Diarrhea remains one of the leading causes of mortality worldwide, especially among children. Recent epidemiological studies conducted in developing countries identified Shigella species as the most predominant pathogenic bacteria responsible for diarrhea. Antimicrobial therapy is necessary for Shigella infections; however, the rapid emergence of resistance against existing antimicrobials in Shigella spp. poses a serious global health problem. To identify alternative antimicrobial compounds with activity against Shigella species, the pathogens responsible for bacterial diarrhea. In this study, we have applied antibacterial phenotypic screening to identify potent anti-Shigella compounds across a broad chemical diversity, including selected acetaminophen derivatives containing benzothiazoles backbone, and their combination with certain antibiotics. In continuation to our effort in searching potent antimicrobial compounds, we have found this time around that two acetaminophen derivatives containing benzothiazoles backbone (4a and 4b) could inhibit the growth of Shigella flexneri with common MIC value of 12.5 µg/ml. These compounds were established through a time-kill kinetics’ study to be bactericidal. Meanwhile, the 2-aminobenzothiazoles (1a and 1b) used for the synthesis of compounds 4 (a & b) were found to be poorly active (MIC: 100 µg/ml) against this pathogen. Combination studies of 4a and 4b with the least susceptible antibiotics (ceftriaxone and cotrimoxazole) demonstrated synergistic anti-Shigella activity. The present study demonstrates that the azobenzothiazole dyes 4 (a & b) can be repurposed as potential anti-Shigella compounds, which can serve as scaffolds for the development of new agents against infectious diarrhea caused by Shigella and other enteric pathogens, especially in developing countries.

Shigellosis is a pathological condition that affects the digestive system to possibly cause diarrhoea. Shigella species, which are responsible for this disease, are highly contagious and spread through contaminated food and water. The increasing development of resistance by Shigella species necessitate the urgent need to search for new therapies against shigellosis causing diarrhoea. The scientific validation of medicinal plants, such as Diospyros gilletii, which is used for the traditional treatment of diarrhoeal conditions is worthwhile. The present study aims to investigate the antibacterial activity of extracts from D. gilletii against selected Shigella species. Extracts from D. gilletii stem bark were prepared by maceration using various solvents. An-tibacterial activity of D. gilletii extracts was evaluated on Shigella dysenteriae, S. flexneri, S. boydii, and S. sonnei using a microdilution method, whereas cytotoxicity test was performed on Vero and Raw cells using resazurin-based colorimetric assay. Bacterial membrane permeability studies were evaluated using pro-pidium iodide (PI) and 1-N-phenylnaphthylamine (NPN) uptake assays, whereas inhibition and eradica-tion tests on bacterial biofilms were carried out by spectrophotometry. As a result, methanol, ethanol and hydroethanol extracts of D. gilletii inhibited the growth of S. boydii, S. flexneri and S. sonnei with MIC values ranging from 125 to 500 µg/mL, without toxicity to Vero and Raw cells. Time kill kinetics revealed bacteri-cidal orientation at 2MIC and 4MIC, and bacteriostatic outcome at 1/2MIC. The mechanistic basis of anti-bacterial action revealed that D. gilletii extracts inhibited and eradicated Shigella biofilms, and promoted the accumulation of NPN and PI within the inner and outer membrane of bacteria to increase membrane per-meability, thereby causing membrane damage. This novel contribution toward the antibacterial and mech-anisms of action of D. gilletii extracts against Shigella species substantiate the use of this plant in the tradi-tional treatment of infectious diarrhoea.