To test a nitric oxide-releasing solution (NORS) as a potential antifungal footbath therapy against Trichophyton mentagrophytes and Trichophyton rubrum during the mycelial and conidial phases.
To test a nitric oxide-releasing solution (NORS) as a potential antifungal footbath therapy against Trichophyton mentagrophytes and Trichophyton rubrum during the mycelial and conidial phases.
NORS (sodium nitrite citric acid) produces nitric oxide verified by gas chromatography and mass spectrometry (GC-MS). Antifungal activity of this solution was tested against mycelia and conidia of T. mentagrophytes and T. rubrum, using 1–20 mmol l−1 nitrites and 10–30 min exposure times. The direct effect of the gas released from the solution on the viability of those fungi was tested. NORS demonstrated strong antifungal activity and was found to be dose and time dependent. NO and nitrogen dioxide (NO2) were the only gases detected from this reaction and are likely responsible for the antifungal effect.
This in vitro research suggests that a single 20-min exposure to NORS could potentially be used as an effective single-dose treatment against fungi that are associated with tinea pedis in both mycelia and spore phase.
This study provides the background for developing a user-friendly footbath treatment for Athlete's Foot that will kill both vegetative fungi and its spores.
If you can't find a tool you're looking for, please click the link at the top of the page to "Go to old article view". Alternatively, view our Knowledge Base articles for additional help. Your feedback is important to us, so please let us know if you have comments or ideas for improvement.
Most cutaneous infections are the work of the homogeneous group of keratinophilic fungi known as dermatophytes. Tinea pedis, known as Athlete's Foot, is the most prevalent form of superficial mycotic infections of the skin and occurs in up to 15% of the human population (Drake et al. 1996). Caused primarily by the dermatophytes infection on the surface of the epidermis, tinea pedis presentations vary from mild scaling to a painful inflammatory process resulting in interdigital fissures (Leyden and Kligman 1978; Drake et al. 1996). Species from the genus Trichophyton are most commonly isolated from clinical samples, with Trichophyton rubrum and Trichophyton mentagrophytes being most common (Drake et al. 1996; Baran and Kaoukhov 2005).
Current treatment for tinea pedis involves daily topical application of creams containing azole-, allylamine- or ciclopiroxolamine-based compounds for up to 6 weeks (Baran and Kaoukhov 2005). Although purportedly effective in placebo-controlled clinical testing, the high costs and the long treatment period needed for these antifungal compounds have resulted in noncompliance of the treatment regimen and ultimately are ineffective in the treatment for tinea pedis (Crawford et al. 2001). Furthermore, recurrence of infection has been reported to occur in up to 70% of patients following topical treatments, especially in cases where T. rubrum is the infective agent (Drake et al. 1996).
Nitric oxide (NO) is a free radical gas molecule that plays a major role in the innate immunity, mammalian host defence against infection, modulation of wound healing, vasodilation, neurotransmission and angiogenesis (Liew and Cox 1991; Rizk et al. 2004). It is believed that nonspecific enzymatic reactions play a vital role in the inflammatory response of macrophages during infection, and the NO released from the macrophage has been reported to alter the pathogen's biochemistry and thought to be responsible for its antimicrobial effect (Jones et al. 2010). NO has been reported to have antimicrobial activity against bacteria, yeast, fungi and viruses both in vitro and in vivo animal studies (Rimmelzwann et al. 1999; Weller et al. 2001; Ghaffari et al. 2006; Regev-Shoshani et al. 2010). Its antifungal activities have been reported to delay mycelium growth and germination of conidia of Colletotrichum coccodes, Aspergillus fumigatus, Aspergillus niger, Monilinia fructicola and Penicillium italicum (Kunert 1995; Wang and Higgins 2005; Lazar et al. 2008).
NO can be produced using the inorganic salt sodium nitrite (NaNO2) or potassium nitrite (KNO2) under acidified conditions according to the reactions shown below (Hardwick et al. 2001).
However, the actual NO produced has not been quantified, and it is unknown how the NO produced correlates to biological or therapeutic concentrations. In 2001, Weller et al. established the antifungal activity of acidified nitrites on dermatophytic fungi at the mycelial growth stage. It still remains unclear whether the same fungicidal effect would be observed on the more robust conidia of dermatophytes. Furthermore, it is not clear whether the antifungal activity is attributed to the acidic environment, nitrite levels or gaseous NO that is produced.
Despite encouraging in vitro data, nitric oxide–producing compounds like acidified nitrite creams have failed because they have been reported to be messy, impractical, causing pain in open wounds, possibly causing further damage to wounds and ineffective against fungal spores (Hardwick et al. 2001). We have discovered that using a nitric oxide-releasing solution (NORS), made from sodium nitrite and citric acid as an aqueous bath, did not cause pain in small lesions and digital ulcerations and was very easy to use by immersing the entire foot into a 5-l container (footbath) of NORS (unpublished data). We wanted to expand the understanding of the antifungal effect of acidified nitrites and to further explore NORS footbath as a potential treatment for tinea pedis. This was accomplished by an in vitro study to evaluate its antifungal effect on T. rubrum and T. mentagrophytes. The effect of concentration and time of exposure on these dermatophytes in conidial and mycelial forms was also investigated. Studies were performed to demonstrate that not only is NO being produced by NORS, but NO itself, and not the nitrites, at a specific concentration appears to be responsible for the antifungal activity observed. Finally, the potential efficacy of NO as an antifungal agent is discussed.
NORS were prepared utilizing sodium nitrite and citric acid, as described in a prior publication, to achieve a specific production of an effective antimicrobial nitric oxide–free gas concentration which was previously identified in the published patent by Stenzler (Stenzler and Miller 2006; Miller and Regev 2011). Specifically, this was done by dissolving solid sodium nitrite (NaNO2) into sterile distilled water (dH2O) to reach a final concentration of 1–20 mmol l−1. Then, those solutions were acidified to pH 3·7 using a predetermined mass of citric acid (up to 3 mmol l−1).
Trichophyton rubrum (18758) and Trichophyton mentagrophytes (114841) were obtained from the American Type Culture Collection (ATCC). Fungi were grown at 30°C in Sabouraud Broth (SAB) for 3 days to a mycelial biomass of 1 mg ml−1. Experiments on mycelial viability were performed with this concentration. Conidia were isolated by shaking (on a Fisher shaker at 100 rev min−1) glass beads (Soda Lime 2 mm, VWR) for 60 s on the surface of mycelia grown on SAB agar plates for a minimum of 7 days. Conidia-covered glass beads were vortexed in sterile saline to suspend conidia in solution.
NORS containing NaNO2 at concentrations of 1, 2, 5, 10 and 20 mmol l−1 were tested for their efficacy as antifungal agents. Sterile water (pH 6) was used as control. Sterile water adjusted to pH 3·7 using citric acid and sterile water at 20 mmol l−1 NaNO2 (pH 6) were tested as well to determine whether either solution possessed a fungicidal effect by themselves. NORS (4 ml) was prepared and added to separate 5-ml sterile plastic tubes. One hundred microlitres of culture containing mycelia at a biomass of 1 mg ml−1 was then added to each tube and incubated for 10, 20 and 30 min. Following incubation, samples from each tube were serially diluted and were plated on SAB agar plates. Plates were incubated at 30°C until growth could be detected and counted (about 3 days for T. rubrum and 2 days for T. mentagrophytes). Each experiment was performed in triplicate and repeated three times.
A set of control experiments were performed to eliminate the potential antifungal effect of the citric acid concentration in the treatment solution. Different concentrations of citric acid (1–50 mmol l−1) were prepared, and pH was raised to 3·7 using NaOH. The same experimental methodologies with water as a control were used to perform these tests.
NORS containing concentrations of 1, 2, 5, 10 and 20 mmol l−1 NaNO2 were tested for their effect on conidial viability. Sterile water (pH 6) was used as control. Again, sterile water adjusted to pH 3·7 using citric acid and sterile water at 20 mmol l−1 NaNO2 (pH 6) were tested as well to eliminate the potential individual effect. NORS (4 ml) or control solution was added to separate 5-ml sterile plastic tubes. Conidia of T. mentagrophytes and T. rubrum (100 μl), at concentrations of 1 × 108 and 1 × 106 CFU ml−1 respectively, were added to each plastic tube. Interestingly, despite multiple attempts, those were the highest concentrations that could be achieved with this methodology. Each tube was then capped, vortexed and incubated at room temperature for 10, 20 and 30 min. Following incubation, samples from each tube were diluted by serial dilutions and plated onto SAB agar plates. Plates were incubated at 30°C until the growth could be detected and counted (about 3 days for T. rubrum and 2 days for T. mentagrophytes). Each experiment was performed in triplicate and repeated three times.
The concentration of NO and other gases released from the NORS into the head space was determined by gas chromatography with a mass spectrometer detector (GC-MS). NORS (20 mmol l−1) was prepared inside the sterile 5-ml plastic tubes described above. Each tube was then sealed for 30 min after which 1 ml of the head space above the solution was analysed by GC-MS. GC-MS (Varian™ CP-3800 Gas Chromatograph connected to a Varian™ 1200 Quadrupole MS) analysis was performed using a standard method that had previously been created and calibrated to separate and quantify NO, NO2 and N2O molecules, using calibration gases. The method was set to a constant temperature of 31°C with a sampling flow rate of 1 ml min−1 with helium gas as the carrier gas. Injector temperature was set to 120°C.
To demonstrate that NO, found in the headspace, is responsible for the fungicidal effect of NORS, mycelia of T. mentagrophytes (10 ml at 1 mg ml−1) were combined with 20 ml of sterile saline inside a sterile glass test tube connected via Teflon tubing to a separate glass apparatus, as illustrated in Fig. 1. Sterile saline (0·9% sodium chloride) was used in replacement of sterile dH2O to ensure any fungicidal activity measured was not the result of osmotic imbalances. NORS (containing 100 mmol l−1 NaNO2 and citric acid) was added to the glass apparatus using a 50-ml syringe through the ‘fill port’ (Fig. 1) then sealed using paraffin laboratory film and plastic wrap. A higher strength NORS was required to produce a sufficient volume of gas to account for the much greater head space volume in the apparatus as opposed to the 5-ml tubes previously described. The apparatus was then left at room temperature for 2, 4, 8, 16 and 24 h (each performed separately) after which samples from the glass test tube were plated onto SAB agar plates, incubated at 30°C for 48 h and fungal viability determined. The growth from the exposed test tube was compared to a control of the same contents kept alongside the exposure in a sealed glass test tube (Fig. 1). Another control study was performed with the same apparatus, using saline instead of NORS. Nitrite concentration in the attached glass test tube was measured after each time point, using Griess reagent (Green et al. 1982).
Data in all the above exposure experiments were expressed as mean standard deviation (SD). Statistical analysis of data obtained in all experiments was performed using a one-way analysis of variance (anova) and Tukey's multiple comparison test. A value of P < 0·05 was considered statistically significant. Data analysis and graphical presentation were performed using a commercial statistics package (Graphpad-Prism ver. 3.0; GraphPad Software Inc., USA).
Trichophyton rubrum and Trichophyton mentagrophytes were grown from conidia for a minimum of 72 h to a mycelial biomass of 1 mg ml−1. Mycelia were added to treatment or control 5-ml tubes and incubated for up to 30 min, after which samples were plated and concentration (CFU ml−1) was determined. As NORS is formulated from nitrites and citric acid (lowering pH to 3·7), the individual exposure effect of water at pH 3·7 and 20 mmol l−1 nitrites at pH 6 was tested and compared to an appropriate water only control. As can be seen in Table 1A, minimal to no effect was detected after a 30-min exposure with either 20 mmol l−1 sodium nitrite (pH 6) or citric acid at pH 3·7.
|Control type||T. rubrum (×103 CFU ml−1)||T. mentagrophytes (×105 CFU ml−1)|
|dH2O (pH 6·0)||3·1 (±0·4)||4·9 (±0·2)|
|dH2O (pH 3·7)||4·0 (±0·4)||5·7 (±0·4)|
|NaNO2 (pH 6·0)||2·1 (±0·6)||4·4 (±0·4)|
|dH2O (pH 6·0)||53 (±2)||54 (±4)|
|dH2O (pH 3·7)||60 (±3)a||63 (±3)a|
|NaNO2 (pH 6·0)||55 (±2)||52 (±3)|
Figure 2 shows the mycelia viability following exposure as a percentage of control. T. mentagrophytes (Fig. 2a) and T. rubrum (Fig. 2b) both demonstrated similar responses to different concentrations of NORS. Both species were tolerant to 1 and 2 mmol l−1 NORS for up to 20 min demonstrating a reduction of <25%. While using a higher concentration of 5 mmol l−1, NORS rendered a time-dependent fungicidal effect starting from a significant 25% reduction after 10 min and reaching a 98% reduction after 30 min for both species. An increase to 10 mmol l−1 NORS was highly effective at eradicating mycelia resulting in a >99% reduction at 10 min and complete kill at 30 min for T. mentagrophytes and a complete kill at all time points for the T. rubrum. Not surprisingly, a concentration of 20 mmol l−1 NORS showed a complete kill, even after 10 min, for both organisms (not shown on graph). Controls with 1–50 mmol l−1 citric acid at pH 3·7, and nitrites alone, had no significant effect on mycelial growth when compared to water control.
Conidia isolated from the mycelia of T. rubrum (106 CFU ml−1) and T. mentagrophytes (108 CFU ml−1) were exposed to NORS at different concentrations for up to 30 min. Again, as the NORS is comprised of nitrites at pH 3·7, the individual inhibitory effect of 20 mmol l−1 NaNO2 at pH 6 and dH2O at pH 3·7 were compared to dH2O using both dermatophytes. As can be seen in Table 1B, nitrites by themselves did not have any significant effect on conidial viability/germination of both species. Surprisingly, a pH of 3·7 alone caused a significant but small increase in T. rubrum growth as compared to the dH2O (pH 6) control. This difference likely reflects the formation of clumps of conidia at pH 6.
Figure 3 shows the inhibitory effect of 5, 10 and 20 mmol l−1 NORS on the conidial viability/germination of T. rubrum and T. mentagrophytes after 10, 20 and 30 min exposures. When treating T. mentagrophytes, NORS showed an inhibitory effect dependent upon time and concentration. Following 10 min of exposure, NORS at 5 mmol l−1 resulted in a 50% reduction, 10 mmol l−1 in a 2 log10 reduction and 20 mmol l−1 showed complete inhibition (Fig. 3a). However, following a 30-min exposure, 5 mmol l−1 NORS resulted in a significant 2 log10 reduction and 10 mmol l−1 resulted in complete inhibition. Conidia from T. rubrum, even at a lower concentration of 106 CFU ml−1, demonstrated a greater tolerance to NORS at all concentrations compared to T. mentagrophytes. Following 10 min of exposure, 5 mmol l−1 NORS showed no significant reduction in conidial viability/germination, 10 mmol l−1 showed a 45% reduction, and 20 mmol l−1 resulted in a 93% reduction (Fig. 3b). Following a 30-min exposure, 5 and 10 mmol l−1 showed a significant one log10 reduction while 20 mmol l−1 resulted in complete inhibition.
A head space sample from the tube (containing 4 ml of 20 mmol l−1 NORS) after 20 min was analysed by GC-MS to determine which gaseous molecules could be detected. Specific detection was set to identify NO, NO2, N2O and CO2, and their respective concentrations were determined. As revealed by the chromatogram in Fig. 4a, three types of gas molecules were detected (excluding water vapour, not shown). NO eluted at 5–4 min, NO2 at 5·98 and CO2 eluted at 6·03 min. No other peaks were detected in a scan program for MW 18–100. Figure 4b shows the molecular weight of 30 for the peak at 5·4 min, which correlates to NO. NO concentration was found to be 170(±30) ppm, and NO2 was 40(±10) ppm. CO2 (coming from ambient air) was found as well but not quantified. N2O was not detected. As a comparison, headspace from a control tube had only ambient levels of CO2 present in it.
To demonstrate that the NO being produced by the NORS is likely the active agent responsible for the antifungal activity observed, an apparatus was constructed to ensure no direct contact occurred between fungal mycelia and the NORS, allowing only for the exchange of headspace gases (Fig. 1). The antifungal activity of the NORS headspace gases was tested on T. mentagrophytes mycelia at 1 mg ml−1. Both mycelial viability and nitrite concentrations were measured after 2, 4, 8, 16 and 24 h. NORS at higher concentration (100 mmol l−1) was used to compensate for the dead space volume between the two compartments of the apparatus. A sample of the headspace gas above the NORS after 16 h, injected into the GC-MS, confirmed the presence of NO and NO2 and the absence of other related species (data not shown). Figure 5 illustrates the antifungal activity of the NORS gases over a 24-h period. Some antifungal effect was observed after 4 and 8 h of exposure, where a one log10 reduction in mycelial viability was observed. Complete kill resulted after 16 h of exposure. Mycelia controls showed no significant change in concentration during these time periods. Nitrite concentrations in the saline tubes (Fig. 1) were shown to inversely correlate with mycelial viability and increased at a rate of roughly 100 μmol l−1 per hour. Following 4 h of exposure, the nitrite concentrations were found to be about 0·23 mmol l−1, while after 16 h, where complete kill of the mycelia was reached, a nitrite concentration of 2 mmol l−1 was measured. Mycelia only controls showed nitrite concentrations to be <5 μmol l−1. When using saline in the apparatus, instead of NORS, no mycelial kill was found, no NO was found in the headspace, and no nitrite in the saline tubes.
This work shows that NORS, a formulation of acidified nitrites at pH 3·7, can produce NO and eradicate fungi associated with Athlete's foot. Data presented here show that the antifungal effect of NORS is likely due to the NO gas produced in the reaction. This has been previously suggested and supported by the chemical equations and a chemiluminescence analysis. Hardwick et al. (2001) showed that NO released from trans-membrane NO-generating system had a bactericidal effect on Staphylococcus aureus and Escherichia coli. However, this was done with bacteria, and the only gas that could be directly measured was NO. Our constructed device, along with the GC-MS analysis, confirms the assumption that NO was produced from NORS (with some concomitant NO2 and minimal ambient CO2 levels) is likely the fungicidal agent as no other gases were detected. Previously, we have published that NO2 alone, even at high concentrations and for extended periods of time, was not responsible for the antimicrobial effects seen during NO exposure (Ghaffari et al. 2007). We have also shown in this study that gas (detected as NO and some NO2) flowing from NORS to saline containing fungi acts as fungicidal agent and causes complete kill of T. mentagrophytes.
The release and measurement (by chemiluminescence) of NO and NO2 from acidified nitrites has been previously reported by Gribbe et al. (2008). This is consistent with our findings, showing that after 20 min of reaction, in a closed system, the headspace contained 170 ± 30 ppm of NO and 40 ± 10 ppm of NO2. This amount of NO2 was found to be reduced to 15 ± 5 after 16 h. Beside those two gases and CO2, no other gases were detected. Although NO2 levels may partially contribute to the fungicidal effect of NORS, we have reported elsewhere that 4 h of continuous exposure to 18 ppm NO2 did not have a measureable effect on the survival of Pseudomonas aeruginosa and S. aureus (McMullin et al. 2005). Although we did not test the fungicidal effect of NO2 independently, we suspect that it would be similar to our observations in these previous published bacterial studies.
The minimal inhibitory concentration (MIC) for a 20-min exposure of NORS was 5 mmol l−1 for both fungal strains at both growth phases tested (mycelial and conidial). The minimal fungicidal concentration (MFC) was found to be 20 mmol l−1 for the same time period. These results are consistent with Anyim et al. (2005), who showed a MFC of 30–60 mmol l−1 after a 10-min exposure with an acidified nitrite solution at a pH of 3·6. Interestingly, when the pH was higher, a higher concentration of nitrites was required to get the same effect.
In this study, we worked with NORS at a pH of 3·7. This pH was found to be an effective pH that will cause an antifungal effect in combination with nitrites, but will not cause an effect by itself. This pH is at the same range as was found to be effective in other studies (Weller et al. 2001; Anyim et al. 2005). Indeed, we can see here that both pH and nitrites on their own did not have any fungicidal effect. On the contrary, we observed that a lower pH environment was found to slightly enhance fungal growth both at the mycelial and at the conidial phase. The same amount of citric acid added to the NORS, when tested on its own, did not cause any fungicidal effect either. Our results are inconsistent with the study by Weller et al.(2001), who claimed that at a pH of 3·6, 30 mmol l−1 nitrites for 30 min do not have any fungicidal effect (though, a bactericidal effect was reached) even though higher mycelia and spore concentrations were used in our study. We speculate that these differences may relate to the specific strains that were used, different NORS formulation, different acid or different methodology.
It has been previously suggested that NO released from human skin might play a part in the prevention of skin infection (Weller et al. 1996), and the effectiveness of acidified nitrite cream as a treatment for tinea pedis has been demonstrated (Weller et al. 1998; Hardwick et al. 2001). However, this effect was reached using a higher nitrite concentration (3%) and lower pH (3% acid), as opposed to 0·14% sodium nitrite and 0·06% citric acid, used in this study. In addition, according to Weller's study, treatment went for 30 days, twice a day, and they reported that clinical use was painful, caused discoloration and was messy. We demonstrate here that a single 30-min exposure to NORS may be as effective and the aqueous footbath with this formulation will hopefully be better tolerated than the acidic cream. It remains to be seen whether these characteristics prove out in controlled clinical studies.
NO gas was shown to have antimicrobial activity against wide variety of bacteria (McMullin et al. 2005; Ghaffari et al. 2006) and fungi (Wang and Higgins 2005; Finnen et al. 2007; Lazar et al. 2008). It has also been reported that a short-term exposure to 50–500 ppm of NO was able to inhibit mycelial growth, sporolation and germination of postharvest horticulture fungi (Lazar et al. 2008). Those concentrations are in the same range as what we found here to be released from NORS over 20 min and cause a complete kill of mycelia and conidia.
The mechanisms through which nitric oxide confers its antifungal activity are poorly understood. However, recent studies in bacteria have suggested that nitric oxide has an affinity for reduced surface thiols and divalent metal centres in intracellular enzymes. It is predicted that nitric oxide will attach to surface cysteines causing the formation of S-nitrosylation (-SNO) sites which perturb enzyme structure and/or catalytic activity (Darling and Evans 2003). It has been previously suggested that the antifungal actions of acidified nitrites are mediated by S-nitrosothiols formed by reaction of thiol groups in the toe-nail with the acidified nitrites (Finnen et al. 2007). Another suggested explanation was that the combination of NO and reactive oxygen species produces intermediates, such as peroxynitrite, which are far more potent microbiocidal agents than their precursors (Fang 1997). By conferring the same mechanisms of action for NO predicted for bacteria onto T. rubrum and T. mentagrophytes, we can speculate on a possible mechanism for the antifungal activity observed here. Zaugg et al.(2008) recently identified and characterized a series of membrane bound and secreted carboxypeptidases produced by T. rubrum. These carboxypeptidases include keratinolytic enzymes, which are responsible for the digestion of keratin (a cysteine and disulphide-rich cytoskeletal filament protein located in the skin), the primary carbon source for dermatophytes in tinea pedis infections (Sharma et al. 2011). TruMcpA, a secreted carboxypeptidase of the M14A family identified by Zaugg et al., was shown to contain a catalytic zinc ion as well as cysteine residues involved in structural disulphide bonds. This may be a potential site for NO to react. Interestingly, Zaugg et al. point out that fungal membrane transporters can only assimilate amino acids and short peptides, indicating that a reduction in keratinolytic and/or extracellular protease activity could result in nitrogen starvation by the fungi and potentially lead to cell death. This mechanism is purely speculative, and more work should be performed to confirm this.
These data, presented herein, are suggestive that a 20-min treatment with NORS might be sufficient to eradicate fungi associated with Athlete's Foot. However, further in vivo studies will be required to be conclusive.
We thank Prof. Yossef Av-Gay for reviewing the manuscript and giving useful edits.
This study was partially funded by Nitric Solutions Inc. (NSI).
C.C.M. is a founder of NSI.