N-acetyl-L-cysteine affects growth, extracellular polysaccharide production, and bacterial biofilm formation on solid surfaces

Abstract

N-Acetyl-L-cysteine (NAC) is used in medical treatment of patients with chronic bronchitis. The positive effects of NAC treatment have primarily been attributed to the mucus-dissolving properties of NAC, as well as its ability to decrease biofilm formation, which reduces bacterial infections. Our results suggest that NAC also may be an interesting candidate for use as an agent to reduce and prevent biofilm formation on stainless steel surfaces in environments typical of paper mill plants. Using 10 different bacterial strains isolated from a paper mill, we found that the mode of action of NAC is chemical, as well as biological, in the case of bacterial adhesion to stainless steel surfaces. The initial adhesion of bacteria is dependent on the wettability of the substratum. NAC was shown to bind to stainless steel, increasing the wettability of the surface. Moreover, NAC decreased bacterial adhesion and even detached bacteria that were adhering to stainless steel surfaces. Growth of various bacteria, as monocultures or in a multispecies community, was inhibited at different concentrations of NAC. We also found that there was no detectable degradation of extracellular polysaccharides (EPS) by NAC, indicating that NAC reduced the production of EPS, in most bacteria tested, even at concentrations at which growth was not affected. Altogether, the presence of NAC changes the texture of the biofilm formed and makes NAC an interesting candidate for use as a general inhibitor of formation of bacterial biofilms on stainless steel surfaces.

FIG. 1.

Effect of NAC on bacteria cultured in 20% LB. Bacterial growth, expressed as optical density at 600 nm [OD_(600nm)], was measured for 24 h. Symbols: •, no NAC;, ×, 0.25 mg of NAC ml⁻¹; □, 0.5 mg of NAC ml⁻¹; ▪, 1.0 mg of NAC ml⁻¹; ○, 2.0 mg of NAC ml⁻¹. The growth of A. lwoffii (a), A. baumannii (b), E. cloacae (c), K. pneumoniae (d), P. mendocina (e), a multispecies community consisting of A. baumannii, P. mendocina, K. pneumoniae, A. lwoffii, E. cloacae,S. warneri, and Bacillus sp. (f), Bacillus sp. (g), B. cereus (h), B. megaterium (i), B. subtilis (j), and S. warneri (k) was examined. The error bars indicate the standard deviations of the means for three experiments.

FIG. 1.

Effect of NAC on bacteria cultured in 20% LB. Bacterial growth, expressed as optical density at 600 nm [OD_(600nm)], was measured for 24 h. Symbols: •, no NAC;, ×, 0.25 mg of NAC ml⁻¹; □, 0.5 mg of NAC ml⁻¹; ▪, 1.0 mg of NAC ml⁻¹; ○, 2.0 mg of NAC ml⁻¹. The growth of A. lwoffii (a), A. baumannii (b), E. cloacae (c), K. pneumoniae (d), P. mendocina (e), a multispecies community consisting of A. baumannii, P. mendocina, K. pneumoniae, A. lwoffii, E. cloacae,S. warneri, and Bacillus sp. (f), Bacillus sp. (g), B. cereus (h), B. megaterium (i), B. subtilis (j), and S. warneri (k) was examined. The error bars indicate the standard deviations of the means for three experiments.

FIG. 2.

Effect of NAC on EPS production. Bacteria were grown in 20% LB overnight in the presence or absence of NAC and then starved in PS for 48 h. Open columns, no NAC; cross-hatched columns, 0.25 mg of NAC ml⁻¹; grey columns, 0.5 mg of NAC ml⁻¹. The bars indicate the standard deviations of the means for four experiments. Abbreviations: K.p, K. pneumoniae; A.b, A. baumannii; B.m, B. megaterium; B.s, B. subtilis; B.c, B. cereus; A.l, A. lwoffii; P.m, P. mendocina; E.c, E. cloacae; S.w, S. warneri; B, Bacillus sp. NM, not measured.

FIG. 3.

Effect of NAC on EPS production by starving bacteria. Bacteria were grown in 20% LB overnight in the absence of NAC, washed, and subsequently exposed to 0.5 mg of NAC ml⁻¹ in PS for 48 h. Open columns, no NAC; cross-hatched columns, 0.5 mg of NAC ml⁻¹. The bars indicate the standard deviations of the means for three experiments. Abbreviations: B.c, B. cereus; A.l, A. lwoffii; P.m, P. mendocina; S.w, S. warneri; B, Bacillus sp.

FIG. 4.

Effect of NAC on degradation of EPS. Isolated EPS from K. pneumoniae, B. megaterium, B. subtilis, B. cereus, E. cloacae, and P. mendocina, as well as dextran, were incubated in the presence or absence of 0.5 mg of NAC ml⁻¹ for 1 h at room temperature. Open columns, no NAC; cross-hatched columns, 0.5 mg of NAC ml⁻¹. The bars indicate the standard deviations of the means for three experiments. The values for dextran (0.5 mg ml⁻¹) were not affected by NAC (the values were too small to show). Abbreviations: K.p, K. pneumoniae; B.m, B. megaterium; B.s, B. subtilis; B.c, B. cereus; E.c, E. cloacae; P.m, P. mendocina.

FIG. 5.

Numbers of cells that adhered to stainless steel surfaces after 24, 48, and 72 h of incubation in 20% LB. The seven-member multispecies community comprised A. baumannii, A. lwoffii, Bacillus sp., E. cloacae, K. pneumoniae, P. mendocina, and S. warneri. Open columns, no NAC; cross-hatched columns, 0.5 mg of NAC ml^−1. The bars indicate the standard deviations of the means for three experiments. Abbreviations: K.p, K. pneumoniae; E.c, E. cloacae; mix, multispecies community.

FIG. 6.

Numbers of cells that adhered to stainless steel surfaces after 24, 48, and 72 h of incubation in process water. The seven-member multispecies community comprised A. baumannii, A. lwoffii, Bacillus sp., E. cloacae, K. pneumoniae, P. mendocina, and S. warneri. Open columns, no NAC; cross-hatched columns, 0.5 mg of NAC ml⁻¹. The bars indicate the standard deviations of the means for three experiments.

FIG. 7.

Acridine orange-stained K. pneumoniae attached to stainless steel surfaces after 72 h of incubation in 20% LB. (a) No NAC in the medium; (b) 0.5 mg of NAC ml⁻¹ in the medium.

FIG. 8.

Attachment of K. pneumoniae to stainless steel surfaces after 72 h of incubation in 20% LB in the absence of NAC (a) and in the presence of 0.5 mg of NAC ml⁻¹ (b). WGA lectin labeled with the fluorescent probe tetramethyl rhodamine isocyanate was used to visualize CPS and EPS.