Use of green fluorescent protein labeled non-tuberculous mycobacteria to evaluate the activity quaternary ammonium compound disinfectants and antibiotics

Abstract

Although infections with NonTuberculous Mycobacteria have become less common in AIDS patients, they are important opportunistic infections after surgical procedures, likely because they are ubiquitous and not efficiently killed by many commonly used disinfectants. In Venezuela there have recently been many non-tuberculous mycobacteria soft tissue infections after minor surgical procedures, some apparently related to the use of a commercial disinfectant based on a Quaternary Ammonium Compound. We studied the activity of this and other quaternary ammonium compounds on different non-tuberculous mycobacteria by transforming the mycobacteria with a dnaA-gfp fusion and then monitoring fluorescence to gauge the capacity of different quaternary ammonium compounds to inhibit bacterial growth. The minimum inhibitory concentration varied for the different quaternary ammonium compounds, but M. chelonae and M. abscessus were consistently more resistant than M. smegmatis, and M. terrae more resistant than M. bovis BCG.

Keywords: Disinfectants; NonTuberculous Mycobacteria (NTM); Post-surgical infections; Quaternary Ammonium Compounds (QACs); Resistance.

Fig. 1

Plasmid pFPV27 (pdnaA-gfp), the dnaA promoter region from M. tuberculosis.

Fig. 2

(A) Comparison of the fluorescence of cultures (RFU-Relative Fluorescent Units) with their Optical Density (OD₆₀₀) for M. smegmatis strains with no gfp fusion (WT) or M. smegmatis with either the multicopy (pdnaA-gfp) or single copy (pintdnaA-gfp) integrated version of the GFP fusion. (B) Comparison of the fluorescence (RFU) with colony counts (CFU – Colony Forming Units) of the same strains shown in (A). All results are from representative experiments that were repeated at least three times.

Fig. 3

Activity of DBLAB against M. abscessus carrying the pdnaA-gfp fusion on a multi-copy plasmid was determined in a 96 well format. Bacterial growth was measured daily over 4 days with a fluorimeter and expressed as Relative Fluorescent Units (RFU) shown on log scale. The experiment was performed in triplicate.

Fig. 4

Activity of four QACs disinfectants tested in decreasing concentrations against M. smegmatis, M. abscessus, M. chelonae, M. terrae and M. bovis BCG, all carrying the dnaA-gfp fusion on a multi-copy plasmid. The assays were performed in a 96 well format and bacterial growth after 4 days of incubation was measured as Relative Fluorescent Units (RFU) and shown on a log scale. The QACs tested were DBLAB, HPC, CTAB and DTAB. All results are averaged from experiments performed at least three times.

Fig. 5

Comparison of the results of three different methods for determining the MICs of four QACs disinfectants (A) DBLAB, (B) HPC, (C) CTAB and (D) DTAB against M. smegmatis, M. abscessus, and M. chelonae. The methods tested were: GFP, monitoring the fluorescence of strains carrying the multicopy dnaA-gfp fusion; AB, Alamar Blue, 96 well plate format; P, plate assays. All results are from representative experiments that were repeated at least three times.

Fig. 6

Activity of five antibiotics against GFP labeled M. abscessus, determined as fluorescence after 72 h of incubation in media containing the drugs at the concentrations indicated. The experiment was performed in triplicate.