Methyl-Hydroxylamine as an Efficacious Antibacterial Agent That Targets the Ribonucleotide Reductase Enzyme

The emergence of multidrug-resistant bacteria has encouraged vigorous efforts to develop antimicrobial agents with new mechanisms of action. Ribonucleotide reductase (RNR) is a key enzyme in DNA replication that acts by converting ribonucleotides into the corresponding deoxyribonucleotides, which are the building blocks of DNA replication and repair. RNR has been extensively studied as an ideal target for DNA inhibition, and several drugs that are already available on the market are used for anticancer and antiviral activity. However, the high toxicity of these current drugs to eukaryotic cells does not permit their use as antibacterial agents. Here, we present a radical scavenger compound that inhibited bacterial RNR, and the compound's activity as an antibacterial agent together with its toxicity in eukaryotic cells were evaluated. First, the efficacy of N-methyl-hydroxylamine (M-HA) in inhibiting the growth of different Gram-positive and Gram-negative bacteria was demonstrated, and no effect on eukaryotic cells was observed. M-HA showed remarkable efficacy against Mycobacterium bovis BCG and Pseudomonas aeruginosa. Thus, given the M-HA activity against these two bacteria, our results showed that M-HA has intracellular antimycobacterial activity against BCG-infected macrophages, and it is efficacious in partially disassembling and inhibiting the further formation of P. aeruginosa biofilms. Furthermore, M-HA and ciprofloxacin showed a synergistic effect that caused a massive reduction in a P. aeruginosa biofilm. Overall, our results suggest the vast potential of M-HA as an antibacterial agent, which acts by specifically targeting a bacterial RNR enzyme.


Introduction
Infectious diseases constitute a tenacious and major public health problem worldwide. For many years, antibiotic-resistant pathogens have been recognized as one of the primary threats to human survival, and some experts predict a return to the pre-antibiotic era. The emergence 35745) was grown on Middlebrook 7H10 agar (Difco Laboratories, Surrey, UK) supplemented with 10% oleic-albumin-dextrose-catalase enrichment medium at 37°C for 2 weeks.

Antibacterial susceptibility testing
To determine the survival of the different strains in the presence of different radical scavengers, each bacterial strain was grown in its specific medium to mid-log phase (A 550 0.5) and plated on solid plates supplemented with different concentrations of each compound.
In the case of BCG, colonies were scraped from Middlebrook 7H10 plates, resuspended in phosphate-buffered saline (PBS), slightly vortexed with glass beads to dissolve clumps, and allowed to settle for 30 minutes. The supernatant was diluted in PBS and adjusted to 1.0 McFarland standard. Serial dilutions were then plated on solid plates containing freshly prepared compounds at the indicated concentrations. Colony-forming units (cfu) were counted after growing.
Inhibitory concentration 50% (MIC 50 ) was defined as the compound concentration that reduced bacterial growth (cfu) by 50%, and MIC 100 was defined as the lowest concentration of drug that visibly inhibited bacterial growth by 100%.

Determining mammalian cytotoxicity
Murine J774 macrophages (6x10 4 per well) were seeded onto 48-well tissue culture plates in complete medium without antibiotics in the presence of different doses of HU, HA and M-HA, or left untreated. After 24, 72 and 120 h of exposure to the different compounds, culture supernatants were removed and cell viability was assessed by using a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay (Sigma Aldrich) (20). Absorbance was measured at 550 nm with an ELISA reader (Infinite M200 Microplate Reader, Tecan). The results were expressed as a percentage of cell survival relative to untreated cells. Each experiment was repeated at least three times.
In another set of experiments, cells were washed at 24 hours after adding the compounds for the first time, and new, freshly made compounds were added. Cell viability was measured each 24 hours as described above.
The 50% cytotoxicity inhibitory concentration (CC 50 ) of each drug was determined from dose-response curves by using Graph Pad Prism v6. The selectivity index (SI) (SI = CC 50 / MIC 50 ) was calculated on the basis of the CC 50 and MIC 50 values as determined after 24 h of exposure.

The anti-BCG intracellular activity of the different compounds
For the infection experiments, BCG suspensions were adjusted to a 1.0 McFarland standard and centrifuged at 2000 g for 10 minutes. The bacterial pellets were re-suspended in complete medium without antibiotics and were further subjected to three consecutive 30 second pulses (45 W) in an ultrasonic water bath to obtain a predominantly single bacterial cell suspension.
Murine J774 macrophages (3x10 4 per well) were seeded onto 48-well plates in complete medium without antibiotics. Twenty-four hours later, they were infected with BCG at a multiplicity of infection (MOI) of 10 for three hours as previously described [20]. The MOI was confirmed by plating serial dilutions of the inoculum on solid media. After three hours, cells were washed to remove extracellular bacteria and incubated with fresh complete medium plus different doses of HU, HA, and M-HA, at 37°C in a 5% CO 2 atmosphere. All infections were performed in triplicate. The cell culture supernatants were removed, macrophages were lysed, and bacterial counts were determined by plating serial dilutions on Middlebrook 7H10 plates at different time points after infection (24,72, and 120 h) [20]. Non-infected control cultures were always included and all experiments were repeated at least three times.

Cytokine analysis and nitric oxide (NO) production
Cell culture supernatants were collected at different time points as indicated above in both experiment types for macrophage cell viability and intracellular BCG susceptibility to the compounds. Interleukin (IL)-10, IL-12 and tumor necrosis factor (TNF)-α levels were determined by using commercially available enzyme-linked immunosorbent assays (ELISA) (IL-10 and IL-12p40 from Mabtech AB, Nacka Strand, Sweden; and TNF-α from R&D Systems Inc., Minneapolis, MN, USA) according to the manufacturer's instructions. All samples were assayed in duplicate.
The NO production was assessed by measuring nitrite concentrations with the Griess reaction (Sigma).

Viability test analysis
An overnight culture of P. aeruginosa PAO1 was diluted in fresh LB medium and grown to the beginning of exponential phase (A 550 * 0.3) to which different concentrations of radical scavenger compounds were added. After 3 or 24 hours of incubation at 37°C, the cells were stained by using the LIVE/DEAD BactLight viability kit (Life Technologies) for 15 minutes at room temperature in the dark. Fluorescent bacteria were visualized with a Nikon E600 microscope (Nikon) coupled with a DP72 Olympus camera.

Viable cell counts in biofilm inhibition after radical scavenger treatment
To investigate the anti-biofilm activity of the different radical scavengers alone or in combination with ciprofloxacin, P. aeruginosa PAO1 biofilms were grown on microtiter plates and a previously described protocol was followed [21]. An overnight-grown culture of P. aeruginosa in TSB was diluted 1:100 in sterile TSB medium supplemented with 0.2% glucose and added to a 96-well microtiter plate with pegs (Nunc-TSP, Thermo Scientific) (200-μl each well). After 24-48 h of incubation at 37°C in a humidified chamber, the culture supernatant was discarded and the pegs were washed three times with sterile PBS to remove non-adherent cells. After being rinsed, the biofilms were treated with different radical scavenger concentrations alone or in combination with ciprofloxacin. After 24 h of treatment, the pegs were transferred to a new plate that had been rinsed with PBS. Adherent bacteria were first fixed with 200 μl of methanol for 10 min and then stained with crystal violet (1%) for 10 min. Excess crystal violet was washed gently with water and the pegs were dried in air for 5 min. The dye that was bound to the cells was dissolved with 150 μl of ethanol 95%, centrifuged at 2000 rpm for 10 min, and read at 570 nm with a microplate reader (Infinite M200).

Biofilm culture in flow cell system and confocal microscopy analysis
To prepare biofilms developed under continuous flow, P. aeruginosa cells (5x10 5 cfu/ml) were cultured in LB medium at 25°C in flow chambers with channel dimension of 1x4x40 mm as described previously [22]. After 96 h of culture, formed biofilms were treated with 40 μg/ml of HA, HU or M-HA, and LB medium was used alone in the control sample. After 24 h of treatment at 25°C, biofilms were stained with 5 μM SYTO 9 at room temperature in the dark for 30 min, according to the specifications of the LIVE/DEAD BacLight Bacterial Viability kit (Molecular Probes, Invitrogen).
Confocal scanning laser microscopy of the biofilms was performed with a Leica TCS-SP5 confocal scanning laser microscope (Leica Microsystems, Wetzlar, Germany), with an excitation wavelength of 477 for SYTO9. To measure biofilm thickness, sections were scanned and Z-stacks were acquired at z step-size of 0.388 μm. Field size was 456 μm x 456 μm at 20X magnification. Microscope images were further processed with ImageJ analysis software (National Institute of Health, USA) and COMSTAT 2 software, specific for biofilm quantitative analysis [23].

Statistical analyses
Data were presented as the means ± standard deviation (SD). The statistical significance of differences between cytokine levels and BCG growth inhibition using the different radical scavenger compounds was assessed by using Student's t-tests (SigmaStat, SPSS, Chicago, IL). Differences were considered significant when P < 0.05. All statistical procedures were performed with SPSS 15.0 software (SPSS Inc., Chicago, IL).

M-HA showed greater antibacterial activity than HU and HA radical scavengers
The antibacterial activity of three radical scavengers (HU, HA and M-HA) was evaluated against four Gram-positive bacteria (S. aureus, S. mutans, S. sanguinis and M. bovis BCG) and two Gram-negative bacteria (P. aeruginosa and B. cenocepacia). As shown in Table 1, the HU compound exhibited moderate activity against S. aureus, S. mutans and S. sanguinis (200-330 μg/mL) and high growth inhibitory activity against P. aeruginosa, M. bovis and B. cenocepacia (7.6 to 13 μg/mL). HA showed better growth inhibitory activity relative to HU in all tested bacteria (2 to 52 μg/mL). M-HA was highly active in M. bovis BCG and P. aeruginosa cultures, with a 1.5 to 4.5-fold lower MIC 50 than HU and HA. In BCG cultures, 7.5 to 43 times lower concentrations of M-HA (MIC 50 = 1.9 μg/mL) were needed to obtain the same results as the other bacteria cultures (from 14.2 μg/mL for B. cenocepacia to 81.9 μg/mL for S. sanguinis) ( Table 1).
To investigate the mechanisms through which M-HA inhibits bacteria growth, we specifically stained bacteria with the Live/Dead BactLight bacterial viability assay (Invitrogen). As shown in Fig. 1, the different radical scavengers (at a MIC 50 concentration) did not apparently modify the bacterial membrane integrity after 3 hours of treatment because all of them were stained green. After 24 h of treatment, the proportion of non-viable cells (red cells) increased, especially when treated with M-HA.

M-HA does not affect eukaryotic cell growth
The antiproliferative activity of the three radical scavengers was evaluated against murine J744 macrophages by MTT staining. As expected, HU and HA interfere with macrophage proliferation even when low concentrations are used (Fig. 2). Only the lowest dose of HA (10 μg/ml) permits macrophage growth. By contrast, concentrations of up to 250 μg/ml M-HA do not inhibit macrophage proliferation (Fig. 2). The same results were obtained when culture medium plus radical scavenger compounds were renewed every 24 hours (data not shown), and even if the cells were treated for up to 120 hours (data not shown). As shown in Table 1, a cytotoxic concentration (CC 50 ) is observed for M-HA when doses higher than 250 μg/mL were used.
The selectivity index (SI) was calculated on the basis of the MIC 50 and CC 50 values that were determined after 24 h of exposure (Table 1). High SI values were obtained for M-HA in Mycobacterium and Pseudomonas growth inhibition (SI = 182.6 for M. bovis and 52.5 for P.  aeruginosa), which were much higher than the SI obtained with HA or HU (SI from 0.08 to 4.9).

M-HA shows intracellular antimycobacterial activity
In view of the results shown in Table 1, M-HA seems to be a promising antimycobacterial candidate because mycobacteria are the intracellular pathogens for which we aimed to demonstrate activity in infected macrophages. As shown in Fig. 3A, M. bovis BCG viability was diminished when infected macrophages were incubated in the presence of the different radical scavenger compounds related to untreated wells. At 72 hours post-infection, HU triggers a BCG growth inhibition of approximately 50%, but only HA treatments exhibited better inhibition values when the dose was greater than 35 μg/ml. As explained before, this finding also corresponds to a concomitantly drastic reduction in macrophage viability (see Table 1, CC and Fig. 2). By contrast, M-HA showed enhanced intracellular M. bovis BCG growth inhibition in comparison with that of HU or HA at a range of concentrations that were not toxic for eukaryotic cells between 74.6% BCG survival at 8 mg/ml and 42.6% at 125 mg/ml (Fig. 3A). Moreover, the M-HA intracellular antimycobacterial activity improved when the culture medium was   (Fig. 3B). In absolute numbers, this finding represents up to a one log reduction, i.e., at 72 hours post-infection, an M-HA concentration of 82 mg/ml was reduced from 6.3x10 4 (untreated wells) to 8.4x10 3 viable BCG cells (cfu). Similar values were observed in both cases (replacing or not replacing the compounds every 24 hours) at 120 hours post-infection (data not shown).

Increased TNF production by M-HA
We investigated the production of two bactericidal products that are able to kill intracellular BCG, namely TNF-α and NO [24], when the macrophages were infected with BCG and treated with the different radical scavenger compounds. As shown in Fig. 4, M-HA-treated macrophages produce higher TNF-α values at 24 hours post-infection than untreated cells (Fig. 4A).
The highest amount of cytokine production was observed when cells were treated with high concentrations of M-HA (Fig. 4A). The amount of cytokine production did not increase after longer periods of incubation (72 or 120 hours after infection) (data not shown). When TNF-α production was evaluated in non-infected cultures, similar values were obtained in radical scavenger-treated and non-treated macrophages (data not shown). Low but detectable amounts of NO production between 1 and 3 μM of NO were found in both BCG-infected radical scavenger-treated and untreated macrophages. No significant differences were observed between HA, HU and M-HA-treated macrophages. These data are consistent with previous data from [25].
When the production of IL-10 and IL-12 was studied in BCG-infected macrophages treated with the different radical scavengers, the results differ between cytokines and treatments. While IL-10 production was not detected in any case (data not shown), IL-12 production was significantly increased in HA-treated macrophages in a dose-dependent manner (Fig. 4B). At the highest HA concentration, however, IL-12 production dramatically diminished, probably due to the reduced presence of viable macrophages (Fig. 2).

M-HA inhibits P. aeruginosa biofilm formation
As explained previously, good M-HA antibacterial activity was also observed against P. aeruginosa (SI = 52.4). Thus, we further investigated the capacity of M-HA in reducing P. aeruginosa biofilms because it is one of the most important forms of persistent bacteria and is a characteristic of chronic P. aeruginosa infections. We used the quantitative microtiter plate method to determine the effect of the different hydroxylamine derivative compounds on biofilm formation. A dose-effect concentration was observed for each compound (Fig. 5). At 20.6 μg/ml HA and 82.5 μg/mL HU and M-HA, P. aeruginosa growth was completely arrested and no biofilm was formed (Fig. 5).
The M-HA effect on a preformed P. aeruginosa biofilm Once a biofilm has been established, the cells are extremely resistant against all types of antibiotics and detergents and it is often challenging to remove. The effects of the different radical scavengers on existing P. aeruginosa biofilms were initially assessed using crystal violet-based biomass staining assay. As Fig. 6 shows, all compounds reduce the amount of biofilm formed, reaching values of approximately 55%, 90% and 70% reduction from HU, HA, and M-HA,    Disassembling the existing P. aeruginosa biofilms by adding HU, HA and M-HA. P. aeruginosa bacteria were allowed to form biofilms in peg plates for 24 h, the medium was removed and fresh medium with different concentrations of radical scavenger compounds were changed every 24 hours over three days (Days 1, 2 and 3). The percentage of biofilm biomass production is represented for each day. The results are existing biofilm can be removed or disaggregated. Moreover, the highest biofilm reductions under M-HA treatment were found after three days of treatment, with 20% less remaining biofilm compared with the first day of treatment (Fig. 6). Further, confocal scanning laser microscopy of a biofilm grown in a flow cell chamber in the presence of the different radical scavengers showed significantly reduced biomass and average thickness of the treated samples (HA, HU and M-HA) compared to the untreated sample ( Table 2 and Fig. 7).

Synergic effects on biofilm reduction by ciprofloxacin plus M-HA
The capacity to remove a pre-existing biofilm was evaluated when treating with ciprofloxacin in combination with M-HA. Ciprofloxacin alone showed a dose-dependent effect on P. aeruginosa biofilm reduction (Fig. 8). However, in comparison with treatments that employed ciprofloxacin alone, the combined use of ciprofloxacin and M-HA was more efficient for removing pre-existing biofilm, yielding 50% reduction values at ciprofloxacin concentrations of 0.016 or 0.008 μg/ml, and 6.6 or 86 μg/m of M-HA, respectively, was added. (for 5 to 8 times lower concentration than ciprofloxacin alone to have the same effect) (Fig. 8).

Discussion
The clinical use of RNR inhibitors has a history of several decades, and this history has demonstrated that RNR inhibitors have antitumor activity alone or in combination with other drugs [13,18,19]. Among RNR inhibitors, some radical scavenger compounds derived from hydroxylamines such as HU have been used for some types of cancer treatment [18]. Both HA and HU dramatically affect eukaryotic viability, and thus little interest has been aroused in studying these compounds as antimicrobial agents. It remains an important goal to develop a novel HA derivative with low toxicity and improved cytostatic action especially for treating bacterial infections. We previously demonstrated the capacity of the HA derivative M-HA in inhibiting RNR enzymatic activity in B. anthracis [11], although its potential as an antimicrobial drug has not been investigated. When exploring the role of M-HA in inhibiting the growth of a wide range of pathogenic Gram-positive or Gram-negative bacteria during a comparison of HA and HU, the intracellular bacterial growth and the formation of biofilms was evaluated.
the means ± SD of three-five replicates from one representative of two independent experiments. A Student's t-test was performed (*, P < 0.05; versus non-treated biofilms). HU, hydroxyurea; HA, hydroxylamine; and M-HA, methyl-hydroxylamine. Our results show that HA and M-HA are more efficacious that HU as antimicrobial agents for both Gram-positive and Gram-negative bacteria (Table 1). For instance, P. aeruginosa and M. tuberculosis use an iron containing class I RNR (class Ib) [26,27] while B. anthracis clearly uses a manganese class I RNR (Class Ib) [28]. Our results suggest that M-HA is an active inhibitor for both iron and manganese forms of RNR.
When eukaryotic cytotoxicity was evaluated, HU and HA exerted high toxicity in murine macrophage cells (CC 50 of 25.8 and 12.9 μg/ml, respectively) as expected (Fig. 2). Our results are consistent with previous results in which HU and resveratrol (a radical scavenger aromatic compound that is not derived from HA) were efficacious against pathogenic bacteria (P. aeruginosa, Propionibacterium acnes, S. aureus, and Enterococcus faecalis) but had high toxicity in eukaryotic cells as well [29,30]. However, M-HA showed a highly reduced toxic effect on macrophage culture (CC 50 of 351 μg/mL), corroborating the low toxicity of M-HA that was also found against human lung fibroblasts [31]. Thus, our first results confirm the promising role of M-HA as an antimicrobial agent.
Because of their small sizes, these compounds would easily cross the cell wall and membrane and exert their antimicrobial activity directly through RNR enzyme inhibition. In previous works, we determined that M-HA specifically inactivates the bacterial enzymatic activity of the essential RNR enzyme by quenching the tyrosyl radical that is necessary for enzymatic activation, thus making it unable to form dNTPs and blocking DNA synthesis [11]. The exact mode The synergistic effect of ciprofloxacin and M-HA on the reduction in P. aeruginosa biofilm formation. P. aeruginosa bacteria were allowed to form biofilms in peg plates for 24 h, the medium was removed and fresh medium with different concentrations of ciprofloxacin with/without M-HA were added. Biofilm formation (crystal violet stain) was evaluated 24 hours later. The biofilm biomass production percentage is represented here. The results are the means ± SD of three-five replicates from one representative of two independent experiments. A Student's t-test was performed (*, P < 0.05; **, P < 0.005 versus ciprofloxacin treated biofilms). CPX, ciprofloxacin; and M-HA, methyl-hydroxylamine.
doi:10.1371/journal.pone.0122049.g008 of M-HA is not completely understood, but some authors hypothesize that this molecule can interact directly in the places where the tyrosyl radical is formed because of its small size [32]. In terms of HU, which is a bigger molecule than M-HA, it seems that the interaction does not occur directly where the tyrosyl radical is generated but is more directed at interrupting the catalytic electron transfer pathway of the small subunit at the interface between the interaction between the small (α) and large RNR (β) subunits [33][34][35]. This finding could explain why M-HA was approximately several orders of magnitude more effective at inhibiting bacterial RNR than HU. Confirmation was established with the use of HA, the smallest HA, with the highest antimicrobial activity that also presents the highest toxicity because this molecule surely interacts with and can reach the tyrosyl radical site easily in both prokaryotic and eukaryotic RNR. Another issue is to understand the precise mechanism of action for the bacterial killing of radical scavengers after blocking the RNR enzyme. In E. coli [36] HU has been recently shown to cause increased superoxide production, and together with increased iron uptake, this increase fuels the formation of hydroxyl radicals that contribute to HU-induced cell death. Based on the HU analogy, we believe that the M-HA mode of action or bacterial killing might be similar.
Interestingly, M-HA has the highest therapeutic index for cytotoxicity (SI) for its inhibition of M. bovis BCG (182.6 μg/ml), which is 3.5 times that of P. aeruginosa (52.5 μg/ml). Because of the global relevance of these two agents, we should search for the possible role of M-HA in treating these agents.
To measure the potential growth inhibitory capacity of these compounds against mycobacteria, we selected BCG as a model in murine macrophages. Although there are significant differences between M. tuberculosis and BCG, they are closer genetically, and previous studies have demonstrated the validity of BCG for the in vitro evaluation of drug candidates against tuberculosis [37,38]. In our case, a comparison of the BCG RNR primary protein structure showed 100% shared identity with that of the M. tuberculosis RNR protein, the causative agent of tuberculosis (see S1 Fig.). This finding indicates a possible identical mode of action for M-HA on the M. tuberculosis RNR. BCG intracellular kinetics in J774 macrophages has been very well characterized [25,39]. Initially, J774 macrophages ingest mycobacteria and internalize BCG cells into phagosomes. An initial killing is observed during the first three days of infection, and then a stable level of viable BCG can be observed in J774 macrophages. Despite the fact that BCG does not grow as exponentially inside macrophages as M. tuberculosis does, BCG is continuously growing and being killed by macrophages [25]. This finding permits the use of this model for evaluating new drug candidates with the capacity to interfere in mycobacterial DNA synthesis.
Our results showed that up to 85% of mycobacterial growth inhibition by M-HA occurs at 72 hours post-infection (Fig. 3). In view of the impressive results obtained here, we aimed to go further in understanding the mechanism. As expected [40], our results showed that BCG-infected macrophages do not induce NO production. Treatment with HA-derivatives did not significantly modify these results. However, enhanced TNF-α production, which is another killing mechanism of macrophages, is observed in M-HA-treated macrophages. The increased production of TNF-α in BCG-infected M-HA-treated macrophages could be explained by different reasons. On the one hand, BCG-infected J774 macrophages release exosomes that contain mycobacterial antigens such as a 19 kDa antigen or phosphatidyl inositol mannosides known to induce pro-inflammatory cytokines such as TFN-α by exerting bystander effects on other cells [41]. The M-HA treatment could induce a higher production of these exosomes than non-treated macrophages. On the other hand, M-HA could directly induce TNF-α production in BCG-infected macrophages. Two reasons led us to support this last option. First, TNF-α production is observed as early as 24 hours post-infection (Fig. 4), whereas the exosome released from BCG-infected J774 macrophages is primarily detected between 48 and 72 hours post-infection [41]. Second, an M-HA dose-dependent response is observed in TNF-α production, leading us to favor the second hypothesis. The fact that TNF production levels in non-infected M-HA-treated macrophages were lower than those detected in M-HA-treated BCGinfected macrophages indicates that a BCG-infection must be related to the capacity of M-HA to induce TNF-α production, as was previously described for other drugs [42,43]. Nevertheless, we cannot rule out other possible mechanisms, such as the influence of such radical scavengers in other routes related to cytokine production. Supporting this idea, significant IL-12 levels were only detected in BCG-infected HA-treated macrophages. Although in vitro and in vivo data indicated that HU or HA treatment triggers chemokines and/or cytokines production [44][45][46][47][48], the mechanism by which these compounds interfere with cytokine-mediated signaling is also unclear. TNF-α and IL-12 are critical cytokines in the control of mycobacterial infections [49]. Synergically with IFN-γ, the compounds activate naive macrophages, which in turn help to control mycobacteria growth. Previous studies demonstrated that TNF-α and IL-12 production is differently regulated in mycobacteria infected macrophages [50]. Thus, our results provide an initial step to further understand the possible role of cytokines production in BCG cell growth inhibition as mediated by these radical scavengers.
To our knowledge, this is the first report describing the use of a radical scavenger and more specifically the first to observe the M-HA effect on mycobacterial inhibition. Among infectious diseases, tuberculosis (TB) is the leading killer with over two million casualties annually worldwide. The WHO considers tuberculosis to the most dangerous chronic disease in the world. In recent years, the emergence and spread of resistant M. tuberculosis strains has fuelled the TB epidemic by making it more difficult to treat. These results make the M-HA a potentially valuable agent, and further analyses must be performed to test it in a combination therapy with existing, well-known antimycobacterial drugs.
Regarding P. aeruginosa, the primary point of interest is an evaluation of the capacity to reduce biofilm formation. Considering that biofilm formation protects bacteria during infections, such as in chronic wounds, hospital-related pneumonia and bacterial chronic lung infections [51], the inhibition of biofilm formation by M-HA is a critically important quality as a potential therapeutic, possibly more so than its anti-microbial activity. Bacterial biofilms generally become 10-1000 times more resistant to the effects of antimicrobial agents than planktonic cells [52].
We were able to demonstrate that M-HA is capable of inhibiting P. aeruginosa biofilm formation (for an approximately 60% reduction at 5.2 μg/mL on the third day of continuous treatment); this study compared favorably with other studies that employed different anti-biofilm strategies [53]. These results were corroborated by growing P. aeruginosa cells to form a continuous biofilm in flow cells and imaging by laser scanning confocal microscopy (Table 2 and Fig. 7). Moreover, the combination of M-HA with the well-known antibiotic ciprofloxacin increased the ability to reduce an existing biofilm 10-20 times better than ciprofloxacin or M-HA alone. Using new and existing antimicrobials may provide a new strategy for bacterial therapy. Drug combinations would decrease the likelihood of resistance [54]. This finding demonstrates the feasibility of combined chemotherapy with known antibiotics for combating multi-resistant bacteria infections.
The global emergence of antibiotic-resistant strains continues unabated, along with an overall increase in the number of infections worldwide, highlighting the urgent need for new agents to treat Mycobacterium and Pseudomonas infections. Non-conventional anti-infective approaches must be explored. Our findings may provide a new basis for the discovery of new and potent radical scavenger inhibitors and improved clinical applications of these compounds in antimicrobial therapy.