Journal: Microbiology Spectrum

Article Title: Combined Host- and Pathogen-Directed Therapy for the Control of Mycobacterium abscessus Infection

doi: 10.1128/spectrum.02546-21

Figure Lengend Snippet: ABL/PA, ABL/PI3P, and ABL/PI5P enhance both internalization and intracellular killing of M. abscessus in dTHP-1 cells and monocyte-derived macrophages (MDM) from healthy donors (HD) with pharmacologically inhibited CFTR. (A to C) dTHP-1 cells (A and B) and primary MDM from healthy donors (C), treated or not treated with INH172, were cultured at 5 × 10 5 cells/well in 24-well plates and 2 × 10 5 cells/well in 96-well plates. (A) Cells were stimulated with selected ABL formulations before infection for 30 min and then infected with the M. abscessus reference strain (ATCC 19977). Bacterial uptake was quantified by CFU assay and indicated as phagocytosis index, calculated as the ratio between the CFU obtained immediately after the infection and those from the inoculum. (B and C) Cells, dTHP-1 cells (B) and MDM from healthy donors (C), were exposed or not exposed to INH172, infected with M. Abscessus , and treated for 18 h with the selected ABL formulations. Bacterial growth was assessed by CFU assay. The replication index was calculated as the ratio between the CFU obtained 18 h after infection, in the presence or absence of ABL formulations, and the CFU obtained before the addition of liposomes. The results are shown as the mean ± standard deviation of the values obtained from triplicates of each condition, and panel C is representative of experiments with cells from four different donors. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by Student’s t test.

Article Snippet: Mice were intratracheally infected with M. abscessus (reference strain ATCC 19977) and, starting on day 7 after infection, were inoculated daily, via the i.p. route, with 100 mg/kg body weight of amikacin, whose dose was chosen in preliminary in vivo experiments (Fig. S7) and/or treated 3 times per week for 4 consecutive weeks by intranasal inoculation of 10 5 liposomes.

Techniques: Derivative Assay, Cell Culture, Infection, Colony-forming Unit Assay, Liposomes, Standard Deviation

Journal: Microbiology Spectrum

Article Title: Combined Host- and Pathogen-Directed Therapy for the Control of Mycobacterium abscessus Infection

doi: 10.1128/spectrum.02546-21

Figure Lengend Snippet: ABL/PI5P promotes both ROS- and phagolysosome acidification-dependent intracellular M. abscessus killing in CF MDM. (A to C) MDM isolated from CF patients ( n = 17) were plated at the concentration of 1 × 10 6 cells/mL, infected with the M. abscessus reference strain (ATCC 19977), and then stimulated for 18 h with ABL carrying PI5P (A) ( n = 17) in the presence or absence of catalase (PEG-Cat) (B) (representative of n = 4) or concanamycin A (Conc A), a specific inhibitor of vacuolar type H + -ATPase activity (C) (representative of n = 4) at the concentration of 100 U/mL or 1 nM, respectively. Bacterial growth was assessed by CFU assay, and the replication index was calculated as the ratio between the CFU obtained 18 h after infection, in the presence or absence of ABL formulations, and the CFU obtained before the addition of liposomes. (A) Statistical analysis was performed using the two-sided Wilcoxon matched-pair signed rank test ( P = 0.0005). (B and C) The results are shown as the mean ± standard deviation of the values obtained from each condition and are representative of experiments with cells from four different CF patients. *, P < 0.05; **, P < 0.01, ***, P < 0.001; ****, P < 0.0001 by Student’s t test.

Article Snippet: Mice were intratracheally infected with M. abscessus (reference strain ATCC 19977) and, starting on day 7 after infection, were inoculated daily, via the i.p. route, with 100 mg/kg body weight of amikacin, whose dose was chosen in preliminary in vivo experiments (Fig. S7) and/or treated 3 times per week for 4 consecutive weeks by intranasal inoculation of 10 5 liposomes.

Techniques: Isolation, Concentration Assay, Infection, Activity Assay, Colony-forming Unit Assay, Liposomes, Standard Deviation

Journal: Microbiology Spectrum

Article Title: Combined Host- and Pathogen-Directed Therapy for the Control of Mycobacterium abscessus Infection

doi: 10.1128/spectrum.02546-21

Figure Lengend Snippet: ABL/PI5P reduces both bacterial burden and leukocyte recruitment in WT mice infected with M. abscessus . (A) C57BL/6N mice were chronically infected with 10 5 CFU of the M. abscessus reference strain (ATCC 19977) by intratracheal (i.t.) injection and treated after 1 week of infection with ABL loaded with PA, PI3P, or PI5P three times a week for 8, 17, and 29 days. (A and B) After these time points mice were sacrificed, and BALF and lungs were processed for microbiological analysis (B) and evaluation of neutrophil, macrophage, and lymphocyte BALF recruitment (C). Data from two independent experiments were pooled. The results are shown as the median of the values, and statistical significance by Mann-Whitney test was determined (*, P < 0.05; **, P < 0.01).

Article Snippet: Mice were intratracheally infected with M. abscessus (reference strain ATCC 19977) and, starting on day 7 after infection, were inoculated daily, via the i.p. route, with 100 mg/kg body weight of amikacin, whose dose was chosen in preliminary in vivo experiments (Fig. S7) and/or treated 3 times per week for 4 consecutive weeks by intranasal inoculation of 10 5 liposomes.

Techniques: Infection, Injection, MANN-WHITNEY

Journal: Microbiology Spectrum

Article Title: Combined Host- and Pathogen-Directed Therapy for the Control of Mycobacterium abscessus Infection

doi: 10.1128/spectrum.02546-21

Figure Lengend Snippet: ABL/PI5P reduces both bacterial burden and leukocyte recruitment in CF mice infected with M. abscessus . (A) WT and CF mice were chronically infected with 10 5 CFU of the M. abscessus reference strain (ATCC 19977) by i.t. injection and treated with ABL/PI5P three times a week for 8 and 29 days. (B and C) Mice were then sacrificed, and BALF and lungs were processed for (B) microbiological analysis and (C) evaluation of neutrophil, macrophage, and lymphocyte BALF recruitment to establish the therapeutic efficacy of liposomes. The results are shown as the median of the values, and statistical significance by Mann-Whitney test was determined (*, P < 0.05; **, P < 0.01).

Article Snippet: Mice were intratracheally infected with M. abscessus (reference strain ATCC 19977) and, starting on day 7 after infection, were inoculated daily, via the i.p. route, with 100 mg/kg body weight of amikacin, whose dose was chosen in preliminary in vivo experiments (Fig. S7) and/or treated 3 times per week for 4 consecutive weeks by intranasal inoculation of 10 5 liposomes.

Techniques: Infection, Injection, Drug discovery, Liposomes, MANN-WHITNEY

Journal: Microbiology Spectrum

Article Title: Combined Host- and Pathogen-Directed Therapy for the Control of Mycobacterium abscessus Infection

doi: 10.1128/spectrum.02546-21

Figure Lengend Snippet: ABL/PI5P-amikacin combined treatment promotes higher reduction of M. abscessus intracellular growth than single treatments in CF MDM. CF MDM were cultured and infected as described in Materials and Methods. (A and B) Finally, supernatant was collected, cells were lysed, and both were analyzed for extracellular (A) and intracellular (B) bacterial growth. The replication index was calculated as the ratio between the CFU obtained 18 h after infection in the presence or absence of ABL/PI5P and/or amikacin (AMK) and those obtained immediately after infection, before the addition of the stimuli. Results are shown as the mean ± standard deviation of the values obtained from triplicates of each condition and are representative of four CF patients. n.s., not significant; *, P < 0.05; ***, P < 0.001; ****, P < 0.0001 by Student’s t test.

Article Snippet: Mice were intratracheally infected with M. abscessus (reference strain ATCC 19977) and, starting on day 7 after infection, were inoculated daily, via the i.p. route, with 100 mg/kg body weight of amikacin, whose dose was chosen in preliminary in vivo experiments (Fig. S7) and/or treated 3 times per week for 4 consecutive weeks by intranasal inoculation of 10 5 liposomes.

Techniques: Cell Culture, Infection, Standard Deviation

Journal: Microbiology Spectrum

Article Title: Combined Host- and Pathogen-Directed Therapy for the Control of Mycobacterium abscessus Infection

doi: 10.1128/spectrum.02546-21

Figure Lengend Snippet: ABL/PI5P-amikacin combined treatment induces both M. abscessus clearance and anti-inflammatory responses in WT mice. (A) WT mice were chronically infected with 10 5 CFU of the M. abscessus reference strain (ATCC 19977) and treated 1 week after infection with AMK and/or ABL/PI5P as described in Materials and Methods. (B and C) After 8 and 29 days of treatment, mice were sacrificed, and BALF and lungs were processed for microbiological analysis (B) and evaluation of neutrophil, macrophage, and lymphocyte BALF recruitment (C). The results are shown as the median of the values, and statistical significance by Mann-Whitney test was determined (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

Article Snippet: Mice were intratracheally infected with M. abscessus (reference strain ATCC 19977) and, starting on day 7 after infection, were inoculated daily, via the i.p. route, with 100 mg/kg body weight of amikacin, whose dose was chosen in preliminary in vivo experiments (Fig. S7) and/or treated 3 times per week for 4 consecutive weeks by intranasal inoculation of 10 5 liposomes.

Techniques: Infection, MANN-WHITNEY

Journal: Microbiology Spectrum

Article Title: Combined Host- and Pathogen-Directed Therapy for the Control of Mycobacterium abscessus Infection

doi: 10.1128/spectrum.02546-21

Figure Lengend Snippet: ABL/PI5P-amikacin combined treatment induces both M. abscessus clearance and anti-inflammatory responses in CF mice. (A) CF mice were chronically infected with 10 5 CFU of the M. abscessus reference strain (ATCC 19977) and treated 1 week after infection with AMK and/or ABL/PI5P as described in Materials and Methods. (B and C) After 29 days of treatment mice were sacrificed, and BALF and lungs were processed for microbiological analysis (B) and evaluation of neutrophil, macrophage, and lymphocyte BALF recruitment (C). The results are shown as the median of the values, and statistical significance by Mann-Whitney test was determined (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

Article Snippet: Mice were intratracheally infected with M. abscessus (reference strain ATCC 19977) and, starting on day 7 after infection, were inoculated daily, via the i.p. route, with 100 mg/kg body weight of amikacin, whose dose was chosen in preliminary in vivo experiments (Fig. S7) and/or treated 3 times per week for 4 consecutive weeks by intranasal inoculation of 10 5 liposomes.

Techniques: Infection, MANN-WHITNEY

Journal: Communications Biology

Article Title: Development of a penem antibiotic against Mycobacteroides abscessus

doi: 10.1038/s42003-020-01475-2

Figure Lengend Snippet: a Structures of the carbapenem Imipenem and the penem Faropenem are shown with red arrows indicating the differences in the ring structures. b Structure of the new penem developed here, T405.

Article Snippet: Next, we determined the MIC of T405 against M. abscessus reference strain ATCC 19977, and found it to be 2 μg/mL.

Techniques:

Journal: Communications Biology

Article Title: Development of a penem antibiotic against Mycobacteroides abscessus

doi: 10.1038/s42003-020-01475-2

Figure Lengend Snippet: MICs of T405, imipenem and faropenem tested against the reference strain (ATCC 19977) and 20 clinical strains of M. abscessus in vitro.

Article Snippet: Next, we determined the MIC of T405 against M. abscessus reference strain ATCC 19977, and found it to be 2 μg/mL.

Techniques:

Journal: Communications Biology

Article Title: Development of a penem antibiotic against Mycobacteroides abscessus

doi: 10.1038/s42003-020-01475-2

Figure Lengend Snippet: Frequency of spontaneous resistant mutants of M. abscessus ATCC 19977 recovered against T405 and imipenem at their individual MICs and when the T405 MIC is combined with avibactam at its individual MIC.

Article Snippet: Next, we determined the MIC of T405 against M. abscessus reference strain ATCC 19977, and found it to be 2 μg/mL.

Techniques:

Journal: Communications Biology

Article Title: Development of a penem antibiotic against Mycobacteroides abscessus

doi: 10.1038/s42003-020-01475-2

Figure Lengend Snippet: a Escalating doses of T405 administered alone. b 450 mg/kg dose of T405 administered alone and in combination with probenecid. c Single-dose plasma PK parameters for total T405 concentration in mice after subcutaneous injection of 25 mg/kg ( n = 3 mice per time point per arm).

Article Snippet: Next, we determined the MIC of T405 against M. abscessus reference strain ATCC 19977, and found it to be 2 μg/mL.

Techniques: Clinical Proteomics, Concentration Assay, Injection