Unique Features of Mycobacterium abscessus Biofilms Formed in Synthetic Cystic Fibrosis Medium

Abstract

Characterizing Mycobacterium abscessus complex (MABSC) biofilms under host-relevant conditions is essential to the design of informed therapeutic strategies targeted to this persistent, drug-tolerant, population of extracellular bacilli. Using synthetic cystic fibrosis medium (SCFM) which we previously reported to closely mimic the conditions encountered by MABSC in actual cystic fibrosis (CF) sputum and a new model of biofilm formation, we show that MABSC biofilms formed under these conditions are substantially different from previously reported biofilms grown in standard laboratory media in terms of their composition, gene expression profile and stress response. Extracellular DNA (eDNA), mannose-and glucose-containing glycans and phospholipids, rather than proteins and mycolic acids, were revealed as key extracellular matrix (ECM) constituents holding clusters of bacilli together. None of the environmental cues previously reported to impact biofilm development had any significant effect on SCFM-grown biofilms, most likely reflecting the fact that SCFM is a nutrient-rich environment in which MABSC finds a variety of ways of coping with stresses. Finally, molecular determinants were identified that may represent attractive new targets for the development of adjunct therapeutics targeting MABSC biofilms in persons with CF.

2-Aminoimidazoles Inhibit Mycobacterium abscessus Biofilms in a Zinc-Dependent Manner

“…To determine whether 2-AI-based small-molecule compounds and related scaffolds inhibited the formation of M. abscessus biofilms, we screened a library of 30 compounds, including twenty-six 2-AI analogs, one 2-AP compound (EL-05-047) and three meridianin analogs (7.079; 7.025 and 8.001), of which two contained the 2-AI moiety ( Table 1 ). The screening was conducted using the Mmas reference strain CIP 108297 grown as submerged biofilms on poly-D-lysine-coated plates in chemically defined synthetic CF medium (SCFM) as previously described [ 11 ]. Recent studies from our laboratory have indeed shown that SCFM closely mimics the nutritional conditions encountered and metabolic adaptation undergone by M. abscessus grown in actual CF sputum [ 26 ], making this model more relevant to infection than other models based on laboratory media.…”

“…Since Zn 2+ ions are naturally absent from SCFM, where M. abscessus forms abundant biofilms, one can exclude that AB-2-29 prevents biofilm formation by acting as a zinc chelator. Likewise, we exclude that the amount of zinc that comes with AB-2-29 (2.52 μM of zinc at its IC 50 value of 20 μM) is responsible for the observed antibiofilm activity of this compound, since we previously established that much higher concentrations of zinc were required to inhibit M. abscessus biofilm formation in SCFM (IC 50 of zinc~150 μM against Mabs NJH12) [ 11 ]. The fact that AB-2-29 comes with small quantities of zinc, however, explains the repression of zur regulon genes in AB-2-29-treated cultures.…”

“…Indeed, the zinc-independent ribosomal proteins S18, S14, L33 and L28 have been involved in biofilm formation in M. smegmatis [ 45 ]. Moreover, we note that a number of genes that are downregulated upon exposure to AB-2-29 ( MAB_1044c , MAB_1046c , MAB_2204 , MAB_2706c , MAB_3438 and the Zur-regulated Mce operon encoded by the gene cluster encompassing MAB_1680 to MAB_1701 ) were found to be upregulated in M. abscessus during biofilm development [ 11 ]. Clearly, further studies are needed to assess the relative contribution of these genes, either individually or combined, to the biofilm-forming capacity of M. abscessus .…”

“…abscessus and M. abscessus subsp. massiliense clinical isolates, NJH9, NJH12 and NJH18 were from persons with CF at National Jewish Health (Denver, CO, USA) [ 11 ]. M. abscessus subsp.…”

“…M. abscessus bacteria are indeed the most antibiotic-resistant and antibiotic-tolerant nontuberculous mycobacteria (NTM) owing to their highly impermeable cell envelope and the variety of efflux pumps and drug- and drug-target-modifying enzymes encoded within their genomes [ 8 , 9 , 10 ]. Further compounding this problem is the propensity of M. abscessus to form biofilms [ 11 ] and the clinical evidence for M. abscessus biofilm formation within the airways and lung cavity of human patients [ 12 , 13 , 14 ]. The presence of biofilms, where bacilli are not only shielded from the effect of antibiotics but may also persist in a drug-tolerant state, may help explain why M. abscessus lung infections are usually incurable with antibiotic therapy alone and why adjunctive surgical resection of cavities may improve treatment outcome [ 4 ].…”