Phenotypic and Genetic Analysis of Antimicrobial Susceptibility in Vibrio cholerae Isolates Collected Between 1970 and 2024

Introduction

Cholera is a rapidly progressing and potentially fatal disease that requires prompt intervention for effective control and treatment. Without timely treatment, cholera can lead to death within hours or days, with case fatality rates exceeding 50%. However, early rehydration and antibiotic therapy can reduce mortality to below 1%.^1–4 Antibiotic resistance is a global public health concern that affects not only well-known pathogens but also less-studied organisms such as V. cholerae. Cholera has traditionally been treated with antibiotics such as tetracycline, doxycycline, and fluoroquinolones. However, the increasing use of these agents has led to the emergence of V. cholerae strains resistant to these drugs, posing significant challenges for disease management and increasing the risk of transmission.⁵

According to the World Health Organization (WHO), V. cholerae has demonstrated increasing resistance to conventional antibiotics, such as ampicillin and tetracycline, in several regions of Africa in recent decades.⁶ A critical aspect of resistance research is understanding the mechanisms that enable V. cholerae to maintain viability under antimicrobial pressure. These mechanisms include both genetic adaptation and the horizontal transfer of resistance genes among bacteria.⁷ For instance, resistance to tetracycline may be associated with genes encoding active efflux pumps or ribosomal protection proteins that prevent antibiotic binding.⁸ Additionally, epidemiological and social factors—such as high population density, inadequate sanitation, and the widespread, unregulated use of antibiotics—play a major role in the dissemination of resistance.⁹

Thus, understanding the causes of V. cholerae resistance and elucidating its underlying mechanisms are essential for developing effective strategies for cholera treatment and prevention.¹⁰

In the Republic of Kazakhstan, cholera is not considered an endemic infection; however, certain regions retain ecological and social conditions that enable the long-term persistence of Vibrio cholerae in environmental reservoirs and its potential spread among the population (particularly in the Caspian Sea region and rivers of the southeastern part of the country). The population density in Kazakhstan is among the lowest in the world—7.5 persons per square kilometer. Among the first-level administrative divisions, the highest population density is observed in the southern regions of the country.

Between 1993 and 2024, more than 400 cholera cases and carriers were registered in Kazakhstan, all of which were imported from abroad.During 2022–2024, a total of 64,420 cases of acute intestinal infections were recorded in the country, of which 39,392 (61.1%) patients were tested for cholera; all laboratory results were negative.

Throughout Kazakhstan, 76,180 water samples from open and wastewater sources were examined during the same period. Vibrio cholerae non-O1 was isolated from 6545 samples (8.59%), and toxigenic V. cholerae O1 was detected in five samples (0.01%)—three Inaba and two Ogawa serovars—from the Turkistan and Mangystau regions.

In 2018, studies were conducted to investigate phenotypic markers of antibiotic resistance among V. cholerae isolates collected in Kazakhstan. Resistant strains isolated from water sources in Almaty city, as well as in the Almaty and Zhambyl regions, shared a common phenotype characterized by resistance to erythromycin.¹¹

For the first time, this study identified and thoroughly examined the role of genetic and biochemical factors contributing to the development of resistance in V. cholerae strains under the conditions of the Republic of Kazakhstan. In this study, we conducted a comprehensive analysis of the antibiotic resistance of V. cholerae strains isolated in Kazakhstan between 1970 and 2024, examining their susceptibility to various classes of antimicrobial agents. Additionally, we investigated the molecular and biochemical mechanisms contributing to resistance, and we assessed their potential impact on the epidemiological situation and public biosafety.

Materials and Methods

Bacterial Strains and Isolates

This study included 26 collection strains of V. cholerae from different serogroups (O1 and non-O1), isolated from diagnostic laboratory samples and surface water in Almaty city and the Almaty, Turkistan, Mangystau, West Kazakhstan, and Karaganda regions of the Republic of Kazakhstan. These strains were collected between 1970 and 2024. Among them, 25 strains belonged to the V. cholerae O1 serogroup, biovar El Tor, and 1 strain was classified as V. cholerae non-O1. Specifically, 5 strains were isolated in Almaty, 1 from the Almaty region, 6 from the Turkistan region, 2 from the Mangystau region, 8 from the West Kazakhstan region, and 4 from the Karaganda region (Table 1).

Table 1 List of Vibrio cholerae Strains Examined by Serogroup, Serovar, Pathogenicity Gene Profile, Kazakhstan Region, Time, and Source of Isolation

Antibiotics Used in the Study

A total of 59 antibiotics belonging to different pharmacological groups were tested, including β-lactams (penicillins, cephalosporins, carbapenems, monobactams), aminoglycosides, tetracyclines, macrolides, lincosamides, fluoroquinolones, glycopeptides, sulfonamides, nitrofurans, and others. The complete list of antibiotics used in the study is presented in Table 2.

Table 2 List of Antibiotics Used in the Study (n = 59)

Antimicrobial Susceptibility Testing

The susceptibility/resistance of V. cholerae strains to antibacterial agents—including doxycycline (30 µg), ciprofloxacin (10 µg), tetracycline (30 µg), cefotaxime (30 µg), kanamycin (30 µg), nalidixic acid (30 µg), trimethoprim (5 µg), furazolidone (50 µg), gentamicin (10 µg), chloramphenicol (10 µg), ampicillin (25 µg), streptomycin (300 µg), and rifampicin (15 µg)—was assessed in accordance with established methodological guidelines.¹² A total of 59 antibiotics in disk form and 50 in E-test strips, representing 25 main groups, were used. Susceptibility testing was performed using the standard disk diffusion method (Kirby–Bauer test) and the E-test. Resistance genes were detected using an extended-spectrum β-lactamase (ESBL) phenotypic method,^13–17 and real-time PCR for screening resistance to glycopeptides and beta-lactams.¹⁷ Reference control strains included V. cholerae KA-37, E. coli ATCC 25922, Salmonella typhimurium ATCC 14025, and P. aeruginosa ATCC 9027. The strains were cultured on Mueller–Hinton agar (pH 7.3 ± 0.2) and Hottinger agar (pH 7.2 ± 0.1) at 37 °C.

For susceptibility testing, bacterial suspensions were prepared from 24-hour agar cultures in 0.85% isotonic sodium chloride solution, standardized using the McFarland standard (R092-1NO LOT0000633797; exp. 02/2026, HiMedia Laboratories Pvt. Ltd., Maharashtra, India), corresponding to 1.5 × 10⁸ CFU/mL (standards R092A and R092B). After 10–15 minutes, antibiotic disks were applied, and cultures were incubated at 37 °C. Preliminary measurements were taken after 12 hours and finalized after 18 hours. Control assays included the reference strains and plates with sterile disks. The diameters of the inhibition zones around the disks were measured to the nearest millimeter using a precision measuring template from HiMedia Laboratories Pvt. Ltd., India.

Molecular Genetic Screening

DNA extraction was performed using the commercial kit “RealBest UniMag” (Series C-8883, expiration date: July 22, 2025), produced by Vector-BEST, Russia, as well as the “RIBO-prep” kit (Cat. No. K2-9-Et-100, Russia), designed for automated DNA/RNA extraction systems.

To detect antibiotic resistance determinants in bacterial lysates, a BacResista GLA Real-Time PCR Detection Kit (DNA-Technology LLC, Moscow, Russia) was used. This kit targets genes encoding resistance to glycopeptide and beta-lactam antibiotics, including vanA/B (vancomycin and teicoplanin); mecA (methicillin and oxacillin); tem, ctx-M-1, and shv (penicillins and cephalosporins); and oxa-40-like, oxa-48-like, oxa-23-like, oxa-51-like, imp, kpc, ges, ndm, and vim (carbapenems).

Results

The results of the laboratory screening for the antimicrobial susceptibility and resistance profiling of V. cholerae strains (n = 26), isolated in the Republic of Kazakhstan between 1970 and 2024 from diagnostic laboratory and environmental sources, are presented in Table 3. Detailed data on inhibition zone diameters for 26 Vibrio cholerae isolates tested with 59 antibiotics on Mueller–Hinton agar are provided in Table 4.

Table 3 Ranges and Diameters (in mm) of Growth Inhibition Zones for 26 Vibrio cholerae Strains Isolated in Kazakhstan Between 1970 and 2024 from Diagnostic Laboratory and Environmental Sources

Table 4 Summary of Inhibition Zone Measurements Obtained During Susceptibility Testing of 26 Vibrio cholerae Isolates with 59 Antibacterial Agents on Mueller–Hinton Agar, in Millimeters

Based on their cultural, morphological, biochemical, and serological characteristics, all strains were identified as typical representatives of the family Vibrionaceae, genus Vibrio, species cholerae, belonging to both O1 and non-O1 serogroups.

Of the 26 V. cholerae O1 and non-O1 strains examined, 100% (26/26) were susceptible to the following antimicrobial agents: doxycycline (30 µg), ciprofloxacin (10 µg), tetracycline (30 µg), cefotaxime (30 µg), and kanamycin (30 µg). With respect to nalidixic acid (30 µg), 65.4% (17/26) of the strains demonstrated high susceptibility, 30.8% (8/26) exhibited intermediate susceptibility, and 3.8% (1/26) showed resistance. For trimethoprim (5 µg), 73.1% (19/26) of the isolates were highly susceptible, while 26.9% (7/26) showed intermediate susceptibility.

For furazolidone (50 µg), 73.1% (19/26) of the V. cholerae strains were highly susceptible, while 26.9% (7/26) exhibited intermediate susceptibility. With respect to gentamicin (10 µg), 96.2% (25/26) of the isolates were highly susceptible, and 3.8% (1/26) showed intermediate susceptibility. Similarly, for chloramphenicol (10 µg) and ampicillin (25 µg), 96.2% (25/26) of the strains demonstrated high susceptibility, with 3.8% (1/26) showing intermediate susceptibility to each antibiotic.

Regarding streptomycin (300 µg), 88.5% (23/26) of the V. cholerae strains were highly susceptible, 7.7% (2/26) showed intermediate susceptibility, and 3.8% (1/26) exhibited low susceptibility. For rifampicin (15 µg), 96.2% (25/26) of the strains demonstrated high susceptibility, while 3.8% (1/26) showed intermediate susceptibility.

The results of the study, including the strain numbers and the names of the antibacterial agents tested on Mueller–Hinton and Hottinger agars, are presented in summary charts in Figures 1 and 2, along with selected individual results in Figures 3 and 4.

Figure 1 Inhibition zone diameters for 26 Vibrio cholerae isolates tested against 59 antibiotics on Hottinger agar, in millimeters.

Figure 2 Inhibition zone diameters for 26 Vibrio cholerae isolates tested against 59 antibiotics on Mueller–Hinton agar, in millimeters.

Figure 3 Inhibition zone diameters obtained using the disk diffusion assay and phenotypic detection of ESBL resistance. (a) Inhibition zones for Doxycycline, Ciprofloxacin, and Nalidixic acid. (b) Inhibition zones for Trimethoprim, Furazolidone, and Gentamicin. (c) Inhibition zones for Kanamycin, Rifampicin, and Streptomycin. (d) Inhibition zones for Tetracycline, Chloramphenicol, Cefotaxime, and Ampicillin. (e) Phenotypic evaluation showing no detectable ESBL resistance genes; assessed with Trimethoprim, Furazolidone, Gentamicin, Tetracycline, and Chloramphenicol. (f) Phenotypic evaluation showing no detectable ESBL resistance genes; assessed with Cefotaxime, Ampicillin, Streptomycin, Kanamycin, and Rifampicin. The Petri dish has a visual diameter of 35 cm.

Figure 4 Results of susceptibility testing using the E-test method. (a–f) show the susceptibility of two different Vibrio cholerae strains to gentamicin, cefotaxime, and ofloxacin. (a–c) correspond to Strain 1, while (d–f) correspond to Strain 2. Multiple strains were tested using the same antibiotic strips, which explains the repeated use of gentamicin, cefotaxime, and ofloxacin. (a) Susceptibility of Strain 1 to Gentamicin. (b) Susceptibility of Strain 1 to Cefotaxime. (c) Susceptibility of Strain 1 to Ofloxacin. (d) Susceptibility of Strain 2 to Gentamicin. (e) Susceptibility of Strain 2 to Cefotaxime. (f) Susceptibility of Strain 2 to Ofloxacin.

The most active antibiotics belonged to the β-lactam group, including cefotaxime, cefixime, chloramphenicol, and cefamandole, followed by cefazolin, tetracycline, and ampicillin. Agents traditionally used for cholera treatment—such as ciprofloxacin, doxycycline, and azithromycin—demonstrated comparatively lower activity. Furazolidone exhibited the weakest activity against all 26 strains, with an average inhibition zone diameter of 11.1 mm (range: 10–18 mm).

An in vitro phenotypic susceptibility analysis of V. cholerae (n = 26) demonstrated 100% sensitivity to β-lactams, tetracyclines, aminoglycosides, amphenicols, glycopeptides, lincosamides, and quinolones. Additionally, 96.5% of the strains were susceptible to antibiotics from other classes.

The results of the susceptibility and resistance testing were confirmed using the standard E-test method, with strips indicating the minimum inhibitory concentration (Figure 4).

The results of the antimicrobial resistance gene detection in 26 V. cholerae strains using the BacResista GLA Real-Time PCR Detection Kit with real-time PCR are presented in Table 5 and Figure 5.

Table 5 Results of Antimicrobial Resistance Gene Detection in 26 Vibrio cholerae Strains Using the BacResista GLA Real-Time PCR Detection Kit and Real-Time PCR

Figure 5 Amplification curves by fluorophore detection channels: (a) FAM; (b) HEX; (c) CY5. Red and green dots represent PCR amplification curves of different samples. Red dots indicate positive amplification signals, while green dots indicate negative or no amplification.

Currently, research on the genetic determinants of bacterial resistance to glycopeptide and beta-lactam antibiotics is receiving significant attention, as these classes of antimicrobial agents are widely used for the treatment of complicated and/or severe infections. Investigating the most common types of beta-lactamases produced by various pathogenic bacteria can aid in interpreting their antibiotic susceptibility profiles, informing therapeutic decision-making, and enhancing infection control practices at the local level.

A molecular genetic analysis aiming to detect antibiotic resistance determinants in the genomes of 26 V. cholerae isolates, including strains originally obtained during diagnostic procedures and from environmental sources in Kazakhstan between 1970 and 2024, revealed no presence of resistance genes to glycopeptides (vanA/B—vancomycin and teicoplanin) or beta-lactam antibiotics (mecA—methicillin and oxacillin; tem, ctx-M-1, and shv—penicillins and cephalosporins; oxa-40-like, oxa-48-like, oxa-23-like, oxa-51-like, imp, kpc, ges, ndm, and vim—carbapenems). The control strains used in this study included E. coli ATCC 25922, S. typhimurium ATCC 14025, and P. aeruginosa ATCC 9027, in which the vanA/B gene (Ct = 9.166, FAM channel) and the tem gene (Ct = 34.60, CY5 channel) were detected in E. coli ATCC 25,922 and in P. aeruginosa ATCC 9027 (Ct = 8.954 and 24.85, respectively).

Thus, the results confirm the absence of genetic markers of resistance to 59 antimicrobial agents in all examined V. cholerae isolates. No resistance was detected to the following major classes of antibiotics: extended-spectrum beta-lactams (penicillins, cephalosporins, and carbapenems), monobactams, macrolides, tetracyclines, aminoglycosides, amphenicols, glycopeptides, lincosamides, fluoroquinolones, and other antibiotic groups. These findings highlight the importance of the regular surveillance of V. cholerae susceptibility to antimicrobial agents, which enables timely adjustments to therapeutic strategies and effective responses to changes in the epidemiological situation.

Discussion

This study provides a comprehensive phenotypic and molecular assessment of antimicrobial susceptibility in Vibrio cholerae isolates preserved in the reference collection of the Masgut Aikimbayev National Scientific Center for Especially Dangerous Infections (NSCEDI). The isolates, obtained between 1970 and 2024 during long-term microbiological surveillance in Kazakhstan, demonstrated consistently high sensitivity to major antibiotic classes and complete absence of β-lactamase or glycopeptide resistance genes. These findings contrast with global patterns of increasing multidrug resistance in V. cholerae and emphasize the unique epidemiological context of Central Asia.

Phenotypic testing confirmed that all V. cholerae isolates remained fully susceptible to doxycycline, ciprofloxacin, tetracycline, cefotaxime, and kanamycin — agents recommended by the WHO for cholera treatment.¹⁸ Only sporadic intermediate susceptibility to nalidixic acid, streptomycin, and trimethoprim was detected. Molecular screening revealed no resistance determinants, including vanA/B, mecA, tem, ctx-M-1, shv, oxa, imp, kpc, or ndm, indicating that these isolates lack mobile genetic elements associated with extended-spectrum β-lactamases (ESBLs) and carbapenemases.

Comparable studies from endemic regions show markedly different results. Investigations in India and China have reported the widespread dissemination of SXT integrative elements, class I integrons, and resistance genes such as sulII, dfrA1, and strB, conferring resistance to trimethoprim–sulfamethoxazole, nalidixic acid, and tetracycline.^19–21 Similarly, in sub-Saharan Africa and Haiti, multidrug-resistant V. cholerae O1 El Tor lineages have emerged, carrying ctxB7, sul2, and floR genes.^4,22,23 These differences may reflect contrasting selective pressures: while clinical environments in endemic regions expose bacteria to continuous antibiotic use, the Kazakhstani isolates were predominantly environmental, recovered from surface water, sediments, and monitoring sites with minimal human antibiotic influence.

The absence of resistance genes in our isolates suggests that V. cholerae populations in Kazakhstan have retained their ancestral antibiotic susceptibility. This may be due to limited antibiotic exposure in aquatic ecosystems, low rates of horizontal gene transfer, and the relatively isolated character of natural foci in arid and semi-arid zones. Comparable findings have been reported from environmental isolates in the Ganges Delta and East Africa, where non-O1/non-O139 strains showed high sensitivity to most antibiotics.^24,25 Such ecological stability supports the hypothesis that environmental Vibrio populations act as reservoirs of sensitive genotypes, contrasting with epidemic clones under strong selective pressure.⁷

Clinically, these results confirm the sustained efficacy of standard first-line agents—doxycycline, ciprofloxacin, and cefotaxime—in cholera management.²⁶ Epidemiologically, the findings highlight the importance of continued laboratory-based resistance monitoring, particularly in regions at risk of pathogen reintroduction through transboundary water systems or human mobility. Although no resistance determinants were detected, vigilance remains essential, as international travel and food trade can introduce resistant Vibrio strains from endemic areas.^27,28

This study has certain limitations. The dataset included 26 archival isolates representing multiple decades, yet it may not capture all genetic diversity of V. cholerae circulating in Central Asia. Whole-genome sequencing of both historical and newly isolated strains would allow for finer resolution of evolutionary trends, virulence determinants, and potential resistance acquisition pathways. Future work should also investigate environmental variables—temperature, salinity, and organic matter content—that influence V. cholerae persistence and potential adaptation to antibiotic stress.^14,29

In conclusion, the Vibrio cholerae isolates collected in Kazakhstan between 1970 and 2024 exhibit no genetic markers of antimicrobial resistance and remain highly susceptible to key therapeutic drugs. These data provide essential reference values for national and regional surveillance programs and reinforce the global need for sustained monitoring to prevent the emergence of resistant V. cholerae lineages.

Conclusions

This study provides the first comprehensive phenotypic and molecular characterization of Vibrio cholerae O1 isolates collected in Kazakhstan between 1970 and 2024. The analysis demonstrated a generally high level of antimicrobial susceptibility, with all isolates remaining sensitive to key therapeutic agents such as doxycycline, ciprofloxacin, tetracycline, cefotaxime, and kanamycin. Occasional intermediate susceptibility was observed to nalidixic acid, trimethoprim, furazolidone, and streptomycin.

No resistance genes to β-lactam or glycopeptide antibiotics were detected using real-time PCR, suggesting that, within this limited sample, genetic determinants of resistance were not present. These findings indicate that the studied isolates largely retained susceptibility to standard cholera treatment drugs; however, the results should be interpreted with caution given the restricted number of strains analyzed and their archival origin.

Although no multidrug-resistant or gene-bearing isolates were identified, the possibility of resistance emergence through horizontal gene transfer or importation from endemic regions cannot be excluded. Therefore, sustained epidemiological and molecular surveillance remains essential to ensure early detection of resistance trends and to support evidence-based cholera control strategies.