The use of quaternary ammonium disinfectants selects for persisters at high frequency from some species of non-tuberculous mycobacteria and may be associated with outbreaks of soft tissue infections

Abstract

Background: Non-tuberculous mycobacteria (NTM) are increasingly important as opportunistic infections after major and minor surgical procedures, likely because they are ubiquitous and not effectively killed by many commonly used disinfectants. Outbreaks of soft tissue infections with NTM appeared related to the use of commercial disinfectants based on quaternary ammonium compounds (QACs).

Methods: We studied the survival of clinical and environmental isolates of Mycobacterium abscessus, Mycobacterium massiliense, Mycobacterium chelonae and Mycobacterium fortuitum after 20 min, 60 min or 24 h exposures to different QACs, and the surviving bacteria were then re-exposed to QACs to see if the percentage of surviving bacteria had increased. The bacteria were labelled with a dnaA-gfp fusion and their level of QAC resistance monitored as increasing fluorescence. The QAC-resistant bacteria were then serially restreaked onto non-selective medium and retested for QAC survival.

Results: The frequency of survivors was <1 in 10(5) bacteria with Mycobacterium smegmatis, but >1 in 100 with the other mycobacteria studied. Different environmental and clinical isolates had similar QAC MICs, but QAC survivors of each strain were resistant. The QAC-surviving strains reverted to the original, non-resistant phenotype after several passages on non-selective medium.

Conclusions: QACs should not be used in settings where even minimally invasive procedures are performed, as they select for a non-genetically determined reversible resistant phenotype that appears at high frequency with several rapidly growing mycobacterial species associated with healthcare-related infections. M. smegmatis behaves differently and is not an adequate model for testing the activity of disinfectants against NTM.

Figure 1.

Different isolates of the M. abscessus-chelonae group and M. fortuitum show similar susceptibilities to DBLAB, while their derivatives, selected after surviving a previous exposure, are resistant to at least 2% DBLAB. The strains were labelled with plasmid pdnaA–gfp and grown in 96-well plates in medium containing DBLAB at the indicated concentrations. Mycobacterial growth was measured as fluorescence after 4 days of incubation at 37°C. All results are from representative experiments that were repeated at least three times. (a) Comparison of the growth in DBLAB of M. massiliense (M. mass. WT 1), M. abscessus (M. absc. WT 2), derivatives of each that were selected as survivors of a previous exposure of DBLAB (M. mass. 1.2 and M. absc. 2.2), M. smegmatis and M. chelonae. (b) Comparison of the growth in DBLAB of M. chelonae (M. chel. WT 2), two isolates of M. fortuitum (M. fort. WT 1 and M. fort. WT 2), derivatives of each that were selected as survivors of a previous exposure to DBLAB (M. fort. 1.2, M. chel. 2.2, and M. fort. 3.2), M. smegmatis and M. abscessus.

Figure 2.

The QAC-resistant phenotype reverts to normal when passaged on medium without disinfectants. An original clinical M. abscessus isolate—M. absc. WT 2.0 (LTE3514), as well as its resistant derivatives and revertants, were plated after 20 or 60 min exposures to 9% CTAB. Survival is expressed as the number of colonies appearing after the CTAB exposures, compared with the number of colonies appearing after a parallel, control treatment without the presence of CTAB. The derivatives of clinical isolate WT 2.0 were selected as survivors after exposures to CTAB (2.1), DBLAB (2.2) or glutaraldehyde (2.3). The revertants were obtained after either three (2.1.3, 2.2.3 and 2.3.3) or six (2.1.6, 2.2.6 and 2.3.6) serial passages on solid medium without disinfectants.