The effect of an innovative thiadiazine-class antibacterial agent on the motility of  Vibrio cholerae

Nadezhda A. Selyanskaya; Селянская Надежда Александровна; Sergey O. Vodopyanov; Водопьянов Сергей Олегович; Elena A. Menshikova; Меньшикова Елена Аркадьевна; Svetlana V. Titova; Титова Светлана Викторовна; Olga V. Duvanova; Дуванова Ольга Викторовна; Polina V. Bodraya; Бодрая Полина Владимировна; Naylia A. Zigangirova; Зигангирова Наиля Ахатовна

doi:10.36233/0372-9311-786

The effect of an innovative thiadiazine-class antibacterial agent on the motility of Vibrio cholerae

Authors: Selyanskaya N.A.¹, Vodopyanov S.O.¹, Menshikova E.A.¹, Titova S.V.¹, Duvanova O.V.¹, Bodraya P.V.¹, Zigangirova N.A.²
Affiliations:
1. Rostov-on-Don Research Institute for Plague Control
2. N.F. Gamaleya National Research Center for Epidemiology and Microbiology
Issue: Vol 103, No 2 (2026)
Pages: 229-235
Section: ORIGINAL RESEARCHES
URL: https://microbiol.crie.ru/jour/article/view/19135
DOI: https://doi.org/10.36233/0372-9311-786
EDN: https://elibrary.ru/LXFYFO
ID: 19135

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Abstract

Introduction. Vibrio cholerae motility plays a key role in the pathogenesis of cholera, so finding ways to suppress it is a promising approach to combating this infection.

Objective: to study the effect of Fluorothiazinone on the motility of V. cholerae.

Materials and methods. Flagellar motility was determined in 0.3% agar, with the motility zone measured, in 40 strains V. cholerae El Tor of varying epidemiological significance. The ability of these strains to acquire resistance to Fluorothiazinone was also assessed through repeated subcultures on alkaline agar containing Fluorothiazinone (100 mg/L).

Results. A significant decrease in the motility of all studied cultures was observed in the presence of Fluorothiazinone, without the development of resistance.

Conclusion. The dose-dependent ability of Fluorothiazinone to reduce the motility of V. cholerae was demonstrated for the first time.

Keywords

Fluorothiazinone, Vibrio cholerae, flagellum, motility

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Introduction

Vibrio cholerae serogroups O1 and O139, which express cholera toxin and toxin-regulated pili, are capable of causing cholera, which could possibly lead to an epidemic [1].

One of the characteristic features of V. cholerae is its high motility, provided by a single polar flagellum. The motility and chemotaxis of V. cholerae play an important role both in the bacterium’s survival in the external environment and in the progression of the infectious process [2, 3]. Motility helps the bacterium overcome the thick mucus barrier covering the small intestine and reach the epithelium, facilitating intestinal colonization and the expression of virulence genes [4, 5]. The flagellum of V. cholerae is sheathed and consists of three main parts: the basal body, a flexible hook, and a rigid filament [6]. The basal bodies of V. cholerae also contain an additional structure consisting of large discs 80–170 nm in diameter, which are susceptible to proteases and associated with the outer membrane, and which may serve as a support or anchoring device for the single flagellum [7]. The flagellar filament of V. cholerae consists of 5 flagellin subunits (FlaA, FlaB, FlaC, FlaD, and FlaE); however, only FlaA is necessary and sufficient for filament synthesis [8]. The flagellum is driven by the proton-soliciting action of sodium ions across the membrane. It rotates both clockwise and counterclockwise, propelling the cell backward and forward, respectively. Due to this, the flagellum can bend, creating a jerky motion that alters the cell’s movement pattern and facilitates chemotaxis. Flagellar motility in vibrios is associated with several cellular processes, such as movement, colonization, adhesion, biofilm formation, and virulence. The V. cholerae flagellum also functions as a secretion channel for virulence factors. For example, the bacterial cytotoxin MakA is secreted through the flagellar channel in a proton-motive force-dependent manner [3]. Transcription of all flagellum-associated genes occurs hierarchically and is regulated by several transcription factors and a multilevel regulon that allows for the integration of extracellular and intracellular signals [9, 10].

Currently, reports of the isolation of V. cholerae strains resistant to antibiotics—which have been successfully used to treat cholera for many years—in various regions of the world are causing concern. For example, in Ethiopia, more than half of the V. cholerae O1 El Tor isolates collected in 2022–2023 were resistant to trimethoprim/sulfamethoxazole (62.4%) and ampicillin (56.8%) [11]. In 2023, M.M. Kabir et al. reported the isolation of V. cholerae strain KBR06 from a cholera patient in Bangladesh; this strain was resistant to 39 antimicrobial agents across 14 classes [12]. The cholera outbreak in India in December 2023 was caused by a strain resistant to ampicillin, tetracycline, azithromycin, chloramphenicol, and ciprofloxacin [13]. At the same time, in Ghana, isolated V. cholerae strains exhibited multidrug resistance in 68–100% of cases, with a predominance of resistance to co-trimoxazole (75–100%) [14]. In 2024, during a cholera outbreak in the Comoros Islands, a strain of V. cholerae O1 El Tor resistant to 10 antibiotics was isolated [15]. Due to the spread of antibiotic resistance among infectious disease pathogens, the search for new drug targets is currently underway. Significant attention is being paid to therapeutic strategies aimed at inhibiting virulence factors and suppressing the pathogenic properties of bacteria. Thus, with regard to V. cholerae, substances have been synthesized that disrupt the production of cholera toxin [16–18], as well as quorum sensing inhibitors [19, 20]. Furthermore, the N.F. Gamaleya National Research Center for Epidemiology and Microbiology has developed an innovative Russian drug, Fluorothiazinone (FT), which targets the ATPase of the type III secretion system as well as flagella, both of which play a crucial role in the pathogenicity of infectious disease pathogens. This antibacterial drug has a broad spectrum of activity, including bacteria resistant to various classes of antibiotics. By specifically inhibiting flagellum-mediated motility, toxin secretion, invasion, colonization, intracellular survival, and biofilm formation, FT has demonstrated in vivo and in vitro activity against Chlamydia trachomatis, Salmonella enterica serovar Typhimurium, as well as multidrug-resistant Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli, Enterococcus spp., and Staphylococcus aureus [21–25]. There are currently no reports in the available literature on the effect of FT on V. cholerae.

The aim of this study was to investigate the effect of FT on the motility of V. cholerae.

Materials and methods

The study utilized a sample of V. cholerae O1 serogroup strains, which included 15 toxigenic strains (ctxAB⁺tcpAB⁺) (5 water-borne strains (Rostov Region, 2020–2025) and 10 clinical strains (Kazan, 2001; Moscow, Tambov, 2023; Ivanovo, Voronezh, Moscow, 2025)) and 20 non-toxigenic strains (ctxAB^–tcpAB^–) (10 water-borne (Rostov and Kherson Regions, Republic of Kalmykia, Krasnodar Krai, 2021–2025) and 10 clinical (Rostov region, 2005; Moscow, 2023; Zaporizhzhia region, 2023; Kherson region, 2023–2024).

The susceptibility/resistance of strains to 11 antimicrobial agents was determined using the serial dilution method on solid culture media in accordance with regulatory documents (MG 4.2.3745-22)¹.

The active pharmaceutical ingredient of the FT drug was provided by the Laboratory of Organic Synthesis of Biologically Active Compounds at the N.F. Gamaleya National Research Center for Epidemiology and Microbiology.

The ability of FT to affect the motility of V. cholerae was evaluated by culturing the bacteria on Petri dishes containing semi-liquid 0.3% Hottinger’s agar with FT at concentrations of 50, 100, and 150 mg/L and without FT (control). The selection of these concentrations was based on their effective suppression of the motility of other members of the Enterobacteriaceae family [24]. A 1.5 μL suspension of V. cholerae at a concentration of 10⁶ CFU/mL was inoculated into the agar by puncture into the center of the dish. Incubation was carried out at 37°C for 24 h, after which the degree of bacterial motility was assessed by the diameter of their radial migration through the agar (the swimming motility method). The viability of bacteria incubated with FT for 24 hours was confirmed by plating on alkaline agar.

To determine the ability of V. cholerae to acquire resistance to FT, multiple subcultures (21 passages) of the strains were performed on alkaline agar containing FT at a concentration of 100 mg/L. Thereafter, the motility of the cultures was determined using the swimming motility method.

Statistical analysis of the results was performed using the Medstatistic online computer program², calculating the arithmetic mean and its standard error (M ± m) based on the results of three experiments. The statistical significance of differences in adhesion values was determined using Student’s t-test. A p-value of ≤ 0.05 was considered statistically significant.

Results

Antibiotic susceptibility testing of the V. cholerae strains examined revealed antibiotic resistance. 62.5% of the cultures were resistant to ampicillin, 87.5% to streptomycin, 50% to nalidixic acid, 50% to tetracyclines, 62.5% to rifampicin, 50% to furazolidone, 10% to ciprofloxacin, and 50% to trimethoprim/sulfamethoxazole. Moreover, more than half of the cultures exhibited multidrug resistance and were resistant to 3–8 antimicrobial agents simultaneously.

The minimum inhibitory concentration (MIC) of FT against V. cholerae could not be determined. Culture growth persisted at drug concentrations in the plates up to 2000 mg/L, indicating no inhibition of bacterial viability in vitro.

All Vibrio strains studied exhibited motility. The mean radial migration diameter in the control group was 58.4–55.8 mm for toxigenic strains and 52.2–49.0 mm for non-toxigenic isolates, with no statistically significant differences (Fig. 1).

Fig. 1. Changes in the diameter of V. cholerae motility zones at various concentrations of FT.

The addition of FT to the culture medium at concentrations of 50–100–150 mg/L significantly reduced the motility of all V. cholerae strains tested, leading to complete inhibition of motility in some strains at a concentration of 150 mg/L (Fig. 1).

At drug concentrations of 50–100 mg/L, the diameter of the motility zones for strains isolated from water and humans decreased, compared to the control, by a factor of 1.2–1.7 for ctxAB⁺tcpAB⁺ cultures, and for strains lacking the cholera toxin gene, by 1.3–2.4 times. Increasing the FT concentration to 150 mg/L resulted in a 2.95–3.70-fold decrease in the motility of non-toxigenic and toxigenic strains. At the same time, the motility of toxigenic strains isolated from water decreased significantly more (up to 6-fold).

Fig. 2 illustrates the effect of various FT concentrations in semi-liquid 0.3% Hottinger’s agar on the motility of the clinical toxigenic isolate V. cholerae O1 El Tor 7875, which is resistant to antibiotics.

Fig. 2. Effect of various concentrations of FT in 0.3% Hottinger’s semi-solid agar on the motility of the clinical toxigenic isolate V. cholerae O1 El Tor 7875, which is resistant to antibiotics. a — control, without flortiazine; b — flortiazine, 50 mg/L; c — flortiazine, 100 mg/L; d — flortiazine, 150 mg/L

Of particular interest was the development of resistance in V. cholerae to the effects of FT, which may manifest as a reduction in the suppression of motility. A comparison of the diameters of motility zones for 10 clinical isolates of V. cholerae O1 El Tor ctxAB⁺tcpAB⁺ after repeated passaging on FT (100 mg/L) revealed no significant differences compared to the control (cultures not passaged on FT).

Discussion

In the study, a dose-dependent effect of FT on the motility of V. cholerae was observed. At concentrations of 50–100 mg/L, this compound was more effective in reducing the motility of non-toxigenic clinical isolates. Increasing the concentration to 150 mg/L led to a more pronounced suppression of motility in toxigenic strains, up to its complete loss. This effect warrants further investigation. According to the literature, FT inhibits flagellar assembly on the surface of Gram-negative pathogenic bacteria, including E. coli, P. aeruginosa, and P. mirabilis [26]. Another study demonstrated a reduction in the number of fimbriae in K. pneumoniae 52TS15 under the influence of this drug [27]. Data on the relationship between motility and virulence are highly contradictory. Vibrio mutants with impaired motility exhibit reduced virulence in a model of newborn mice [28] and lower reactogenicity compared to the original motile strains [29]. On the other hand, V. cholerae mutants with impaired flagellar motility show a significant increase in the transcription of the tcpA and ctxA genes, as confirmed by quantitative reverse transcription-polymerase chain reaction (RT-qPCR) [30].

FT has already demonstrated its efficacy in vivo for the prevention of model pneumonia caused by a multidrug-resistant clinical isolate of K. pneumoniae [27], in a model of chronic chlamydial infection [31], oral salmonellosis [24], infection caused by P. aeruginosa [32], and Acinetobacter baumannii-induced septicemia [33], as well as in clinical trials. Successful use of FT in combination with cefepime has been reported in patients with complicated urinary tract infections caused by uropathogens with multidrug resistance [34]. This drug disrupts the mechanisms that contribute to the persistence of pathogens in the development of chronic urinary tract infections [35].

Conclusion

Thus, for the first time, the dose-dependent ability of FT to reduce the motility of V. cholerae without inhibiting microbial growth or inducing resistance has been demonstrated, which highlights the potential of this drug both for studying specific stages of cholera pathogenesis and for possible application in etiological therapy.

¹ Methods of laboratory diagnosis of cholera. Guidelines. MUK 4.2.3745-22. Moscow; 2022.

² URL: https://medstatistic.ru/calculators/calcvaries.html

About the authors

Nadezhda A. Selyanskaya

Rostov-on-Don Research Institute for Plague Control

Author for correspondence.
Email: selyanskaya_na@antiplague.ru
ORCID iD: 0000-0002-0008-4705

Cand. Sci. (Med.), senior researcher, Department of Microbiology of cholera and other acute intestinal infections