INTRODUCTION

Tuberculosis (TB) is caused by Mycobacterium tuberculosis and affects one-third of the world’s population; approximately 10 million people were infected and 1.4 million died in 2019 ¹. Treatment is usually insufficient due to the emergence of multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) cases, which is why this disease is considered a major global health problem ². According to the World Tuberculosis Report ¹, Peru is the country most affected by MDR-TB in Latin America, with 30% of the cases, which have been increasing in recent years and include a high incidence of XDR-TB cases in more than half of the regions throughout the country. This has led to TB being considered a health priority, and for this reason it is diagnosed and treated free of charge by the Peruvian state health institutions ³.

The emergence of multiple drug resistance pathways has prompted the study and search for new compounds for the effective treatment of TB in order to control drug resistance ⁴. Different natural products have been discovered that possess activity against different resistant strains of M. tuberculosis, among which alkaloids stand out ⁵. Ambiguine, derived from Fischerella ambigua, significantly inhibits M. tuberculosis H37RV with a minimum inhibitory concentration (MIC) of 6.6 µM ⁶. On the other hand, cryptolepine, an indoloquinoline alkaloid extracted from Cryptolepis sanguinolenta have shown activity against M. fortuitum, with an MIC of 16 ug/mL ⁷. Other alkaloids, including indoles, pyrroles, carbazoles, indoloquinolines, manzamines, quinolines and isoquinolines, have antimicrobial effect on different species of mycobacteria ⁵. Among these compounds are the quinolizidine alkaloids, which have demonstrated anti-TB activity ⁸. Sparteine, in particular, has shown bacteriostatic activity on Mycobacterium phlei ⁹, a bacterium with high phylogenetic similarity to M. tuberculosis, as revealed by 23S ribosomal RNA analysis, with an identity of 90-100% and an expected BLAST value (E value) = 0 ¹⁰.

Given the antimicrobial activity of sparteine on M. phlei, and after in vivo and clinical trials, this alkaloid, or its derivatives, could be used against M. tuberculosis. Therefore, this study aimed to evaluate the antimicrobial in vitro effect of sparteine on the growth of four ATCC strains of Mycobacterium tuberculosis with different degrees of antibiotic resistance, using culture and susceptibility to antituberculosis drugs by microscopic observation (MODS) assay.

THE STUDY

Preparation of the culture medium and the solutions with sparteine sulfate

The evaluation of the bactericidal activity of sparteine sulfate (SS) was carried out using an adaptation of the culture and susceptibility to antituberculosis drugs assay method by MODS, according to the procedure described in the technical guideline prepared by the Instituto Nacional de Salud ¹¹. This guideline is based on the observation of the characteristic cords of M. tuberculosis when it grows in liquid medium, by means of an inverted light microscope. The flow chart of the SS bactericidal activity evaluation by microscopic observation is described in Figure 1.

Figure 1 Flow chart of the evaluation of the bactericidal activity of sparteine sulfate against M. tuberculosis by microscopic observation (MODS). Middlebrook 7H9 base broth enriched with OADC (Oleic Albumin Dextrose Catalase) was prepared. The base broth was used to prepare the controls. Sparteine concentrations were obtained from dilution of a 1000 mMol stock solution to produce concentrations of 1; 2.5; 5; 10; 25; 50 and 100 mMol. The bacterial suspension was added to the culture media by adjusting the turbidity to Mc Farland scale 1 (3 x 10⁸ CFU/mL). 

Middlebrook 7H9 broth enriched with 10% OADC (Oleic Albumin Dextrose Catalase) was used as the base for the culture medium. Based on the concentrations used in the Wink study ⁹, SS was added to the medium in microplates, to obtain an intermediate concentration of SS equivalent to: 1000; 500; 250; 100; 50; 25 and 10 milliMol. Finally, in another microplate, we placed an aliquot of 100 µL of each previously prepared concentration. Each strain was evaluated in a microplate, in which an aliquot was placed in triplicate (Figure 1). The microplates were placed inside a Ziplock®-type transparent polyethylene bag and protected from light until further evaluation according to the MODS protocol ¹¹.

FINDINGS

Our results show normal growth of the four M. tuberculosis strains (ATCC 27294; ATCC 35822; ATCC 35838; ATCC 35821) in the wells without SS, and CFUs could be identified (Table 2). The results detailed below were observed in the three evaluated replicates.

Likewise, during microscopic observation, CFU growth was detected in all M. tuberculosis strains (ATCC 27294; ATCC 35822; ATCC 35838; ATCC 35821) at concentrations of 1; 2.5; 5 and 10 mM of sparteine sulfate. In contrast, we did not detect the cords that indicate the presence of CFU (supplementary material) when evaluating the growth of the same strains in the wells with SS concentrations of 25; 50 and 100 mM. This lack of growth showed the inhibition of Mycobacterium tuberculosis growth by the antimicrobial effect of SS. Microscopic findings are shown in Figure 2.

Figure 2 Broth growth of M. tuberculosis MDR strain (ATCC 35821) subjected to different concentrations of sparteine sulfate. The control photograph shows the characteristic cords which indicate growth in liquid medium of M. tuberculosis. The photos with concentrations of 10, 25, 50 and 100 mmol show inhibition of M. tuberculosis growth. The photographs were taken using an Olympus inverted light microscope with 1000x magnification. 

DISCUSSION

All the evaluated M. tuberculosis strains in the wells with SS concentrations of 1 mM; 2.5 mM; 5 mM and 10 mM showed growth. However, growth was inhibited by SS at concentrations of 25mM, 50mM and 100mM, which have antimicrobial action against the following M. tuberculosis strains grown in broth: ATCC 27294, ATCC 35822, ATCC 35838 and ATCC 35821. The acquisition of antimicrobial resistance is a natural biological occurrence, which derives from specific chromosomal mutations and is a different and irreversible process for each drug ². However, the emergence of MDR strains has generated the need to search for new diagnostic and treatment methods ¹².

These results contrast with those obtained for the quinolizidine alkaloid bis-1-oxaquinolizidine, obtained from Xestospongia exigua, evaluated with the Microplate Alamar Blue Assay (MABA) colorimetric micromethod which showed an MIC of 3.4 uM against M. tuberculosis H37Rv ¹³. On the other hand, sparteine at 20 mM has achieved 100% growth inhibition against M. phlei ⁹. Therefore, its antimicrobial activity against M. tuberculosis opens the possibility of using it against other species of the genus Mycobacterium. This could be of vital importance, because in humans, the rate of infection by non-tuberculous mycobacteria has increased in recent years, as can be evidenced in the following prevalence rates reported by Mora et al. ^{( 14}., M. avium (52%), M. abscessus (34%), M. chelonae (18%), M. fortuitum (16%) and M. kansasii (9.1%).

The MODS method has been validated by several studies to detect the presence of M. tuberculosis and its susceptibility to antituberculosis drugs such as rifampicin, isoniazid and others such as pyrazinamide, reaching sensitivity and specificity values higher than 90% in TB and MDR-TB compared to the standard agar plate ratio test (APRT) ¹⁵. It has been successfully used in the Regional Referral Public Health Laboratory of La Libertad, and is the basis for the development of a tuberculosis telediagnosis system and determination of multidrug resistance ¹⁶.

The limitations of this study include the need for validation of the results by APRT, the evaluation of toxicity in human cells and the comparison with existing drugs recommended for the treatment of TB, such as isoniazid or rifampicin, which have minimum inhibitory concentrations of 0.25 and 2.0 ug/mL, as reported by Schönfeld et al. ¹⁷. In addition, the mechanism of action of sparteine on M. tuberculosis should be further studied, since it is a quinolizidine alkaloid and its action could be related to quinolones, with which they share elements of their chemical structure. The bactericidal activity of these drugs is linked to the inhibition of bacterial DNA replication by blocking the ligase domain of DNA gyrase, causing supercoiling and preventing DNA repair, which is why they can be used in cases of MDR-TB ¹⁸.

In conclusion, this study reports a possible antimicrobial effect of sparteine sulfate on four ATCC strains of M. tuberculosis with different degrees of antibiotic resistance (sensitive, isoniazid-resistant, rifampicin-resistant and MDR), under in vitro conditions. On the other hand, more standardized assays are required to determine the in vivo MIC, as well as clinical studies to determine its effect against TB.