(W1030-04-22) Antimicrobial Effects of S-nitrosothiols on Mycobacterium abscessus Biofilms

Purpose: Mycobacterium abscessus (M. abscessus) is an opportunistic gram-positive bacterium in cystic fibrosis (CF). The number of CF patients who are diagnosed with M. abscessus infection has been increasing annually due to several factors, including growing medical attention and more advanced diagnostic technologies being developed. Biofilms of M. abscessus are notoriously difficult to treat because of its low permeability, high tolerance to acidic environment, and the bacteria’s inherent high resistance to antibiotics. Nitric oxide (NO) has gained tremendous attention since the late 1990s for the treatment of CF and related diseases based on its strong antimicrobial and antibiofilm effects, which are partially owing to the generation of reactive nitrogen species (RNS) following pulmonary NO administration. Moreover, NO has been shown not to induce resistance in most bacteria strains. However, delivering NO gas is challenging due to its high reactivity in biological environments. As a result, more stable NO donor molecules (i.e., NO prodrugs) have garnered interest from researchers and pharmaceutical companies. S-nitrosothiols (RSNOs) represent a class of NO donors in which NO moiety conjugated the sulfur. Despite some research on some common bacteria, such as keratitis, P. aeruginosa, S. marcescens, and V. cholerae, the efficacy of RSNOs against M. abscessus biofilms has not been explored. This study examined the antimicrobial effects of three common RSNOs against M. abscessus biofilms and investigated the cause for differences observed among these RSNOs. This work aims to improve our understanding the NO’s antimicrobial effect against M. abscessus biofilm and guide the design and development of NO-based drugs to treat M. abscessus infections.

Methods: S-nitroso-cysteine (CysNO) was prepared freshly by nitrosation of cysteine with acidified nitrite before each use. S-nitroso-glutathione (GSNO) and S-nitroso-N-acetylpenicillamine (SNAP) were obtained from Pharmablock USA (Hatfield, PA), and directly used. The ATCC 19977 strain was utilized to culture M. abscessus biofilm. Synthetic cystic fibrosis medium (SCFM) was used as broth to mimic CF extracellular matrix in the human lung. Biofilms were grown for 5 days and treated with RSNOs (including CysNO, GSNO, SNAP) dissolved in SCFM on day 5 by replacing the original medium. After 24 h, the biofilm mass was washed and assayed by MTT assay to determine the remaining active bacterial metabolic activity. The NO release kinetics of RSNOs in SCFM at 37 °C was measured for 2 h by sparging the RSNO solutions with N₂, and the released NO was measured with an ozone-chemiluminescent NO analyzer (NOA Sievers 280i).

Results: The biofilm eradication efficacy of RSNOs is shown in Figure 1A. All three RSNOs required concentrations in the millimolar range to achieve complete biofilm eradication. At 10 mM (Figure 1B), CysNO and SNAP demonstrated comparable efficacy, while GSNO showed significant less ability to kill bacteria in biofilm. Therefore, the more potent CysNO and SNAP at 10 mM were used for further study. The release kinetics of NO from RSNOs can be modulated by either inhibiting it with 10 µM ethylenediamine tetraacetic acid (EDTA), which chelates trace amounts of transition metal ions in the release solution or accelerating it by adding copper (II) chloride (CuCl₂). As seen in Figure 2, EDTA did not affect the antibiofilm effect of CysNO and SNAP, while CuCl₂ significantly boosted the efficacy of SNAP. To understand this difference, release kinetics of RSNOs was measured as shown in Figure 3. CysNO released all NO within 30 min, whereas GSNO and SNAP required much longer time. Adding CuCl₂ resulted in a burst release of NO from SNAP, although this burst was still much lower than that of the CysNO. The short NO release time from CysNO may explain the lack of effect from adding EDTA or CuCl₂. The short NO release time from CysNO may explain the lack of effect from adding EDTA or CuCl₂. It is speculated that the hydrophobicity of SNAP allows it to enter the cells more effectively and then to release NO and induce stronger nitrosative stress intracellularly. Another possible reason is that SNAP has better transnitrosation efficiency to M. abscessus protein than GSNO and CysNO, a post-translational modification that can defunctionalize proteins.

Conclusion: All three RSNOs require very high media concentrations to eliminate M. abscessus biofilms completely without antibiotics. Among the three RSNOs tested, GSNO shows the least efficacy compared to the comparably more potent CysNO and SNAP. However, the addition of Cu (II) can boost the efficacy of SNAP but not CysNO probably due to the initial burst release followed by the sustained NO release. To determine the exact mechanisms behind RSNOs’ antimicrobial effects, future work will test the protein nitrosation following the administration of RSNOs. Future work will test the synergy effects of RSNOs with antibiotics to achieve better efficacy against M. abscessus biofilms as well.

Figure 1. (A) Potency of RSNOs for eradication of M. abscessus biofilms (n=6). (B) Comparison of RSNOs’ anti-biofilm efficacy at 10 mM (n=3). Values are mean ± SD. Statistical analysis by 1-way ANOVA with Tukey test, *** p < 0.001.

Figure 2. Impact of the stability of RSNOs on anti-biofilm efficacy (n=3). Values are mean ± SD. Statistical analysis by 1-way ANOVA with Tukey test, *** p < 0.001.

Figure 3. Direct measurements of total NO released from 3 mL of 10 mM of (A) CysNO (part of curve peak was cut out due to that the detection limit of Sievers 280i is ~450 ppm), (B) SNAP, and (C) GSNO dissolved in SCFM at pH 6.8. Sampling rate of NO to NOA was 208 standard mL/min.