Role of porins in the susceptibility of Mycobacterium smegmatis and Mycobacterium chelonae to aldehyde-based disinfectants and drugs

Abstract

Nosocomial outbreaks attributable to glutaraldehyde-resistant, rapidly growing mycobacteria are increasing. Here, evidence is provided that defects in porin expression dramatically increase the resistance of Mycobacterium smegmatis and Mycobacterium chelonae to glutaraldehyde and another aldehyde disinfectant, ortho-phthalaldehyde. Since defects in porin activity also dramatically increased the resistance of M. chelonae to drugs, there is thus some concern that the widespread use of glutaraldehyde and ortho-phthalaldehyde in clinical settings may select for drug-resistant bacteria.

FIG. 1.

Chemical structures of monomeric GTA and OPA.

FIG. 2.

GTA and OPA susceptibility of defined isogenic mutants of M. smegmatis mc²155. Results are expressed as CFU counts upon exposure of the test organisms to the indicated concentrations of disinfectants for 0 to 15 min. (A) mc²155ΔembA (closed circles), mc²155ΔembB (closed triangles), and mc²155ΔMSMEG4245 (closed diamonds) mutants are slightly more sensitive to GTA than is their WT parent, mc²155 (open triangle). GTA (B) and OPA (C) sensitivity of the porin mutants MN01 (closed rectangles, solid line) and ML10 (closed triangles, solid line); the complemented porin mutants MN01/pMN013 (open rectangles, dashed line) and ML10/pMN013 (open triangles, dotted line); and their WT parent, SMR5 (closed circles, solid line).

FIG. 3.

Susceptibility of the M. chelonae strains ATCC 35752 and 9917 to GTA and OPA, and effect of expressing the mspA porin gene from M. smegmatis. Results are expressed as CFU counts upon exposure of the test organisms (M. chelonae ATCC 35752 [closed rectangles, solid line]; M. chelonae 9917 [open triangles, solid line]; M. chelonae ATCC 35752/pZS01 [closed rectangles, dotted line]; 9917/pZS01 [open triangles, dotted line]) to the indicated concentrations of GTA (A and B) or OPA (C) for 0 to 30 min. The increased susceptibility of 9917/pZS01 to GTA was consistently visible at the highest concentration of disinfectant (B) after 5 min of exposure, indicative of a more rapid killing of the M. chelonae recombinant isolate expressing the mspA porin gene.

FIG. 4.

Growth rates and glucose uptake by M. chelonae ATCC 35752 and 9917. (A) Growth rates in 7H9-oleic acid-albumin-dextrose-catalase broth at 30°C. Abs600nm, absorbance at 600 nm. (B) Glucose uptake. The accumulation of [U-¹⁴C]glucose by the two strains over time was measured as described previously (27) and expressed as nmol mg⁻¹ (dry weight) cells. Uptake experiments were performed in triplicates, and the results are shown with their standard deviations. M. chelonae ATCC 35752 (closed rectangles, solid line); 9917 (open triangles, solid line); M. chelonae ATCC 35752/pZS01 (closed rectangles, dotted line); 9917/pZS01 (open triangles, dotted line).