Diarrhoea remains an important public health concern, particularly in developing countries, and has become difficult to treat because of antibacterial resistance. The development of synergistic antimicrobial agents appears to be a promising alternative treatment against diarrhoeic infections. In this study, the combined effect of tetracycline together with either nitroxoline, sanguinarine, or zinc pyrithione (representing various classes of plant-based compounds) was evaluated in vitro against selected diarrhoeic bacteria (Enterococcus faecalis, Escherichia coli, Listeria monocytogenes, Shigella flexneri, Vibrio parahaemolyticus, and Yersinia enterocolitica). The chequerboard method in 96-well microtiter plates was used to determine the sum of the fractional inhibitory concentration indices (FICIs). Three independent experiments were performed per combination, each in triplicate. It was observed that the combination of tetracycline with either nitroxoline, sanguinarine, or zinc pyrithione produced synergistic effects against most of the pathogenic bacteria tested, with FICI values ranging from 0.086 to 0.5. Tetracycline-nitroxoline combinations produced the greatest synergistic action against S. flexneri at a FICI value of 0.086. The combinations of the agents tested in this study can thus be used for the development of new anti-diarrhoeic medications. However, studies focusing on their in vivo anti-diarrhoeic activity and safety are required before any consideration for utilization in human medicine.

• Keywords
• antibacterial resistance, antimicrobial agents, combination therapy, diarrhoea, pathogens, synergistic effect,
• MeSH
• Alkaloids pharmacology   MeSH
• Anti-Bacterial Agents * pharmacology   MeSH
• Bacteria drug effects   MeSH
• Humans MeSH
• Microbial Sensitivity Tests * MeSH
• Nitroquinolines pharmacology   MeSH
• Organometallic Compounds MeSH
• Diarrhea microbiology   drug therapy   MeSH
• Pyridines pharmacology   MeSH
• Drug Synergism * MeSH
• Tetracycline * pharmacology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Alkaloids MeSH
• Anti-Bacterial Agents * MeSH
• Nitroquinolines MeSH
• nitroxoline MeSH Browser
• Organometallic Compounds MeSH
• Pyridines MeSH
• pyrithione zinc MeSH Browser
• Tetracycline * MeSH

Antibiotic resistance in diarrhea-causing bacteria and its disruption of gut microbiota composition are health problems worldwide. The development of combinatory agents that increase the selective inhibitory effect (synergism) against diarrheagenic pathogens and, simultaneously, have a lowered impact (antagonism) or no negative action on the gut microbiota is therefore proposed as a new strategy efficient for chemotherapy against diarrheal conditions. In this study, the in vitro selective combinatory effect of ciprofloxacin with nitroxoline, sanguinarine, and zinc pyrithione, representing various classes of alkaloid-related compounds (nitroquinolines, benzylisoquinolines and metal-pyridine derivative complexes) against selected standard diarrhea-causing (Bacillus cereus, Enterococcus faecalis, Listeria monocytogenes, Shigella flexneri, and Vibrio parahaemolyticus) and gut-beneficial (Bifidobacterium adolescentis, Bifidobacterium animalis subsp. lactis, Bifidobacterium breve, Lactobacillus casei, and Lactobacillus rhamnosus) bacteria, was evaluated according to the sum of fractional inhibitory concentration indices (FICIs) obtained by the checkerboard method. The results showed that the individual combination of ciprofloxacin with nitroxoline, sanguinarine, and zinc pyrithione produced a synergistic effect against the pathogenic bacteria, with FICI values ranging from 0.071 to 0.5, whereas their antagonistic interaction toward the Bifidobacterium strains (with FICI values ranging from 4.012 to 8.023) was observed. Ciprofloxacin-zinc pyrithione produced significant synergistic action against S. flexneri, whereas a strong antagonistic interaction was observed toward B. breve for the ciprofloxacin-nitroxoline combination. These findings suggest that certain combinations of agents tested in this study can be used for the development of antidiarrheal therapeutic agents with reduced harmful action on the gastrointestinal microbiome. However, further studies focused on their pharmacological efficacy and safety are needed before they are considered for clinical trials. IMPORTANCE Diarrheal infections, which are commonly treated by antibiotics, are still responsible for over 4 to 5 million cases of human deaths annually. Moreover, the rising incidence of antibiotic resistance and its negative effect on beneficial bacteria (e.g., Bifidobacteria) of the gut microbial community are another problem. Thus, the development of selective agents able to inhibit diarrheal bacteria and, simultaneously, that have no negative impact on the gut microbiota, is important. Our results showed that individual combinations of ciprofloxacin with nitroxoline, sanguinarine, and zinc pyrithione produced synergism against the pathogenic bacteria, whereas their antagonistic interaction toward the beneficial strains was observed. The antagonism can be considered a positive effect contributing to the safety of the therapeutic agents, whereas their synergism against diarrheal bacteria significantly potentiates total antimicrobial efficacy. The certain combinations tested in this study can be used for the development of antidiarrheal agents with reduced harmful action on the gastrointestinal microbiome.

• Keywords
• antagonism, antimicrobial agents, diarrhea, gut microbiota, selectivity, synergism,
• MeSH
• Anti-Bacterial Agents pharmacology   MeSH
• Bacteria MeSH
• Benzylisoquinolines * MeSH
• Bifidobacterium MeSH
• Ciprofloxacin pharmacology   MeSH
• Humans MeSH
• Nitroquinolines * MeSH
• Antidiarrheals MeSH
• Diarrhea drug therapy   MeSH
• Pyridines pharmacology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Anti-Bacterial Agents MeSH
• Benzylisoquinolines * MeSH
• Ciprofloxacin MeSH
• Nitroquinolines * MeSH
• nitroxoline MeSH Browser
• Antidiarrheals MeSH
• Pyridines MeSH
• pyrithione zinc MeSH Browser
• sanguinarine MeSH Browser

Bacterial diarrhea remains a global health problem, especially in developing tropical countries. Moreover, dysbiosis caused by diarrheagenic bacteria and inappropriate antimicrobial treatment has been associated with intestinal carcinogenesis. Despite the rich tradition of the use of herbs for the treatment of gastrointestinal disorders in Cambodian and Philippine folk medicine, many of them have not yet been systematically studied for their in vitro selective inhibitory effects on intestinal bacteria and cells. In the present study, in vitro inhibitory activities of 35 ethanolic extracts derived from 32 Cambodian and Philippine medicinal plants were determined by broth microdilution method against 12 pathogenic bacteria. Furthermore, cytotoxicity against intestinal cancer cells (Caco-2 and HT-29) using thiazolyl blue tetrazolium bromide cytotoxicity assay and safety to six beneficial intestinal bacteria (bifidobacteria and lactobacilli) and intestinal normal cells (FHs 74 Int) were determined for the antimicrobially active extracts. Selectivity indices (SIs) were calculated among the averages of minimum inhibitory concentrations (MICs), half-maximal inhibitory concentrations (IC50), and 80% inhibitory concentrations of proliferation (IC80) for each type of the tested agents. The extracts of Artocarpus blancoi (Elmer) Merr. (Moraceae), Ancistrocladus tectorius (Lour.) Merr. (Ancistrocladaceae), and Pentacme siamensis (Miq.) Kurz (Dipterocarpaceae) produced significant growth-inhibitory effects (MICs = 32-512 μg/ml) against intestinal pathogenic bacteria at the concentrations nontoxic to normal intestinal cells (IC80 values >512 μg/ml; SIs = 0.11-0.2). Moreover, the extract of P. siamensis (Miq.) Kurz was relatively safe to beneficial bacteria (MICs ≥512 μg/ml; SI = 0.1), and together with A. blancoi (Elmer) Merr., they selectively inhibited intestinal cancer cells (IC50 values ≥51.98 ± 19.79 μg/ml; SIs = 0.3 and 0.6). Finally, a strong selective antiproliferative effect on cancer cells (IC50 values 37.89 ± 2.68 to 130.89 ± 13.99 μg/ml; SIs = 0.5) was exerted by Ehretia microphylla Lam. (Boraginaceae), Lagerstroemia cochinchinensis Pierre ex Gagnep. (Lythraceae), and Melastoma saigonense (Kuntze) Merr. (Melastomataceae) (leaves with flower buds). The results suggest that the above-mentioned species are promising materials for the development of new selective antibacterial and antiproliferative agents for the treatment of infectious diarrhea and associated intestinal cancer diseases. However, further research is needed regarding the isolation and identification of their active constituents.

• Keywords
• Cambodia, Philippines, antibacterial, anticancer, diarrhea, medicinal plant,
• Publication type
• Journal Article MeSH