Infection model of THP-1 cells, growth dynamics, and antimicrobial susceptibility of clinical Mycobacterium abscessus isolates from cystic fibrosis patients: Results from a multicentre study

Abstract

Mycobacterium abscessus (MABS) is an emerging pathogen causing severe infections, particularly in cystic fibrosis (CF) patients. A prospective multicentre study included CF patients from four hospitals in Madrid between January 2022 and January 2024. Respiratory samples were collected, and MABS isolates were analysed to determine their antibiotic resistance profiles, growth dynamics, infection kinetics, intracellular behaviour, and pathogenicity. Intracellular bacterial growth and macrophage viability were evaluated through THP-1 cell infection experiments, with and without amikacin. Phenotypic susceptibility testing and genotypic susceptibility testing were also conducted. Among 148 patients, 28 MABS isolates were detected from 16 patients (10.8%), and the first isolate from each patient was analysed. Isolation was more prevalent in younger individuals (median age 24.4 vs. 28.4 years, p = 0.049), and most isolates (81.25%) were identified as M. abscessus subsp. abscessus (MABSa). MABS isolates exhibited high resistance rates (>85%) to doxycycline, tobramycin, ciprofloxacin, moxifloxacin (75%) and cotrimoxazole (56.3%). Amikacin resistance (18.8%) was higher than expected, and inducible (10/16 isolates) or acquired (1/16 isolate) macrolide resistance was found in 68.8% of strains. Phenotypic and genotypic testing results were fully concordant. Tigecycline demonstrated strong in vitro activity, and resistance to imipenem, linezolid, and cefoxitin remained low. Rough strains displayed lower optical density values in later growth stages, probably due to their increased aggregation. In THP-1 cell infection experiments, rough strains showed higher intracellular bacterial loads with statistically significant differences observed at 2 hours (both with and without amikacin) and at 72 hours (with amikacin) post infection. Notably, rough strains also exhibited a higher internalisation index and greater impact on THP-1 cell viability, especially in the absence of amikacin.

Fig 1. Example of morphotypes: A rough; B: smooth; C: mixed (strain 14).

Fig 2. Growth dynamics of smooth and rough strains over time by measuring OD.

Panel A represents data for all strains. B: smooth strains; C: rough strains with rescaled OD values (see arrows). Although smooth (B) and rough (C) strains manifested similar growth dynamics, smooth strains reached higher OD values. Data are mean and SD from three independent experiments.

Fig 3. Internalisation indices for each MABS strain, compared with the ATCC strain.

A: Error bars indicate the SD, based on the results from three independent experiments. *  p < 0.05. One-way ANOVA with Dunnett’s post hoc test. B: Differences in means for internalisation indices, comparing each strain to the ATCC strain. C: 95% confidence intervals for the differences in group means, using Dunnett’s method for multiple comparisons. ¥ Strains exhibiting mixed morphology.

Fig 4. Intracellular growth of smooth and rough strains under amikacin or amikacin-free conditions.

A, B: Intracellular growth of smooth and rough strains, respectively, in the absence of amikacin. C, D: Intracellular growth of smooth and rough strains, respectively, in the presence of amikacin. Error bars indicate the standard deviation, based on the results from three independent experiments. *  Amikacin-resistant strains. ¥ Strains exhibiting mixed morphology.

Fig 5. Effect of infection conditions on THP-1 cell viability.

Data represent the mean ±  95% confidence interval of three independent experiments. Panels A, B: comparison of the viability of THP-1 cells infected with rough and smooth strains in amikacin-free medium (A) or amikacin-containing medium (B). Panels C, D: comparison of THP-1 cell viability in the absence or presence of amikacin for infections with S (C) and R (D) strains. Statistical significance was determined using a two-tailed Student’s t-test. *  p <  0.05; p < 0.01; ***p <  0.001.

Fig 6. Impact of MABS strains on THP-1 cell viability under untreated and amikacin-treated conditions.

Viability percentages are shown relative to the uninfected control at 2 hpi, allowing for the observation of natural viability loss in uninfected THP-1 cells up to 72 hours. Statistical analyses compared the viability of THP-1 cells infected with each strain to the corresponding control at each time point. A, B: Viability of THP-1 cells infected with smooth and rough strains, respectively, in the absence of amikacin. C, D: Viability of THP-1 cells infected with smooth and rough strains, respectively, in the presence of amikacin. Error bars indicate the SD, based on the results from three independent experiments. Two-way ANOVA with Dunnett’s post hoc test. *  p < 0.05, ** p < 0.01. ^a Amikacin-resistant strains (numbers 9, 13 and 16) were excluded from this statistical analysis. ¥ Strains exhibiting mixed morphology.

Fig 7. Example of cord-like aggregates produced by the rough strain number 15 at 48 hpi (A) and 72 hpi (B) in the experiment without amikacin.

These cords were not observed in smooth strains at 48 hpi (C) or 72 hpi (D).