Mycobacterium abscessus Smooth and Rough Morphotypes Form Antimicrobial-Tolerant Biofilm Phenotypes but Are Killed by Acetic Acid

Abstract

Mycobacterium abscessus has emerged as an important pathogen in people with chronic inflammatory lung diseases such as cystic fibrosis, and recent reports suggest that it may be transmissible by fomites. M. abscessus exhibits two major colony morphology variants: a smooth morphotype (MaSm) and a rough morphotype (MaRg). Biofilm formation, prolonged intracellular survival, and colony variant diversity can each contribute to the persistence of M. abscessus and other bacterial pathogens in chronic pulmonary diseases. A prevailing paradigm of chronic M. abscessus infection is that MaSm is a noninvasive, biofilm-forming, persistent phenotype and MaRg an invasive phenotype that is unable to form biofilms. We show that MaRg is hyperaggregative and forms biofilm-like aggregates, which, like MaSm biofilm aggregates, are significantly more tolerant than planktonic variants to acidic pHs, hydrogen peroxide (H2O2), and treatment with amikacin or azithromycin. We further show that both variants are recalcitrant to antibiotic treatment inside human macrophage-like cells and that MaRg is more refractory than MaSm to azithromycin. Our results indicate that biofilm-like aggregation and protracted intracellular survival may each contribute to the persistence of this problematic pathogen in the face of antimicrobial agents regardless of morphotype. Biofilms of each M. abscessus variant are rapidly killed, however, by acetic acid, which may help to prevent local fomite transmission.

Biofilm-associatedMycobacterium abscessuscells have altered antibiotic tolerance and surface glycolipids in Artificial Cystic Fibrosis Sputum Media

“…Our results imply that the modest degree of antibiotic tolerance in biofilms that has been observed before (19) may be a function of the culture conditions. Our data show that mature biofilms grown in ACFS media afford considerable protection against three clinical antibiotics from different classes.…”

“…Because nutrient availability affects the physiology of cells within a biofilm, we sought to develop methods that would allow us to disentangle responses to nutrients from responses to biofilm formation. Other groups solved this problem by moving biofilms to fresh media each day (12, 19). We assumed that nutrients would not be unlimited during infection, and so worked to assess biofilm physiology in nutrient-limited culture (Fig.…”

“…While we currently lack a comprehensive model of the chemical and genetic basis of Mab biofilm formation, it is clear that surface glycolipids are important. Glycopeptidolipids, which are found on the outer leaflet of the mycobacterial outer membrane, have been shown to affect biofilm structure (19) and are thought to modulate the course of infection (20, 21). Trehalose dimycolate (TDM), which is known to contribute to virulence in Mtb (22) are also found on the surface of some Mab strains, though its contribution to biofilm formation and virulence in Mab has not been studied.…”

“…However, the rough variants only form in some infections, and it is thought that most infections are established by smooth strains (24). Smooth strains are known to form biofilms in vitro (12, 19) and are thought to also form biofilms during infection (13). Antibiotic-recalcitrant Mab infections are likely to involve mature biofilms growing in complex nutrient environments.…”

Children with cystic fibrosis are infected with multiple subpopulations of Mycobacterium abscessus with different antimicrobial resistance profiles

“…There is also, of course, phenotypic diversity due to expression. For example, colony morphotype has previously been suggested as providing a distinction between a 'non-invasive, biofilm-forming, persistent phenotype' (smooth) and an 'invasive phenotype that is unable to form biofilms' (rough), although recent work suggests both morphotypes are capable of aggregation and intracellular survival 29 . Further diversity may come from the subpopulations harboured at different locations within the patient's lung, which may therefore be an important source of diversity for clinically significant phenotypes.…”