechanism of action of the antifungal drug naftifine against Mycobacterium abscessus infection

doi: 10.1128/aac.01105-25

Figure Lengend Snippet: Synergistic interactions between naftifine and β-lactam antibiotics against M. abscessus . Checkerboard assays were performed to evaluate drug interactions between naftifine ( y -axis) and various antibiotics ( x -axis) against M. abscessus ATCC 19977 T smooth variant. Heat maps display the percentage of bacterial growth, with darker blue indicating greater growth. Twofold serial dilutions of each drug were tested in combination, and growth inhibition was assessed after incubation. Naftifine showed indifferent interactions with clarithromycin ( A ) and tigecycline ( E ), additive/partial synergistic effects with amikacin ( B ), but demonstrated synergistic effects when combined with the β-lactam antibiotics cefoxitin ( C ) and imipenem ( D ), particularly at sub-inhibitory concentrations. Synergy was defined as FICI ≤ 0.5, where the combined effect significantly exceeded the sum of individual drug activities. Data were analyzed and plotted using GraphPad Prism version 10.

Article Snippet: For this purpose, we engineered the M. abscessus ATCC 19977 type strain (ATCC 19977 T ) to express the red fluorescent protein tdTomato, enabling quantification of intracellular bacterial burden via fluorescence intensity.

Techniques: Variant Assay, Inhibition, Incubation

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Protective mechanism of action of the antifungal drug naftifine against Mycobacterium abscessus infection

doi: 10.1128/aac.01105-25

Figure Lengend Snippet: Naftifine inhibits intracellular M. abscessus growth in a concentration- and time-dependent manner. THP-1 macrophages were infected with ( A ) ATCC 19977 T smooth variant, ( B ) ATCC 19977 T rough variant, and clinical isolates ( C ) 23-S-10, ( D ) 23-S-12, and ( E ) 23-S-13. The ATCC 19977 T strains are sensitive to both clarithromycin and amikacin, while the clinical isolates are resistant to both antibiotics. Following infection, cells were treated with increasing concentrations of naftifine (3–90 μM) or 10 μM clarithromycin as a control. Intracellular bacterial burden was quantified by CFU enumeration at 24, 48, and 72 hours post-treatment. Data represent mean ± SD from three independent experiments. Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons test for comparisons between each naftifine concentration and the DMSO control at corresponding time points. ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: The reference strain M. abscessus ATCC 19977 T was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Concentration Assay, Infection, Variant Assay, Control

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Protective mechanism of action of the antifungal drug naftifine against Mycobacterium abscessus infection

doi: 10.1128/aac.01105-25

Figure Lengend Snippet: Dose-dependent cytotoxicity analysis in uninfected and M. abscessus -infected cells. ( A ) Representative flow cytometry dot plots show Annexin V and 7-AAD staining patterns in uninfected (UI) and infected (IN) cells treated with increasing concentrations (0, 10, 30, and 90 μM) of the test compound. THP-1 cells were infected with M. abscessus ATCC 19977 T smooth variants and treated with naftifine for 3 days. Gated regions indicate dead cells (Annexin V + , including early and late apoptotic and necrotic populations). ( B and C ) Quantification of cell death shows the percentage of Annexin V + population (both 7-AAD + and 7-AAD⁻) in uninfected ( B ) and infected ( C ) cells. Data represent mean ± SEM from at least three independent experiments. Statistical significance was determined by one-way ANOVA followed by Dunnett’s post hoc test for multiple comparisons against the control. ** P < 0.01 and **** P < 0.0001 compared to untreated control (0 μM).

Article Snippet: The reference strain M. abscessus ATCC 19977 T was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Infection, Flow Cytometry, Staining, Control

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Protective mechanism of action of the antifungal drug naftifine against Mycobacterium abscessus infection

doi: 10.1128/aac.01105-25

Figure Lengend Snippet: Naftifine exhibits bacteriostatic activity against M. abscessus in axenic culture. Time-kill assays were performed to evaluate the antimicrobial activity of naftifine against different M. abscessus strains and morphological variants. ( A ) ATCC 19977 T smooth variant, ( B ) ATCC 19977 T rough variant, and clinical isolates ( C ) 23-S-10, ( D ) 23-S-12, and ( E ) 23-S-13. The ATCC 19977 T strains are sensitive to both clarithromycin and amikacin, while the clinical isolates are resistant to both antibiotics. Bacterial cultures in logarithmic growth phase were treated with naftifine at concentrations ranging from 2 to 64 µg/mL (equivalent to 6–198 μM) or DMSO vehicle control. Samples were collected at 24-hour intervals over a 96-hour incubation period, and bacterial viability was assessed by CFU enumeration following serial dilution plating. Data represent mean ± SEM from two independent experiments performed in triplicate. Statistical comparisons between naftifine-treated and DMSO control at each time point were performed using two-way ANOVA with Tukey’s multiple comparisons test. Statistical significance is indicated as * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: The reference strain M. abscessus ATCC 19977 T was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Activity Assay, Variant Assay, Control, Incubation, Serial Dilution

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Protective mechanism of action of the antifungal drug naftifine against Mycobacterium abscessus infection

doi: 10.1128/aac.01105-25

Figure Lengend Snippet: Naftifine reduces M. abscessus burden in the lungs and spleens of immunosuppressed mice. BALB/c mice were immunosuppressed with dexamethasone and subsequently infected intranasally with 1,000 CFU of M. abscessus ATCC 19977 T rough variant. Treatment began 3 days post-infection and continued once daily for 14 days. Mice received either naftifine HCl (50 mg/kg of body weight, intraperitoneal), cefoxitin (200 mg/kg of body weight, subcutaneous, positive control), or PBS vehicle control (intraperitoneal). After the treatment period, mice were euthanized, and bacterial loads in the ( A ) lungs and ( B ) spleens were quantified by CFU enumeration and expressed as log 10 CFU per organ. Data represent individual mice with mean ± SEM. ( C ) Representative hematoxylin and eosin-stained lung sections from infected mice treated with PBS vehicle control (top panels) or naftifine (bottom panels) are shown at 4×, 10×, and 40× magnifications to illustrate histopathological changes. Statistical comparisons between treatment groups were performed with the Kruskal–Wallis test followed by Dunn’s multiple comparisons test. * P < 0.05; ** P < 0.01; *** P < 0.001 and ns, not significant.

Article Snippet: The reference strain M. abscessus ATCC 19977 T was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Infection, Variant Assay, Positive Control, Control, Staining

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Protective mechanism of action of the antifungal drug naftifine against Mycobacterium abscessus infection

doi: 10.1128/aac.01105-25

Figure Lengend Snippet: Autophagy contributes to the intracellular efficacy of naftifine against M. abscessus . Intracellular bacterial survival (log 10 CFU) of ( A ) naftifine-sensitive M. abscessus ATCC 19977 T S (Mab S, blue) strain and naftifine-resistant M. abscessus strain (Mab-Naft R1, red) in wild-type RAW 264.7 macrophages (filled circles) or ( B ) ATG16L1 knockdown macrophages (open circles) treated with naftifine at 0, 5, 10, and 20 µM. Bacterial loads were quantified on days 1 (D1) and 2 (D2) post-infection. ( C and D ) Representative immunoblots showing LC3-I/LC3-II expression and β-actin (loading control) in macrophage lysates at day 1 post-infection with ( C ) Mab-Naft S or ( D ) Mab-Naft R1, treated with indicated naftifine concentrations (μM). Data represent mean ± SEM. Statistical significance was determined by two-way ANOVA. ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: The reference strain M. abscessus ATCC 19977 T was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Knockdown, Infection, Western Blot, Expressing, Control

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Protective mechanism of action of the antifungal drug naftifine against Mycobacterium abscessus infection

doi: 10.1128/aac.01105-25

Figure Lengend Snippet: Synergistic interactions between naftifine and β-lactam antibiotics against M. abscessus . Checkerboard assays were performed to evaluate drug interactions between naftifine ( y -axis) and various antibiotics ( x -axis) against M. abscessus ATCC 19977 T smooth variant. Heat maps display the percentage of bacterial growth, with darker blue indicating greater growth. Twofold serial dilutions of each drug were tested in combination, and growth inhibition was assessed after incubation. Naftifine showed indifferent interactions with clarithromycin ( A ) and tigecycline ( E ), additive/partial synergistic effects with amikacin ( B ), but demonstrated synergistic effects when combined with the β-lactam antibiotics cefoxitin ( C ) and imipenem ( D ), particularly at sub-inhibitory concentrations. Synergy was defined as FICI ≤ 0.5, where the combined effect significantly exceeded the sum of individual drug activities. Data were analyzed and plotted using GraphPad Prism version 10.

Article Snippet: The reference strain M. abscessus ATCC 19977 T was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Variant Assay, Inhibition, Incubation