Structural analysis of biofilm formation by rapidly and slowly growing nontuberculous mycobacteria

Abstract

Mycobacterium avium complex (MAC) and rapidly growing mycobacteria (RGM) such as M. abscessus, M. mucogenicum, M. chelonae, and M. fortuitum, implicated in health care-associated infections, are often isolated from potable water supplies as part of the microbial flora. To understand factors that influence growth in their environmental source, clinical RGM and slowly growing MAC isolates were grown as biofilm in a laboratory batch system. High and low nutrient levels were compared, as well as stainless steel and polycarbonate surfaces. Biofilm growth was measured after 72 h of incubation by enumeration of bacteria from disrupted biofilms and by direct quantitative image analysis of biofilm microcolony structure. RGM biofilm development was influenced more by nutrient level than by substrate material, though both affected biofilm growth for most of the isolates tested. Microcolony structure revealed that RGM develop several different biofilm structures under high-nutrient growth conditions, including pillars of various shapes (M. abscessus and M. fortuitum) and extensive cording (M. abscessus and M. chelonae). Although it is a slowly growing species in the laboratory, a clinical isolate of M. avium developed more culturable biofilm in potable water in 72 h than any of the 10 RGM examined. This indicates that M. avium is better adapted for growth in potable water systems than in laboratory incubation conditions and suggests some advantage that MAC has over RGM in low-nutrient environments.

FIG. 1.

Culturable biofilm of Mycobacterium clinical isolates and reference strains after growth for 3 days at 35°C in R2A medium (a) or PW (b) on PC or SS disks. Data were transformed by addition of 1 and converting values to log₁₀ values. The detection limit was 100 CFU/disk or approximately 24 CFU/cm² (n = 3). Strain identifiers are as follows: Mab23, M. abscessus ATCC 23007; MabBF6, M. abscessus BF6; Mab4AU, M. abscessus 4AU; Mch35, M. chelonae ATCC 35752; Mch34, M. chelonae 34; Mch56, M. chelonae 56; Mfo32, M. fortuitum 32; Mfo89, M. fortuitum 89; Msm19, M. smegmatis ATCC 19420; Mav91, M. avium 91; Mav61, M. avium EPA 61151; Mav26, M. avium EPA 88126; and Min44, M. intracellulare EPA 88144.

FIG. 2.

Compiled biofilm images of six mycobacteria isolates grown in R2A medium on PC disks. Biofilm was stained with Sybr green I before image stacks were obtained. (A) M. abscessus ATCC 23007; (B) M. abscessus BF6; (C) M. chelonae ATCC 35752; (D) M. smegmatis ATCC 19420; (E) M. fortuitum 32; (F) M. avium 91.

FIG. 3.

Maximum biofilm thickness of 10 RGM and two M. avium isolates incubated at two nutrient levels and on two substratum materials. Maximum thickness was significantly higher for all RGM grown on PC substratum (P < 0.05) in R2A (high-nutrient condition) than for those grown on PC in PW (low-nutrient condition). Substratum material was significant for all RGM grown in R2A, except for M. chelonae 56 and for M. avium isolates 91 and EPA 61151, at either nutrient level. Nutrient level did not significantly affect M. avium biofilm thickness (n = 15 for most isolates). Legend abbreviations for biofilm grown: R2A-PC, R2A broth on PC; R2A-SS, R2A broth on SS; PW-PC, PW on PC; PW-SS, PW on SS. Strain identifiers on the x axis are defined in the legend to Fig. 1.