Tuberculosis (TB) and paracoccidioidomycosis (PCM) are responsible for important systemic lung diseases and are distributed throughout the world and Latin America, respectively, especially in Brazil (World Health Organization, 2018). PCM is has a large presence in Latin America, most frequently in Argentina, Brazil, Colombia, Venezuela, Ecuador, and Paraguay, with an occurrence of 80% of cases in Brazil and a high incidence in the states of São Paulo, Paraná, Rio Grande do Sul, Goiás, and Rondônia (Martinez, 2017; Shikanai-Yasuda et al., 2017). Both diseases are important public health problems, with high mortality and morbidity indices (Ostrosky-Zeichner, 2012; Martín-Peña et al., 2014). Mycobacterium tuberculosis and Paracoccidioides brasiliensis, the etiological agents of these diseases, can be present as comorbidities, as has been documented in the literature, with an approximate occurrence of 2–20% (Shikanai-Yasuda et al., 2017). Currently, this infection is present in endemic and nonendemic areas due to migratory processes (Guery et al., 2015). In addition, this comorbidity can affect approximately 15–20% of immunocompromised patients (Quagliato et al., 2007; Martinez, 2017). There are few clinically reported cases of both diseases because the lack of medical records and similar symptoms can lead to misdiagnosis (Quagliato et al., 2007; Guery et al., 2015). Misdiagnosis can cause the death of the patient due to the lack of adequate treatment, which can be exacerbated by treatment resistance. This resistance can be caused by the formation of a mixed biofilm of these diseases (Sardi et al., 2014; Guery et al., 2015; Martinez, 2017). Thus, biofilms are organized and dynamic communities of microorganisms that strongly adhere to biological and non-biological surfaces, and serve as a niche for microorganisms and defense mechanisms against hostile environments, in addition to being reservoirs of persistent substances that can cause fatal infections (Costa-Orlandi et al., 2014; Del Pozo, 2018).

Mycobacterium spp. can form biofilms in vitro and in vivo with increased virulence and resistance to antibiotics, due to the difficulty of infusing the drug through the biofilm matrix. (Flores-Valdez et al., 2015; de la Cruz et al., 2020). According to Flores-Valdez et al., 2015, evaluations of chronic infections in rats have shown a tolerance to antibiotic drugs, which have been frequently related to the presence of biofilms in these infections (Flores-Valdez et al., 2015; Flores-Valdez, 2016).

According to Chakraborty et al. (2021), the formation of the M. tuberculosis biofilm was demonstrated in animal models in rats and primates as well as in sections of lung tissue obtained from patients with tuberculosis, demonstrating that the biofilm protects the bacilli from the immune system of the host, in addition to showing that isoniazid and rifampicin antimicrobial activity were reduced in infected mice, indicating the role of biofilms in phenotypic tolerance to drugs (Chakraborty et al., 2021).

The formation of the M. tuberculosis biofilm is facilitated by the aggregate-driven hydrophobicity which is associated with the presence of mycolic acids, GroEL1 chaperone, and glycopeptideglycans (Ojha et al., 2008; Nayak, 2015; Esteban and García-Coca, 2018).

Nayak et al. (2015) demonstrated the ability of Mycobacterium avium to form biofilms due to the presence of certain phospholipases (Nayak, 2015). Our research group demonstrated that P. brasiliensis is capable of forming biofilms in vitro; one of the factors that influence biofilm formation is the presence of hydrolytic enzymes and specific adhesins in the yeast biofilm (de Cássia Orlandi Sardi et al., 2015). Cattana and collaborators (2017), demonstrated the presence of a biofilm ofP. brasiliensis in vivo in a 63-year-old patient with aorto-bifemural vascular prosthesis (Cattana et al., 2017).

Bacteria and Candida albicans are part of the intestinal microbiota, which can induce the formation of mixed biofilms in the blood circulation. According to a study conducted by Pornpimol et al. (2020), the formation of several monospecies biofilms such as Acinetobacter baumannii and Pseudomonas aeruginosa (PA) showed a formation less and more prominent, respectively. A mixed formation of C. albicans with P. aeruginosa (PA + CA) was shown to induce the formation of larger biofilms among the groups of mixed organisms and was even larger in the simultaneous formation of the same (Pornpimol et al., 2020).

Biofilm tolerance of antibiotics is multifactorial and is attributed to the low penetration of antibiotics, restricted growth with low oxygen tension, regulation of specific genes, and the presence of persistent cells (Ciofu et al., 2017). Thus, there is a need for the discovery of new compounds as chalcones, which are precursor substances for the synthesis of flavonoids and isoflavonoids with antitumor, antifungal, antibacterial, and anti-inflammatory properties (Pinho-Ribeiro et al., 2015; Wang et al., 2016).

Previous studies of our research group have demonstrated the potent antifungal activity of 2’hidroxychalcone against the fungus P. brasiliensis (Medina-Alarcón et al., 2020). According to Ramesh and collaborators, (2020) the indole chalcones presented antitubercular activity against the H37Rv strain of M. tuberculosis with MIC values of 210, 197, and 236 μM (Ramesh et al., 2020). The compound 3´hydroxychalcone (3’chalc) shows potent antifungal activity against Cryptococcus gattii in planktonic and biofilm forms in vitro and in vivo (Palanco et al., 2017). However, low solubility and instability allow chalcones to be incorporated into drug delivery systems to increase bioavailability, increase antimicrobial properties, and minimize the collateral effects (Talluri et al., 2016; Medina-Alarcón et al., 2017a; Yonashiro Marcelino et al., 2018). Among systems for chalcone release, nanoemulsions (NE) are widely used to improve their physicochemical properties (Čerpnjak et al., 2013; Kalepu et al., 2013; C. de Lima et al., 2020).

NE are heterogeneous systems that consist of an oil phase dispersed in an aqueous solution phase and stabilized by a surfactant or emulsifying agent; they have diameters that can vary from 50–200 nm. Those with smaller droplet sizes provide a large surface area to volume ratio, with faster absorption and greater uniformity in drug administration (Ganta et al., 2014; Medina-Alarcón et al., 2017b; dos Santos Ramos et al., 2019).

The NE of Cymbopogon flexuosus exhibited significant antimicrobial activity against Mycobacterium fortuitum, Mycobacterium massiliense, and Mycobacterium abscessus and was efficient against extant biofilm, while the free oil inhibited mycobacterial biofilm formation (Rossi et al., 2017).

A study was performed by Quatrin et al. on a NE containing Eucalyptus gloubulus oil, which did not present antimicrobial activity against P. aeruginosa. However, the NE was more efficient for Candida spp. biofilms than for oil-free (Quatrin et al., 2017).

It has been demonstrated that the microorganisms under study can form biofilms in vitro and in vivo; P. brasiliensis can form biofilms in patients, and M. tuberculosis can form biofilms in primates and lung tissue of patients, as has been reported previously. Although there are still no studies on the formation of mixed biofilms of these microorganisms, the comorbidity of PCM and tuberculosis is an alarming reality. Therefore, the present research aims to show for the first time the formation of the mixed biofilm of M. tuberculosis and P. brasiliensis in vitro, as well as to evaluate the action of NE loaded on 3’chalc (NE3’chalc) against monospecies and mixed biofilms.

M. tuberculosis biofilms play an important role in the process of caseous necrosis and cavitation formation in the lung tissue (Kulka et al., 2012b; Basaraba and Ojha, 2017). In addition, M. tuberculosis can coexist with other mycobacteria, bacteria, and fungi, and its ability to form biofilms probably gives it the ability to communicate with other microorganisms to form complex biofilms (Flores-Valdez, 2016; Brennan, 2017). It can coat medical devices in hospitalized and immunologically compromised patients, enabling the formation of coinfection. P. brasiliensis is increasingly recognized by clinicians as comorbidities can occur simultaneously in up to 20% of patients (Quagliato et al., 2007; Shikanai-Yasuda et al., 2017). These interactions may be the key to understanding the development of new treatments (Flores-Valdez, 2016). Thus, we evaluated the formation and treatment of mixed biofilms of M. tuberculosis and P. brasiliensis. Colorimetric assays were initially performed to evaluate the metabolic activity, quantification of biomass and extracellular matrix, and essential parameters for biofilm formation, which is an important tool for assessing the viability of eukaryotic and prokaryotic cells (Costa-Orlandi et al., 2014). The P. brasiliensis monospecies biofilm showed high metabolic activity, biomass, and extracellular matrix within 144 h of formation ( Figures 2A, B ). These results are corroborated by previous studies (de Cássia Orlandi Sardi et al., 2015; Cattana et al., 2017; Oliveira et al., 2020). The evaluation of metabolic activity, biomass, and extracellular matrix of M. tuberculosis H37Rv (ATCC) and the 40 Rv clinical strain presented a strong and mature biofilm within 45 days of formation, showing a high survival and leading 45 days to be recognized as the optimal amount of time to allow for formation. This evaluation also showed that it did not present a significant difference (* = p < 0.05) in the three evaluations carried out when compared with ATCC and the clinical strain, showing a similar pattern of behavior between all three ( Figures 2C and 3A, C ). Previous studies have demonstrated the formation of a biofilm of M. tuberculosis at 5 weeks and observed a strong propensity to grow in organized multicellular structures, with biomass production being directly related to biofilm survival (Kulka et al., 2012; Trivedi et al., 2016). The mixed biofilms formed in the first association formed with M. tuberculosis H37Rv (ATCC27294) and 40Rv (clinical strain) were initially incubated following the subsequent assistance of P.brasiliensis forming the (H37Rv + Pb18) and (40Rv + Pb18) biofilms ( Figure 1B.1 ). This association was considered in cases of comorbidity reported in patients who had TB and with compromised immune systems who acquired PCM, which is an opportunistic disease (Torres-Pereira et al., 2009; Martinez, 2017). These mixed biofilms also showed high metabolic activity, biomass production, and extracellular matrices. In general, the curves increased constantly from day 20 after formation until day 45 at the complete formation of the mature mixed biofilm. The results of these evaluations were comparable to those of the monospecies biofilms and showed no significant difference (^* = p <0.05) in metabolic activity assessment and biomass quantification. However, the extracellular matrix did show a significant variation between mixed biofilms and H37+Pb18 and 40Rv monospecies biofilms as shown in Figures 2C , 3A, C , implying that the microorganisms present in the mixed biofilms did not interfere completely with the development of each other in this interaction. Thus, the multispecies biofilms presented different behaviors and m benefit from co-aggregation, which enables them to compete, or have antagonistic interactions (Rodrigues et al., 2020). In the development of polymicrobial biofilms, consisting of fungi and bacteria, C. albicans can develop together with Staphylococcus aureus and Staphylococcus epidermidis can also develop together with Enterococcus spp. in the course of blood-borne infections (Harriott and Noverr, 2010; Kim et al., 2012).

The second condition of biofilm (PB18 + H37Rv) formation consists of the initial formation of Pb18 with the subsequent addition of M. tuberculosis ( Figure 1B.2 ). This association was due to the initial comorbidity cases of patients with PCM who acquired tuberculosis due to their critical condition (Nunura et al., 2010; Guery et al., 2015). The results showed a high quantification of metabolic activity, biomass, and extracellular matrix during the 30 days of formation, with a decay in production occurring within 35 days that continued until the last day of formation at 45 days. This association showed no significant difference ^* = p < 0.05 when compared to their monospecies biofilms in the three evaluations; however, the metabolic activity showed a significant difference in a single time within 40 days of formation (^** = p < 0.01). This association did not significantly interfere with the development of one or both microorganisms present in the mixed biofilm in ( Figures 2D , 3B, D ). Therefore, the order of formation of microorganisms in a biofilm is an important factor of formation, whether it is bacteria or fungi. The production of biomass and the extracellular matrix can change, with greater or lesser production of these components by each species individually (Foster and Bell, 2012; Ren et al., 2015; Tan et al., 2017; Wijesinghe et al., 2019). A study conducted by Rodrıguez-Sevilla et al. (2018) demonstrated the formation of monospecies and dual-species of M. abscessus and P. aeruginosa. This evaluation showed a difference between the monospecies mycobacterial biofilm and its dual-species after 24 h of formation, slightly modifying the formation (Rodríguez-Sevilla et al., 2018).

Following the SEM analysis, it was shown that clinical biofilm strains of M. tuberculosis H37Rv in ( Figures 4A, B ) and 40Rv in ( Figure 4C, D ) formed biofilms on the polystyrene surface for 45 days in the presence of high biomass, with a consistent and dense network of characteristic bacilli growing in all strain directions and covered by an extracellular polysaccharide (EPS) matrix that constitutes the ultrastructure of a mature biofilm. M. tuberculosis 40Rv proved to be a more compact biofilm than H37Rv and showed a greater amount of EPS, especially in some points where greater accumulation was observed. The 40Rv bacilli showed different sizes and dimensions, unlike the H37Rv bacilli which were more homogeneous. Biofilms formed by mycobacteria have the same definition as other biofilms and can develop these structures on surfaces as well as at aerial interfaces (Ojha et al., 2008). Canetti discovered a biofilm of M. tuberculosis in vivo which exhibited dense layers of bacilli that did not adhere to the surface within the granuloma (Canetti, 1965; Bacon et al., 2014). Among the factors involved in the formation of M. tuberculosis biofilm, PE11 is a lipase associated with the cell wall expressed by Mtb both in vitro and in vivo, modulating its lipid profile and cell wall architecture, such as adding mycolic acids and threolase esters (Islam et al., 2012; Rastogi et al., 2017). In addition, the monospecies P. brasiliensis biofilm in ( Figures 4E, F ) showed a solid network of yeast embedded with an extracellular polysaccharide matrix (EPS), which has been shown by our research group, indicating that P. brasiliensis can form a biofilm (de Cássia Orlandi Sardi et al., 2015). The biofilm of Paracoccidioides spp. was demonstrated in vivo in a 63-year-old patient in the aorto-bifemural vascular prosthesis after implantation in the patient, as confirmed using SEM (Cattana et al., 2017; Lorente-Leal et al., 2019).

The mixed biofilms (H37Rv + Pb18) in ( Figures 5A–C ) and (40Rv + Pb18) in ( Figures 5D–F ) presented a dense structure where the M. tuberculosis bacilli unit formed a highly organized network of bacilli that connect with the yeasts, creating a coexistence between the two microorganisms. However, the mixed biofilm formed with the clinical strain (40Rv + Pb18) revealed a more compact biofilm formation, with a greater accumulation of EPS at some points. This appeared in larger blocks, and the yeasts were more intertwined than in the mixed biofilm (H37Rv + Pb18). According to Costa et al. (2020) the study was carried out in hemodialysis samples from hospital environments; biofilms of single and double species of Candida parapsilosis and M. smegmatis were found where this bacterium benefits from its association with C. parapsilosis, which retards the growth of the fungus and allows the coexistence of microorganisms (Costa et al., 2020). Another study demonstrated the interaction of C. albicans with Streptococcus mutans microorganisms present in periodontitis lesions, which showed synergism with an increase in the virulence of biofilm (Falsetta et al., 2014; Chevalier et al., 2018).

In contrast, in the second combination of mixed biofilm (Pb18 + H37Rv) in ( Figures 5G–I ), a large number of open yeasts of P. brasiliensis was observed at the base of the biofilm, connecting to the fragmented bacilli and forming a compact and complex structure; thus, characterizing the mature biofilm. Although the quantification results did not show a great interference in biofilm formation, in this evaluation, this interference becomes more evident. This is due to the previous formation of the biofilm, P. brasileinsis, which can produce adhesins, such as GP43 and 30 kDa, and virulence proteins, such as glyceraldehyde-3-phosphate-dehydrogenase (GAPDH), involved in the pathogenesis of the fungus, and are important factors for its formation (de Cássia Orlandi Sardi et al., 2015). Other studies have shown that C. albicans and P. aeruginosa exhibit competitive action and antagonistic behavior; this interaction has a distinct clinical implication if they occur in an immunocompromised host (Shirtliff et al., 2009; Bernard et al., 2019).

In agreement with previous results, the order of formation is an important parameter for determining the coexistence or prevalence of one of the microorganisms present in the mixed biofilm. Polymerase chain reaction (PCR) was performed to identify the microorganisms present in the mixed biofilm in all associations. According to our results, multiplex PCR confirmed the presence of M. tuberculosis H37Rv (ATCC 27294), 40Rv (clinical strains), and P. brasiliensis, which are involved in the formation of mixed biofilms ( Figure 6 ). According to Díez et al. (1999), PCR specifically targets the gp43 protein that is widely used in the identification and diagnosis of adhesion and biofilm formation in P. brasiliensis (Correa et al., 2007; Reis et al., 2008; de Cássia Orlandi Sardi et al., 2015).

Amini et al. (2015) evaluated samples from 100 patients using bronchoalveolar lavage samples from patients with suspected TB using multiplex PCR, with a specific gyR gene target used in the identification of M. tuberculosis. They obtained a specificity of up to 80 and a sensitivity of 97.8% (Amini et al., 2015; Sharma et al., 2018), confirming the presence of both microorganisms studied in the mixed biofilm.

Owing to the lack of treatment for this coinfection and the toxic effects of the standard drugs used in the treatment of TB and PCM, there is a need for new treatment alternatives to reduce side effects and increase their biological activity (Brime et al., 2004; Ferrara et al., 2011; Combrink et al., 2020). NE are controlled drug release systems that allow greater bioavailability and stability of the administered drug (Souza et al., 2015; Medina-Alarcón et al., 2017b). The main reason why 3’chalc was incorporated into NE was to improve its physicochemical characteristics and increase its antibiofilm activity (Medina-Alarcón et al., 2020).

The results of the present study showed that the formulations evaluated were thermodynamically stable and translucent, and all are within the parameters of normality observed in Table 1 . The nanoemulsions (NE) had a particle size of 145.0 nm and NE3’chalc (nanoemulsion loaded with 3’hydroxichalone) had a diameter of 151.25 nm. Both formulations were within the range of 10-200 nm, hence the formulations studied are within the defined value (Goyal et al., 2012; Medina-Alarcón et al., 2017b; Haider et al., 2020). Another parameter of physicochemical characterization is the polydispersity index (PDI), which is calculated by dividing the average droplet size by the average number of droplets measured for each formulation. The value obtained for all formulations was 0.20 for (NE) and 0.16 for NE3’chalc. Several studies have reported that the ideal distribution of the (PDI) is the value (< 0.5); therefore, these results reflect the homogeneity in the formulations (da Silva et al., 2014). The potential zeta of the formulations under study are within the parameters of normality: the values were -58.20 and -56.10 mV for NE and NE3’chalc respectively, and the reference values range from -30–30 mV. This parameter allows forecasts on the stability of colloidal dispersion storage (Wang et al., 2006; Araújo et al., 2011). Another physicochemical characterization parameter was morphological and distribution evaluation through AFM, a widely used technique for nanostructured lipid system (SNL) evaluation (Starostina et al., 2008). The evaluated formulations of NE and NE3’chalc showed homogeneous systems with lamellar characteristics, and particle size distribution for NE within 145 nm and NE3’chalc within 153.85 nm ( Figures 7A–D ), which is consistent with previous results, showing formulations within the normality parameters in the evaluation of NE and nanoparticles (Marquele-Oliveira et al., 2016).

In order to reduce the cytotoxicity and increase its antimicrobial activity, the cytotoxicity assay was performed in which our results showed that NE and NE3’chalc have no toxic effect and NE3’chalc reduces the cytotoxicity of the free drug, increasing its cellular viability by 48%. These results ( Figure 8 ) show that the NE are biocompatible formulations and that the lipids used in the formulation are well tolerated by eukaryotic cells (Medina-Alarcón et al., 2020). In addition to the reduction in cell uptake, their toxic effects were reduced by reducing the dose to the host (Nakamura et al., 2018).

Subsequently, the antifungal and antitubercular activity of NE3’chalc presented MIC values at concentrations lower than that of 3’chalc, thus presenting a better antifungal and antitubercular action than the free drug and showed a better antifungal action than the control RIF used in the treatment. The NE was also evaluated, and it did not show antifungal or antitubercular activity, which demonstrates that it does not interfere with the action of 3’chalc ( Table 2 ). This was due to the efficient increase in the release of drugs when incorporated into the NE, thereby increasing drug solubility, biological activity, and bioavailability (Kawakami et al., 2002; Thakur et al., 2013; Hertiani et al., 2019). This result can be facilitated by the lipid composition of NE, such as cholesterol and phosphatidylcholine, which easily interact with the fungal or bacterial membrane, thus destabilizing their integrity and allowing an increase in drug permeability and the consequent death of microorganisms (Sha et al., 2005; Nirmala et al., 2013; Nikonenko et al., 2014; do Carmo Silva et al., 2020). Medina-Alarcón et al. (2020) demonstrated an increase in the antifungal activity of 2’hydroxychalcone when incorporated into a NE, managing to significantly increase its activity against Paracoccidioides spp. (Medina-Alarcón et al., 2020).

Owing to the antibacterial and antifungal activity presented by NE3’chalc, the antibiofilm activity of this formulation was evaluated against the monospecies biofilms of P. brasiliensis and M. tuberculosis (ATCC H37Rv and Rv40 clinical strain) and the mixed biofilms formed in the first condition of association (H37Rv + Pb18 and 40Rv + Pb18). According to our results, NE3’chalc showed the best results in evaluating the reduction in metabolic activity, amount of biomass, and extracellular matrix of monospecies and mixed biofilms. Their SMIC₅₀ values showed a reduction in their concentrations by 4–8 fold for monospecies biofilms and 2-8 fold for mixed biofilms when compared to free 3’chalc ( Table 3 ). In addition, NE3’chalc showed a greater reduction in metabolic activity, biomass, and extracellular matrix significantly (^#p < 0.001), managing to reduce the metabolic activity of the mixed biofilm up to 42% more when compared to 3’chalc free and with the RIF treatment control ( Figure 9 ). The high lipophilicity of the NE components allows it to effectively interact with the biofilm cell membrane, which is highly lipophilic and allows for a change in cell permeability and consequent death of the microorganisms (Andrade-Ochoa et al., 2015).

Similarly, Rossi et al. (2017) evaluated Cymbopogon flexuosus NE against M. fortuitum (ATCC 6841), M. massiliense (ATCC 48898), and M. abscessus (ATCC 19977) in planktonic and biofilm form, showing antibiofilm action mainly in its nanostructured form (Rossi et al., 2017).

Finally, the SEM evaluation results for NE3’chalc corroborate our previous results and show that NE3’chalc obtained the best results when compared to 3’chalc and RIF, with the near elimination of the biofilm with biofilm rupture, leakage of components cells, and almost complete elimination of monospecies and mixed biofilm, and with some structural alterations of the yeast membrane in theP. brasiliensis biofilm and the mixed biofilm ( Figures 10 – 12 ).

Because the systems can act as drug reservoirs, which allow the release of the active principle from the internal to the external phases, the process of adhesion of the microorganism is possible due to the oil phase of the system and is involved in the interaction with the cell membrane components of the microorganism, such as ergosterol, mycolic acids, and other membrane lipids, allowing the destabilization of the integrity and consequent lysis and death of the microorganism in planktonic form or biofilm (Aiemsaard et al., 2011; Nirmala et al., 2013; Rossi et al., 2017). In addition, Song et al. (2016) showed that MRSA cells were analyzed using microscopy scanning electronics, where bacteria treated with CHX (nanoemulsion) at a concentration of 0.5 µg/mL showed homogeneously distributed cytoplasm after treatment with NE, and a change was observed in the cell that led to a change in the permeability of the cell membrane (Song et al., 2016). NE3’chalc had the highest antibiofilm activity and demonstrated that the system is extremely promising for the treatment of monospecies biofilms and especially mixed biofilms.

Given the above, we can conclude that our results are extremely important, as they show for the first time the formation of mixed biofilms of M. tuberculosis and P. brasiliensis, which are microorganisms responsible for serious pulmonary infections that can lead to the death of patients due to lack of adequate treatment. In addition, our research highlights NE3’chalc, which has been shown to be an effective alternative, increasing its solubility, stability, and antibiofilm activity, and may become a promising new alternative for the treatment of mixed biofilms of these microorganisms. This is of great relevance, since there is a lack of treatment for these microorganisms that present themselves as comorbidities, especially in immunocompromised patients. More trials will be needed in the future to complement our research.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

KM-A performed the tests: monospecies and mixed biofilm formation assay, characterization of all biofilms, preparation of NLS, characterization of the physicochemical properties of the nanoemulsion, evaluation of antifungal, antibacterial, and antibiofilm activity, and writing of the manuscript. IT and GF evaluated the antifungal and antibiofilm activity of P. brasiliensis. MP-d-S performed the AFM analysis. CM performed the PCR analysis. MS prepared and characterized the 3’hydroxychalcone. LR professor guided in the preparation and characterization of the 3’hydroxychalcone. MC professor of nanotechnology oriented in the preparation and characterization of the nanoemulsion and corrected the manuscript. FP professor guided in mycobacterial assays. MM-G professor guided in the biological tests and corrected the manuscript. AF-A professor guided in the biological assays and corrected the manuscript. All authors contributed to the article and approved the submitted version.

This research was funded by the Coordenacão de Aperfeiçoamento Pessoal de Nıvel Superior (CAPES). Pró-Reitoria de Pós-Graduação (PROPG), PDI-UNESP, Conselho Nacional de Pesquisa e Desenvolvimento (CNPq), Programa de Apoio ao Desenvolvimento Científico e Tecnologico (PADC- UNESP), Fundaçao de Amparo a Pesquisa do Estado de São Paulo (FAPESP; grant number: 2013/05853-1).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.