Synthesis of Supports

The highest purity reagents commercially available, In₂O₃ (99.9% Rare Metallic Co., Ltd.) and TiO₂ (99.9% Rare Metallic Co., Ltd.), were mixed in 1:1 molar ratio, and grounded in an agate mortar. The mixture was molded into pellets and annealed in air at 1250 °C in a Thermolyne^TM 1300 furnace for 48 h. Then, In₂TiO₅ was obtained through conventional solid state reaction similar to previously-reported methods (^{Muñoz et al., 2016}; ^{Senegas and Galy, 1975}). Comercially-available hydroxyapatite (HA reagent grade 36.2 % Ca Aldrich) was used to form a HA+In₂TiO₅ mixture at a 1:1 mass proportion. A total mixture mass of 50 mg of HA+In₂TiO₅ were prepared in a cylindrical shape in a Carver® model C hydraulic press using 0.5 ton for 10 s. Next, supports were synthetized at 1200 °C for 48 h, resulting in rigid supports.

Characterization

X-ray powder diffraction (XRD) patterns were recorded at room temperature on an X-ray powder diffractometer Rigaku® Geigerflex D/MaxB with a Cu anode and a graphite monochromator operated at 40 kV, 20 mA, a scan speed of 2°·min^-1 in 2 tetha on the interval of 10° to 70°. Scanning electron microscopy (SEM) images and energy dispersive spectra were acquired using a JEOL JSM-5410LV scanning electron microscope, equipped with an Oxford X-ray Energy Dispersive Spectroscopy (EDS) analyzer operating at 20 kV. The oxygen concentration was analyzed in a Fisher Scientific^TM Oxygen Meter using aerated Middlebrook (MDB) 7H9 medium as a control.

Ms Growth on Ceramic Mixture Supports with MDB 7H9/OADC

The ceramic supports, were placed in each well of a sterile flat-bottom 96-well plate with 100 µL of Middlebrook 7H9 broth supplemented with OADC (oleic acid, albumin, dextrose, and catalase) (7H9-S) as culture media, and 100 µL of bacterial inoculum adjusted using the turbidity McFarland standard 1 (3×10⁸ CFU/mL), further diluted at a 1:50 ratio in 7H9-S broth for the test. The plate was covered, sealed in a plastic bag, and incubated at 37 °C under normal atmosphere. After 2 days of incubation, the supports were fixed with formaldehyde at 10% for 2 hours and then were analyzed by SEM.

Resazurin Microtiter Assay Plate (REMA)

The resazurin reduction and the M. smegmatis growth were determined as previously described (^{Khalifa et al., 2013}) with modifications. The final drug concentration tested was 64 μg/mL for gentamicin. After 2 days of incubation, 30 μL of resazurin solution was added to each well, and the plate was reincubated overnight. A change from blue to pink indicates resazurin reduction and, therefore, bacterial growth. The minimal inhibitory concentration (MIC) was defined as the lowest concentration of the drug that prevented this change in color. The assays were performed using the supports with solar light exposure during 30 min and 60 min, prior to being inoculated with M. smegmatis. The sunlight exposition was carried out in Hermosillo Sonora, México; during spring, at an ambient temperature of around 38 °C and UV index ranging between 8 and 10.

Supports Characterization

The ceramic supports were prepared with HA and In₂TiO₅ in 1:1 mass proportion. The supports are white color and extremely fragile before synthesis (Figure 1), but after they become more rigid and their color turns slightly gray. Its dimensions are reduced in area and height to 4 mm in diameter and 2.5 mm in height. In Figure 2, the powder XRD of a sample of cylindrical support synthetized at 1200 °C for 48 h is shown. According to X-rays patterns, the supports were composed by Ca₃(PO₄)₂ [01-070-2075, Rombohedric]; In₂TiO₅ [01-082-0326, Orthorombic]; CaTiO₃ [01-076-2400, Orthorombic] and In₂O₃ [01-074-1990, Cubic C-Type].

Figure 1 Ceramics supports before and after the 48 h-synthesis at 1200 °C.
Figura 1. Soportes cerámicos antes y después de la síntesis a 1200 °C durante 48 h. 

Figure 2 X-ray diffraction of a ceramic support synthetized at 1200 °C for 48 h, compared with Ca₃(PO₄)₂ [ICCD card 01-070-2065], In₂TiO₅ [ICCD card 01-082-0326], CaTiO₃ [ICCD card 01-086-1393], and In₂O₃ [ICCD card 01-076-0152].
Figura 2. Difracción de rayos X de un soporte sintetizado a 1200°C durante 48 h comparado con las tarjetas de la base de datos de ICCD de Ca₃(PO₄)₂ [ICCD 01-070-2065], In₂TiO₅ [ICCD 01-082-0326], CaTiO₃ [ICCD 01-086-1393], y In₂O₃ [ICCD 01-076-0152]. 

To elucidate these results, we propose that HA (25 mg) and In₂TiO₅ (25 mg) undergo a reaction during its annealing at 1200 °C/48 h:

Reaction 1 Ca₅O₁₃P₃ + In₂TiO₅ → In₂TiO₅ + In₂O₃ + Ca₃(PO₄)₂ + CaTiO₃

The stoichiometry of the reaction: 4.99×10^-5 mol of HA and 6.99×10^-5 mol of In₂TiO₅ reveals HA as the limiting reagent, which is consistent with the proposed reaction. The phases shown by XRD were confirmed by SEM and EDS. Figure 3 presents a microphotograph of the sample obtained by backscattered electrons. We can see syntered bodies with at least four different contrasts corresponding to four different phases. Based on the EDS data, these correspond to phases Ca₃(PO₄)₂, In₂TiO₅, In₂O₃ and CaTiO₃.

Figure 3 Micrography X1000 of a ceramic support synthetized at 1200 °C for 48 h. Present phases: Ca₃(PO₄)₂, In₂TiO₅, CaTiO₃, and In₂O₃.
Figura 3. Micrografía X1000 de un soporte cerámico sintetizado a 1200 °C durante 48 h. Fases presentes: Ca₃(PO₄)₂, In₂TiO₅, CaTiO₃, y In₂O₃. 

Ms Growth on Ceramic Mixture Supports

Growth in communities appears to be a preferred survival strategy of microbes and is achieved through genetic components that regulate surface attachment, intercellular communications, and synthesis of extracellular polymeric substances (^{O’Toole and Kolter 1998}; ^{Henke and Bassler 2004}; ^{Syed et al., 2014}). There are some reports regarding the strong cellular aggregation and rapid formation of the Ms biofilm (^{Shi Fu et al., 2011}; ^{Bonkat et al., 2012}; ^{Ojha et al., 2005}). Scanning electron microscopy (SEM) revealed that Ms exhibits strong cellular aggregation and dense material (biofilm) formation (^{Syed et al., 2014}; Tingyu et al., 2011). The micrographs obtained here from supports after the incubation and M. smegmatis growth, revealed biofilm formation attached to the surface of the ceramic support (Figures 4a and 4b). These micrographs were compared with micrographs of ceramic supports without the addition of bacterial inoculums, in which a porous surface was observed. The porosity has been reported as a promotor of bacterial growth and cell adhesion to some surfaces (^{Annaz et al., 2004}; ^{Bagambisa and Joos, 1990}; ^{Salem, 2002}).

Figure 4 Micrographs of ceramic support X5000: general view (a); M. smegmatis biofilm over a support (b); M. smegmatis bacillus embedded into the biofilm (c); M. smegmatis bacterial conglomerates and biofilm formation over ceramic mixture support (d). Samples were incubated at 37 °C/48 h.
Figura 4. Micrografías de soportes cerámicos X5000: vista general (a); biopelícula de M. smegmatis sobre un soporte (b); bacilos de M. smegmatis embebidos dentro de la biopelícula (c); conglomerado de bacterias y formación de biopelícula de M. smegmatis sobre el soporte de mezcla cerámica (d). Las muestras fueron incubadas a 37 °C/48 h. 

Figure 4c shows the biofilm where bacteria are embedded in a matrix of extracellular polymeric compounds. The bacterial conglomerates were observed inside the pores and spread throughout the surface of the support (Figure 4d). Ms is known to form well-organized colonies and microcolonies which have been described by Danese et al. as a type of biofilm (qtd. in ^{Chakraborti and Kumar, 2019}). These colonies are composed of microbial cells encapsulated by a large amount of exopolysaccharides (^{Chakraborti and Kumar, 2019}).

Resazurin Microtiter Assay Plate (REMA)

Isolates of M. smegmatis were assessed using REMA on supports for resistance to gentamicin in Middlebrook 7H9 broth by cultivation on plates containing serial dilutions of gentamicin.

The MICs obtained were compared to REMA assays with and without supports of ceramic mixture and assays with Ca₃(PO₄)₂ supports (Table 1). An increment on the MIC of gentamicin that inhibits M. smegmatis is related with a favorable growth impact, attributed to the ceramic mixture supports. The MIC of assays with supports was higher by a factor of 4 compared to the MIC of assays without supports, and also twice as high as the MIC with Ca₃(PO₄)₂ supports.

Photocatalysis Induced by Sunlight

In order to evaluate the photocatalytic effect induced by the supports on M. smegmatis growth, REMA assays were performed with 30 and 60 min of exposure to sunlight prior to the bacterial inoculum. The MICs of assays employing supports with different sunlight exposure times, were the same, but these were higher by a factor of 8 compared to the MIC obtained on assays without supports (Table 1). An increase of oxygen concentration has been reported on different systems where titanium-containing materials, such as In₂TiO_5, have had a photocatalytic effect (^{Subrahmanyam et al., 2014}). Mycobacteria are strictly aerobic organisms and the oxygen generated by the material might have been used by the bacteria and favored their growth and the formation of biofilm. How does the mycobacterial biofilms form and what induces their formation have remained important questions in the field. In the last two decades, understanding the mycobacterial biofilms has evolved into a niche area for research (^{Chakraborti and Kumar, 2019}).

The dissolved oxygen in MDB 7H9 was determined using Winkler bottles and ceramic supports, after the sunlight exposure. The dissolved oxygen concentration increased by 35% on the ceramic supports-containing culture medium after being exposed to sunlight for 30 min. The maximum concentration of dissolved oxygen was 20% higher than the support-less control and it is attributed to the photocatalytic effect of In₂TiO₅, a component of the supports immersed in the culture medium (Figure 5).

Figure 5 Dissolved oxygen versus time of sun light exposure of ceramic mixture support samples in MDB 7H9 culture.
Figura 5. Oxígeno disuelto con respecto al tiempo de exposición a la luz solar de los soportes de mezcla cerámica inmersos en medio MDB 7H9.