Characterization of
            <i>Mycobacterium abscessus</i>
            colony-biofilms based on bi-dimensional images

Aguilera-Correa, John Jairo; Boudehen, Yves-Marie; Kremer, Laurent

doi:10.1128/aac.00402-23

Cited by 3 publications

(12 citation statements)

References 42 publications

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“…An alternative method for growing biofilms in liquid medium is by using a colony-forming model , in which the biofilm is grown on a solid surface—typically by placing an inoculated semi-permeable membrane on top of solid agar ( Rodríguez-Sevilla et al, 2018a ; Gloag et al, 2021 ; Aguilera-Correa et al, 2023 ). Biofilms can be detached and collected via sonicating and vortexing the membrane.…”

Section: Building the Biofilm Research Fundament—laboratory Models Us...mentioning

confidence: 99%

“…Colony-forming biofilms share morphological traits of liquid grown biofilms and tend to have more biomass, enabling the study of various aspects, such as its biomechanical properties ( Gloag et al, 2021 ). In addition, the colony-forming biofilm model has been used to study the interaction of M. abscessus and Pseudomonas aeruginosa in a polymicrobial biofilm, and to analyse the growth of smooth and rough M. abscessus biofilms in response to antibiotic exposure ( Rodríguez-Sevilla et al, 2018a , 2019 ; Idosa et al, 2022 ; Aguilera-Correa et al, 2023 ).…”

Section: Building the Biofilm Research Fundament—laboratory Models Us...mentioning

confidence: 99%

“…Initially it was thought that only smooth M. abscessus morphotypes were capable of forming biofilms ( Howard et al, 2006 ; Greendyke and Byrd, 2008 ; Bernut et al, 2016 ). However, subsequent studies demonstrated that the rough morphotype, which differs from the smooth morphotype by absence of glycopeptidolipids in the cell wall, was also able to form biofilms ( Howard et al, 2006 ; Williams et al, 2009 ; Clary et al, 2018 ; Gloag et al, 2021 ; Aguilera-Correa et al, 2023 ; Veigyabati Devi and Singh, 2023 ; Oschmann-Kadenbach et al, 2024 ). This discrepancy is likely due to differences in experimental conditions, experimental duration and strains, further emphasizing the importance of using different experimental variables in M. abscessus biofilm research ( Clary et al, 2018 ).…”

Section: Culture Condition Complexities—an Overview Of Experimental V...mentioning

confidence: 99%

“…This allows the measurement of the relative fluorescence intensity of mycobacteria within biofilms, not only providing insight into the density of bacterial populations, but also into the spatial distribution of the bacteria. Recently, mWasabi transfected M. abscessus in a colony-biofilm was photographed using a fluorescence microscope, and the intensity of fluorescence in different areas of the images was measured over time ( Aguilera-Correa et al, 2023 ). Subsequently, the volume of each colony was calculated with a mathematical formula based on the signal intensity and colony size.…”

Section: Exploring the Toolbox—commonly Used Techniques For Studying ...mentioning

confidence: 99%

“…Subsequently, the volume of each colony was calculated with a mathematical formula based on the signal intensity and colony size. This approach mirrored the CFU counts, and thus provided a rapid estimation of the number of bacteria in the biofilm ( Aguilera-Correa et al, 2023 ).…”

Section: Exploring the Toolbox—commonly Used Techniques For Studying ...mentioning

confidence: 99%

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A laboratory perspective on Mycobacterium abscessus biofilm culture, characterization and drug activity testing

Meliefste,

Mudde,

Ammerman

et al. 2024

Front. Microbiol.

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Mycobacterium abscessus is an emerging opportunistic pathogen causing severe pulmonary infections in patients with underlying lung disease and cystic fibrosis in particular. The rising prevalence of M. abscessus infections poses an alarming threat, as the success rates of available treatment options are limited. Central to this challenge is the absence of preclinical in vitro models that accurately mimic in vivo conditions and that can reliably predict treatment outcomes in patients. M. abscessus is notorious for its association with biofilm formation within the lung. Bacteria in biofilms are more recalcitrant to antibiotic treatment compared to planktonic bacteria, which likely contributes to the lack of correlation between preclinical drug activity testing (typically performed on planktonic bacteria) and treatment outcome. In recent years, there has been a growing interest in M. abscessus biofilm research. However, the absence of standardized methods for biofilm culture, biofilm characterization and drug activity testing has led to a wide spectrum of, sometimes inconsistent, findings across various studies. Factors such as strain selection, culture medium, and incubation time hugely impact biofilm development, phenotypical characteristics and antibiotic susceptibility. Additionally, a broad range of techniques are used to study M. abscessus biofilms, including quantification of colony-forming units, crystal violet staining and fluorescence microscopy. Yet, limitations of these techniques and the selected readouts for analysis affect study outcomes. Currently, research on the activity of conventional antibiotics, such as clarithromycin and amikacin, against M. abscessus biofilms yield ambiguous results, underscoring the substantial impact of experimental conditions on drug activity assessment. Beyond traditional drug activity testing, the exploration of novel anti-biofilm compounds and the improvement of in vitro biofilm models are ongoing. In this review, we outline the laboratory models, experimental variables and techniques that are used to study M. abscessus biofilms. We elaborate on the current insights of M. abscessus biofilm characteristics and describe the present understanding of the activity of traditional antibiotics, as well as potential novel compounds, against M. abscessus biofilms. Ultimately, this work contributes to the advancement of fundamental knowledge and practical applications of accurate preclinical M. abscessus models, thereby facilitating progress towards improved therapies for M. abscessus infections.

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Section: Building the Biofilm Research Fundament—laboratory Models Us...mentioning

confidence: 99%

Section: Building the Biofilm Research Fundament—laboratory Models Us...mentioning

confidence: 99%

Section: Culture Condition Complexities—an Overview Of Experimental V...mentioning

confidence: 99%

Section: Exploring the Toolbox—commonly Used Techniques For Studying ...mentioning

confidence: 99%

Section: Exploring the Toolbox—commonly Used Techniques For Studying ...mentioning

confidence: 99%

See 3 more Smart Citations

A laboratory perspective on Mycobacterium abscessus biofilm culture, characterization and drug activity testing

Meliefste,

Mudde,

Ammerman

et al. 2024

Front. Microbiol.

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A dTDP-L-Rhamnose 4-epimerase required for glycopeptidolipid biosynthesis in Mycobacterium abscessus

Aguilera-Correa,

Wei,

Leclercq

et al. 2024

Journal of Biological Chemistry

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Silencing essential gene expression in Mycobacterium abscessus during infection

Boudehen,

Tasrini,

Aguilera-Correa

et al. 2023

Microbiol Spectr

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Mycobacterium abscessus is a multi-drug-resistant non-tuberculous mycobacterial species causing tuberculosis-like lung infections. The functional characterization of the extensive repertoire of genes encoding virulence factors and drug targets has been largely restricted by the lack of powerful genetic tools. In this study, we evaluated the performances of a Tet-OFF system, previously optimized for M. tuberculosis and M. smegmatis . Fluorescence reporter M. abscessus strains were developed where mCherry was under the control of the Tet-OFF regulatory system on an integrative plasmid. The addition of anhydrotetracycline (ATc) correlated with a decrease in fluorescence intensity not only in solid and broth medium but also in colony biofilms, for both smooth and rough variants of M. abscessus . Next, unmarked mmpL3 conditional knockdown mutants were engineered in both smooth and rough M. abscessus . Biochemical studies indicated that the addition of ATc was associated with a dose-dependent decrease in (i) the production of the MmpL3 protein, (ii) the biosynthesis of trehalose dimycolate and mycolylated arabinogalactan, (iii) bacterial viability on agar and liquid medium as well as in biofilms. Importantly, intravenous injection of ATc in zebrafish embryos infected with the rough mmpL3 conditional mutant impeded bacterial growth and correlated with a significant gain in embryo survival. These results suggest that mmpL3 is essential for M. abscessus growth in vitro and in the infected host, thus validating MmpL3 as an attractive drug target. Together, this study demonstrates the potential of ATc-dependent repression to modulate the expression of M. abscessus genes in the infected host. IMPORTANCE Mycobacterium abscessus represents the most common rapidly growing mycobacterial pathogen in cystic fibrosis and is extremely difficult to eradicate. Essential genes are required for growth, often participate in pathogenesis, and encode valid drug targets for further chemotherapeutic developments. However, assessing the function of essential genes in M. abscessus remains challenging due to the limited spectrum of efficient genetic tools. Herein, we generated a Tet-OFF-based system allowing to knock down the expression of mmpL3 , encoding the mycolic acid transporter in mycobacteria. Using this conditional mutant, we confirm the essentiality of mmpL3 in planktonic cultures, in biofilms, and during infection in zebrafish embryos. Thus, in this study, we developed a robust and reliable method to silence the expression of any M. abscessus gene during host infection.

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Characterization of Mycobacterium abscessus colony-biofilms based on bi-dimensional images

Cited by 3 publications

References 42 publications

A laboratory perspective on Mycobacterium abscessus biofilm culture, characterization and drug activity testing

A laboratory perspective on Mycobacterium abscessus biofilm culture, characterization and drug activity testing

A dTDP-L-Rhamnose 4-epimerase required for glycopeptidolipid biosynthesis in Mycobacterium abscessus

Silencing essential gene expression in Mycobacterium abscessus during infection

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