Genetic characteristics of Vibrio cholerae non-O1/non-O139 and Vibrio paracholerae strains circulating in the Volga River near Saratov

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Introduction

Cholera vibrios of non-O1/non-O139 serogroups (non-agglutinating (NAG) vibrios) are natural inhabitants of aquatic ecosystems and are widespread in various countries around the world. When they enter the human body, they are not capable of causing the dangerous infectious disease cholera, but they can cause sporadic cases and outbreaks of acute intestinal infections, as well as otitis, wound infections and septicemia. It has been suggested that differences in the clinical manifestation of the disease are due to the presence of various pathogenicity factors in these strains [1–3]. In recent years, due to changes in climatic and environmental conditions, as well as active water use, there has been an increase in diseases caused by these vibrios. It is believed that, since these diseases are not subject to mandatory registration, the number of unregistered cases of infections caused by NAG vibrios is significant and, in the last decade, has exceeded the number of officially registered cases of cholera caused by vibrios of the O1 and O139 serogroups. At the same time, the increase in cases of bacteremia caused by Vibrio cholerae strains of non-O1/non-O139 serogroups in elderly citizens, people with weakened immune systems, and those with chronic diseases is alarming [2–5].

NAG vibrios are widespread in Russia and are isolated annually from open water bodies during monitoring studies for cholera, occasionally from sick people [6–9]. When studying the genetic organization of V. cholerae non-O1/non-O139 strains detected in Russia, it was found that most isolates do not contain the CTXφ prophage with ctxAB genes encoding cholera toxin biosynthesis, as well as preCTX, which does not have these genes but contains other CTXφ prophage genes. The VPI-1 pathogenicity island, which includes the tcpA-F locus responsible for the biosynthesis of toxin-coregulated pilus (the main factor in the colonization of the intestine by vibrios), and the VSP-I pandemic islands, VSP-II, necessary for the pandemic spread of V. cholerae strains. At the same time, NAG vibrios possess a sufficient number of pathogenicity determinants involved in the development of the infectious process. These may contain genes of the MARTX toxin cluster, cholix toxin, contact-dependent secretion systems of types 6 (T6SS) and 3 (T3SS), hemolysin, proteases, Cef cytotoxic toxin, as well as genes for thermostable direct and related hemolysins (tdh, trh) V. parahaemolyticus [8, 10]. It should be noted that in terms of genetic organization, strains circulating in Russia are similar to strains isolated in other countries [11, 12]. Occasionally, toxigenic strains of V. cholerae non-O1/non-O139 are isolated in Russia, as well as isolates containing the ICE SXT element without antibiotic resistance genes [13, 9]. According to a number of researchers, NAG vibrios are natural reservoirs of virulence and antibiotic resistance genes and can transfer them to other bacteria, including the pathogen of cholera [4, 6, 14–16]. Despite the active isolation and study of V. cholerae non-O1/non-O139 strains in various regions of our country, there is no information on the genetic organization of these vibrios circulating in the Volga River near the city of Saratov.

In addition to V. cholerae non-O1/non-O139 serogroups, other representatives of the Vibrio genus reside in the aquatic environment, including V. paracholerae. This new species was officially registered in 2022 [17]. It is interesting to note that these vibrios were first isolated in 1916 in Egypt from a British soldier with cholera-like diarrhea. In 1935, A.D. Gardner and K.V. Venkatraman proposed naming these vibrios V. paracholerae and the disease they cause paracholera [18]. In 2019, strain NCTC30, isolated in 1916, was sequenced [19]. In memory of the scientists who first proposed the term V. paracholerae, it was decided to retain this designation for the new species [20]. Currently, the NCB GenBank database contains 63 nucleotide sequences of complete V. paracholerae genomes, with strain NCTC30 being the oldest isolate. It has been shown that these vibrios circulate together with the pathogen of cholera in such cholera-endemic countries as Bangladesh, Mozambique, and the Republic of Haiti and are also isolated in non-endemic areas (Serbia, the United States, Brazil, Germany, China and Thailand) [5, 20, 21].

Paracholerae vibrios are characterized as motile, containing a single polar flagellum, oxidase-positive, curved Gram-negative bacteria. They grow at a temperature of 30°C in the presence of NaCl in the range of 0 to 6.0%; at a salt concentration of 10%, growth is absent. Strains of V. paracholerae, like V. cholerae, ferment D-glucose, D-fructose, sucrose, maltose, D-galactose, maltotriose, D-mannitol, D-trehalose, D-glucose-6-phosphate, N-acetyl-D-glucosamine, glycerol, succinic acid, L-lactic acid, L-glutamic acid, fumaric acid, acetic acid, L-proline, D-alanine, L-asparagine, 2-deoxyadenosine, adenosine, inosine, and L-serine, dextrin, gelatin, glycogen, D-glucosamine, and methyl ester of D-lactic acid, but do not break down N-acetyl-D-galactosamine and D-glucuronic acid. They form acetoin from glucose in the Voges-Proskauer test. About 60% of strains have β-lactamase activity. They form yellow colonies on TCBS agar [20]. Thus, in terms of most biochemical properties, V. paracholerae strains correspond to V. cholerae. Differences include the ability of V. paracholerae to produce amylase and pectinase, which break down α-cyclodextrin and pectin, respectively, the biosynthesis of which has not been detected in V. cholerae. Only 25% of the studied V. paracholerae strains are capable of using D-mannose for growth, which is fermented by virtually all V. cholerae strains. There are also a number of genetic differences between these two species. The average nucleotide identity (ANI) between V. cholerae and V. paracholerae varies from 95% to 96%, while the ANI within the V. paracholerae group is 97–100%. In phylogenetic analysis, V. paracholerae strains form a separate branch from V. cholerae [20]. Despite active research on V. paracholerae strains isolated in different countries, there is no information in the literature about the detection of these vibrios in Russia.

The aim of this study is to investigate the genetic characteristics of V. cholerae non-O1/non-O139 serogroups and V. paracholerae strains isolated from the Volga River near the city of Saratov.

Materials and methods

Twenty-one strains of vibrios isolated in 2024 during monitoring studies for cholera in water samples from the Volga River in the Saratov region were used as the object of study. The strains were grown on alkaline agar for 18–24 hours at 37°C.

Samples were collected from July 1 to August 30, 2024, once a week in the morning. One liter of water was taken from each section of the Volga River in sterile bottles, placed in a stainless steel box, and transported to the laboratory. Water samples were collected and examined in accordance with MG 4.2.3745-22 “Control Methods. Biological and Microbiological Factors. Methods for Laboratory Diagnosis of Cholera” (approved on May 12, 2022).

Colonies suspected of V. cholerae were examined using standard serological tests with sera produced by the Russian Anti-Plague Institute “Microbe”: “Diagnostic cholera serum O1 adsorbed dry for agglutination reaction (AR)” (diagnostic titer 1:2000, p. 113, valid until 02.2025); “Ogawa diagnostic cholera serum, adsorbed dry, for agglutination reaction (AR)” (diagnostic titer 1:400, p. 115, valid until 03.2025); “Inaba adsorbed dry diagnostic cholera serum for agglutination reaction (AR)” (diagnostic titer 1:400, p. 115, valid until 03.2025); “Adsorbed dry diagnostic cholera RO serum for agglutination reaction (AR)” (diagnostic titer 1:800, p. 120, valid until 03.2025); “Diagnostic cholera serum, non-O1 group, O139, adsorbed rabbit for agglutination reaction on glass” (diagnostic titer 1:2000, p. 121, valid until 03.2025).

To study the biochemical properties, we used “api 20 E — Enterobacteriaceae Identification Kit” (bioMerieux). Hemolytic activity was determined by culturing isolated colonies of the studied strains on alkaline agar containing 1% sheep erythrocyte suspension, and the size of the erythrocyte hemolysis zone around the colony was measured.

The isolation of V. cholerae was confirmed by MALDI-TOF mass spectrometry. Mass spectra were recorded automatically on a Microflex LT mass spectrometer (Bruker Daltonics).

Axy Prep Bacterial Genomic DNA Miniprep Kit (Axygen Biosciences) was used to isolate DNA from the bacterial suspension. The bacteria were pretreated with sodium merthiolate to a final dilution of 1:10,000 (0.01%) and incubated at 56°C for 30 min.

V. cholerae strains were identified by the presence of the hly gene using real-time PCR with the Amplisens Vibrio cholerae-FL kit (registration certificate No. FSR 2011/11139), virulence was determined by the presence/absence of the ctxA, tcpA genes, and membership in the O1 and O139 serogroups was determined by testing the wbeT and wbfR genes, respectively.

Sequencing was performed on the MGI DNBSEQ-G50 (MGI) platform. Libraries were prepared according to the standard protocol using DNBSEQ-G50RS (FCLPE150) and MGI EasyFastPCR-FREE FS Library PrepSet (MGI) kits. As a result of sequencing, paired-end reads with a length of 150 bp were obtained for each sample. The quality control of the obtained reads was performed using the fastp v. 0.23 program, and contigs were assembled using unicycler v. 0.4.7. For further analysis, we used assembled genomes with an average read depth of at least 50 and an N50 value > 80,000 bp.

For bioinformatics analysis, we used the freely available programs BLAST v. 2.15.0 and BioEdit v. 7.0.9.0. The presence of a number of genes in the studied strains was determined using the SeqAnalyzer program (Rostov-on-Don Research Anti-Plague Institute).

The annotation of the collected genomes was performed using the prokka v. 1.15.6 program. Pangenomic analysis was performed using the panaroo v.1.5.2 program. The study used 64 nucleotide sequences of strains, including 21 nucleotide sequences of V. cholerae non-O1/non-O139 strains isolated from the Volga River in 2024, 43 nucleotide sequences deposited in the NCBI GenBank database and represented by 23 isolates of V. paracholerae, 13 strains of V. cholerae non-O1/non-O139 isolated in Russia and a number of endemic countries, 3 non-toxigenic strains of V. cholerae O1 El Tor, the toxigenic reference strain V. cholerae N16961 O1 El Tor, and 3 strains of V. metoecus added as an outgroup. The snippy v. 4.6.0 program was used to search for core SNPs, and recombination sites were removed from the alignment using the gubbins v. 3.4.1 program.

Phylogenetic trees were constructed using the maximum likelihood method with the iqtree v. 2.4.0 program and visualized on the iTOL platform (https://itol.embl.de). The fastANI v.1.34 program was used to calculate the ANI between the studied genomes.

Results

Sampling

Samples were taken from the Volga River at six points: No. 1 — Pristannoye village (200 m upstream from the new bridge); No. 2 — Makhanny ravine, wastewater discharge site; No. 3 — Zaton beach; No. 4 — Saratov embankment (50 m downstream from the Glebuchev Ravine wastewater discharge point); No. 5 — Saratov embankment (50 m downstream from the Beloglinsky Ravine wastewater discharge point); No. 6 — “Beach of the Volga Conquerors” recreational area (Fig. 1).

 

Fig. 1. Sampling sites on the Volga River in 2024 during monitoring studies for cholera.

 

As a result of the study, 21 cultures were isolated, and the sampling point number and the isolation time were used to designate the strains (for example: No. 3 3007 — strain isolated in the Zaton beach area July 30).

Based on their growth characteristics on nutrient media, motility, and biochemical tests, the strains were classified as V. cholerae (production of β-galactosidase, lysine decarboxylase, ornithine decarboxylase, gelatinase, fermentation of citrates, glucose, mannitol, sucrose, reduction of nitrates to nitrites, indole formation, cytochrome oxidase production, glucose oxidation in the Hugh-Leifson medium, absence of urea, inositol, rhamnose, arabinose, and sorbitol fermentation). MALDI-Tof mass spectrometry confirmed that the isolated strains belonged to V. cholerae. The strains did not agglutinate with O1 and O139 antisera. PCR did not detect the wbeT, wbfR, ctxA or tcpA genes. As a result of comprehensive analysis, the isolated strains were identified as non-toxigenic V. cholerae non-O1/non-O139.

Pangenomic and phylogenetic analyses

In the first stage of the study, research was conducted to identify V. paracholerae strains among the isolated isolates. According to the literature, V. paracholerae isolated in different countries form a separate phylogenetic branch from the toxigenic and non-toxigenic strains of V. cholerae O1 El Tor, as well as V. cholerae non-O1/non-O139 [19, 20].

To align core genes and construct a phylogenetic tree based on them, a pan-genome analysis was performed. The resulting alignment, approximately 2.7 million nucleotides long, included 2,506 genes present in at least 99% (63) of the genomes in the sample. The GTR+G+F model was used to construct a phylogenetic tree using the Maximum Likelihood method. V. metoecus was used as an outgroup for rooting the tree. As a result, the genomes of V. cholerae and V. paracholerae formed two separate branches on the tree, sister to each other. The V. paracholerae branch also included five studied strains of V. cholerae non-O1/non-O139, isolated from the Volga River in 2024. — No. 5-2 0207, No. 3 1808, No. 6 2207, No. 5 2207, No. 5 3007 (Fig. 2). The data obtained may indicate their relationship to the V. paracholerae species. The strains were isolated in the area of the Zaton natural beach (point No. 3), from the wastewater of the Beloglinsky ravine (point No. 5), as well as in the recreational area “Beach of the Volga Conquerors” (point No. 6). It is worth noting that the identified V. paracholerae were related to V. paracholerae strains isolated in Bangladesh (2016), the USA (2017), Mozambique (2008), Thailand (1992), and Haiti (2010) (Fig. 2).

 

Fig. 2. Phylogenetic analysis of strains.

The cluster containing V. paracholerae strains is marked in yellow. Strains isolated from the Volga River in 2024 are marked in bold. Bootstrap values are given for large nodes within clusters.

 

The remaining analyzed NAG vibrios formed a cluster with other non-O1/non-O139 V. cholerae isolates (Fig. 2). Some of them were also related to strains circulating in endemic areas. For example, V. cholerae No. 4 2207 formed a subcluster with the V. cholerae EDC_715 strain isolated from the external environment in Bangladesh in 2015, and V. cholerae No. 2 2207 formed a subcluster with V. cholerae EM-1676A, isolated in Bangladesh in 2011 (Fig. 2).

To confirm their belonging to the V. paracholerae species, the ANI matrix between the genomes of 21 studied strains was calculated using the fastANI program for the 5 studied genomes. The average ANI value between the groups of genomes defined as V. cholerae non-O1/non-139 (n = 16) and V. paracholerae (n = 5) was 96.18% (95.95–96.46%), and within these groups — 98.07% (97.8–99.9%) and 98.01% (97.71–98.23%), respectively. Thus, based on the results of phylogenetic analysis and comparison of ANI values between the two groups of genomes, it can be concluded that the five isolates studied belong to the species V. paracholerae.

For the groups of genomes included in the V. cholerae (n = 33) and V. paracholerae (n = 28 genomes) branches, the number of genes found in all genomes of the group was calculated. For V. cholerae, it was 2858, for V. paracholerae — 2675, of which 2605 genes were common to both species. Thus, the number of unique genes for these groups was 328, of which 253 are found only in V. cholerae genomes and 75 are found only in V. paracholerae genomes.

To assess the clonality of the origin of the isolates obtained, an additional phylogenetic analysis was performed. Using snippy for the V. paracholerae and V. cholerae groups, core SNP alignments were obtained, using V. paracholerae EDC-792 (NZ_WYCC00000000.1) and sample N1_2207 as reference genomes, respectively. Recombination sites were removed from the SNP alignments using the gubbins tool. As a result, the alignments contained 113,643 (V. paracholerae) and 168,889 (V. cholerae) polymorphic sites, and two unrooted phylogenetic trees were constructed based on them using the Maximum Likelihood method with the GTR+G+F model (Fig. 3). Pairwise distance matrices were also calculated based on the obtained alignments. For the group of V. paracholerae isolates, the distance matrix contained values in the range of 41,752–56,741 SNPs, and, as expected, the resulting tree does not contain groups of genetically closely related isolates, but consists of separate, approximately equidistant nodes (Fig. 3, a).

 

Fig. 3. Intraspecies phylogenetic trees constructed based on core SNPs for isolates of V. paracholerae (a) and V. cholerae (b).

 

The tree for the group of V. cholerae isolates has a similar topology, but includes two groups of extremely close sequences: N6_0608-N4_1808 and N6_2008-N5_0608 (Fig. 3, b), with a distance of 5 and 6 SNPs, respectively (while the average distance between all studied V. cholerae genomes is 48,528 SNPs). These isolates also have virtually identical genetic properties (Table 1), which also supports their clonal origin. Isolate N4_1808 was isolated 12 days later and more than 2.5 km upstream from N6_0808, indicating the persistence of this clone and the possible existence of an ecological reservoir in which it has taken root.

 

Table 1. Genetic properties of V. cholerae non-O1/non-O139 and V. paracholerae strains isolated in 2024 from the Volga River near Saratov

Strain designation*	hlyA	cas3	toxR	toxS	MARTX			VPI–2		T3SS				T6SS			MSHA		ompW vca0867	ompW vca0867
					rtxA vc1451	rtxC vc1450	vc 1447	nanH vc1784	vc 1757	vcsN2	vcsV2	vcsC2	vspD	vgrG3 vca0123	hcp vc1415	vasK vca0120	mshA vc0409	mshD vc0411
No. 4-2 0207	micr029864	94.5	–	98.4	98.3	97.2	100	98.8	96.7	94.4	99.4	99.5	98.8	99.3	97.7	98.2	98.6	–	98.0	99.9
No. 5-2 0207	micr029869	97.0	96.4	88.5	89.6	–	98.9	98.0	–	96.9	–	–	–	–	94.7	98.7	98.2	–	97.5	98.5
No. 4 0907	micr029870	99.0	94.6	98.6	98.5	97.7	99.6	99.1	96.9	98.2	99.4	99.4	97.9	99.0	94.6	99.0	99.0	–	95.0	100
No. 1 2207	micr029871	100	96.5	98.5	100	–	99.8	98.9	–	96.5	–	–	–	–	97.6	89.2	98.4	–	98.6	88.5
No. 2 2207	micr029872	94.5	96.4	98.6	98.1	97.6	99.4	97.5	–	96.8	–	–	–	–	94.1	99.6	98.9	–	94.9	99.9
No. 3 2207	micr029873	99.0	–	98.6	97.9	97.1	99.4	98.6	–	97.0	–	–	–	–	85.8	92.1	98.6	–	94.6	99.0
No. 4 2207	micr029874	100	–	98.1	99.8	–	99.6	99.4	–	98.0	–	–	–	–	93.4	87.8	98.6		97.5	89.1
No. 5 2207	micr029866	100	–	88.6	98.9	–	98.9	97.9	–	96.6	–	–	–	–	97.4	99.2	98.1	–	98.0	99.0
No. 6 2207	micr029865	97.0	–	88.9	89.6	–	98.9	97.2	–	96.6	–	–	–	–	94.9	–	98.2	–	98.0	97.9
No. 1 3007	micr029875	100	–	99.21	99.8	–	99.4	97.5	–	97.2	–	–	–	–	94.4	98.1	98.6	–	94.4	99.5
No. 2 3007	micr029876	100	–	99.6	99.4	97.1	99.6	98.5	96.9	95.9	99.1	99.2	97.8	99.0	94.3	99.0	98.9	–	98.5	99.5
No. 3 3007	micr029877	94.5	–	98.3	99.0	–	98.7	99.2	96.1	98.0	–	–	–	–	94.0	98.7	98.7	–	97.7	89.2
No. 5 3007	micr029867	97.0	–	90.3	89.6	–	98.9	97.2	–	96.6	–	–	–	–	94.9	96.1	98.6	–	98.0	97.9
No. 6 3007	micr029878	100	–	98.6	98.5	97.2	99.6	98.7	–	97.1	–	–	–	–	94.1	98.2	98.7	–	95.0	98.5
No. 4 0608	micr029879	98.0	–	98.3	98.5	97.6	99.6	98.5	96.1	97.1	99.7	99.5	96.4	99.4	93.9	98.6	98.6	99.3	98.5	99.7
No. 5 0608	micr029880	100	–	99.0	99.2	–	99.1	98.4	–	94.7	–	–	–	–	93.8	98.7	98.9	–	98.4	99.2
No. 6 0608	micr029881	100	–	98.3	98.5	97.4	100	98.6	96.7	94.3	99.5	99.5	98.6	99.1	94.9	99.0	98.6	99.4	94.3	97.6
No. 3 1808	micr029868	97.0	–	88.7	89.6	–	99.8	96.7	–	97.0	–	–	–	–	–	98.5	–	–	93.5	97.9
No. 4 1808	micr029882	100	–	98.3	98.5	97.4	100	98.6	96.7	94.3	99.5	99.5	98.6	99.4	94.9	99.0	98.6	99.4	94.3	97.6
No. 2 2008	micr029883	100	–	98.1	98.5	97.4	99.6	99.2	–	97.4	–	–	–	–	97.5	98.3	98.8	99.3	98.5	99.9
No. 6 2008	micr029884	100	–	99.0	99.2	–	99.1	98.4	–	94.7	–	–	–	–	93.8	98.7	98.9	–	98.4	99.2

Note. «–» — gene is missing; numbers indicate the percentage of similarity to the corresponding gene of the reference strain V. cholerae N16961 O1 El Tor; * — V. paracholerae strains are highlighted in bold.

 

Study of the genetic characteristics of isolated strains of V. cholerae non-O1/non-O139 and V. paracholerae

Bioinformatics analysis of the nucleotide sequences of the studied strains did not reveal the following mobile genetic elements with genes of pathogenicity, pandemicity, and antibiotic resistance: prophages CTXφ (ctxA^–, ctxB^–, cep^–, ace^–, zot^–, orfU^–) and RSIφ (rstR, rstA, rstB, rstC), TLC element, pathogenicity island VPI-1 (tcpA^–, toxT^–, aldA^–, acfB^–, mop^–), pandemic island VSP-I (vc0175, vc0178, vc0180, vc0183, vc0185), ICE SXT element. The absence of CTXφ and VPI-1, containing, respectively, the ctxAB and tcpA genes encoding the biosynthesis of cholera toxin and the major subunit of toxin-coregulated pilus, indicates the avirulence of the strains and their inability to cause cholera. However, the genomes of all isolates studied contain the toxR/toxS genes responsible for the biosynthesis of important regulatory proteins that control the production of the main virulence factors of the cholera pathogen. Their similarity to the toxR/toxS genes of the reference strain V. cholerae N16961 O1 El Tor was 88.5–99.8% (Table 1).

Most strains lack the VSP-II pandemic island genes (vc0490, vc0496, vc0502, vc0512), with the exception of two strains of NAG vibrios — No. 1 3007 and No. 4 0608, as well as V. paracholerae No. 5 2207, which have the vc0496 gene located in the central part of VSP-II (identity with the similar gene of the reference strain — 97.7–98.8%; Table 2).

 

Table 2. Comparative analysis of the genetic properties of V. cholerae and V. paracholerae strains

Strain	Place of isolation	Source	Year of isolation	Tor operon		Potassium pump regulated by glutathione
				vc1692–vc1694	vc1719–vc1720	vc2606–vc2607
V. cholerae N16961AE003852 О1 El Tor*	Bangladesh	Clinic	1971	+	+	+
Non-toxigenic strains of V. cholerae О1 El Tor
2012ENV-9JSTH01	Haiti	EO	2012	+	+	+
12129ACFQ01	Australia	EO	1985	+	+	+
29VUAB01	Elista, Russia	EO	2013	+	+	+
V. cholerae non-O1/non-O139 strains
C197VUAG01	Stavropol, Russia	EO	1974	+	+	–
1587AAUR01 О12	Peru	Clinic	2000	+	+	+
MZO-03AAUU01 О37	Bangladesh	Clinic	2001	+	+	+
CP1035AJRM01	Mexico	Clinic	2004	+	+	+
HE48AFOR01	Haiti	EO	2010	+	+	+
EM-1676AAPFY01	Bangladesh	EO	2011	+	+	+
2012EL-1759JNEW01, 2012ENV-92 JSTJ01	Haiti	EO	2012	+	+	+
EDC_689WYCR01, EDC_715WYCJ01	Bangladesh	EO	2015	+	+	+
EDC_ 800WYCA01	Bangladesh	EO	2016	+	+	+
41JAJPEH01	Moscow, Russia	Clinic	2018	+	+	+
118JAJPEC01	Chelyabinsk, Russia	Clinic	2020	+	+	+
No. 4-2 0207micr029864, No. 4 0907micr029870, No. 1 2207micr029871, No. 2 2207micr029872, No. 3 2207micr029873, No. 4 2207micr029874, No. 1 3007micr029875, No. 2 3007micr029876, No. 3 3007micr029877, No. 6 3007micr029878, No. 4 0608micr029879, No. 5 0608micr029880, No. 6 0608micr029881, No. 4 1808micr029882, No. 2 2008micr029883, No. 6 2008micr029884	Saratov, Russia	EO	2024	+	+	+
V. paracholerae strains
NCTC30NZ_LS997867	Egypt	Clinic	1916	–	–	–
490-93 DA89JIDQ01	Thailand	Clinic	1992	–	–	+
VCC19ATEV02	Brazil	Wastewater	1994	–	–	–
877-163LBNV01	Bangladesh	EO	2002	–	–	–
SIOMIPJ01	USA	EO	2003	–	–	–
CISM300506NZ_MWFL01	Mozambique	Clinic	2008	–	–	–
HE-16ALEB01, HE-09AFOP01	Haiti	EO	2010	–	–	–
CISM1163068NZ_MWFO01	Mozambique	Clinic	2012	–	–	–
2014V-1107QKKP01	USA	Clinic	2014	–	–	–
EDC-690WUWI01, EDC-716WYBZ01, EDC-717WYBY01	Bangladesh	EO	2015	+	–	–
**EDC-792WYCC01	Bangladesh	EO	2016	–	–	–
2016V-1091QKKQ01, 2016V-1111QKKR01, 2016V-1114 QKKS01	USA	Clinic	2016	–	–	–
2017V-1105QKKT01, 2017V-1110QKKU01	USA	Wound	2017	–	–	–
2017V-1144QKKV01	USA	Clinic	2017	–	–	–
2017V-1176QKKW01	USA	Animal feed	2017	–	–	–
07-2425QKKO01, 87395APFL01	Unknown			–	–	–
No. 5-2 0207micr029869, No. 5 2207micr029866, No. 6 2207micr029865, No. 5 3007micr029867, No. 3 1808micr029868	Saratov, Russia	EO	2024	–	–	–

Note. EO — environmental objects. *Toxigenic reference strain V. cholerae O1 El Tor; **type strain V. paracholerae; «+» — gene is present; «–» — gene is missing.

 

Analysis of the VPI-2 pathogenicity island revealed that all strains have altered marginal genes (vc1757 and vc1810) of this mobile genetic element (identity 94.3–98.2% and 94.9–98.5%, respectively), but only 6 (28.6%) strains were found to have the nanH gene, which encodes neuraminidase and is located in the central part of this island. The structure of this gene was also variable (Table 1). The nanH gene was not detected in V. paracholerae strains. As is known, neuraminidase is an important factor in pathogenicity and participates in the cleavage of sialic acids that are part of intestinal mucus, which facilitates the penetration of vibrios into intestinal epithelial cells for their subsequent colonization. Although T3SS is an established pathogenicity factor of NAG vibrios that promotes colonization [11], altered genes of this system were detected in only 6 (28.6%) of the strains we studied. Previous studies have also shown its presence in the genome of only some isolates [8, 20]. At the same time, five verified genes (vasA, vasF, vasK, vgrG3, hcp) that are part of T6SS were found in almost all strains. The exceptions were strains V. paracholerae No. 3 1808 and No. 6 2207, which, respectively, had (one of all studied) and lacked the locus encoding hemolysin Hcp (Table 1). As is known, T6SS is a contact-dependent mechanism by which vibrios infect various prokaryotic and eukaryotic organisms by translocating toxic effector proteins into target cells. The elimination of competitors increases the survival of V. cholerae in the environment and in macroorganisms [22]. It has been suggested that the presence of genes encoding ToxR/ToxS proteins, hemolysins, lipases, and T6SS effectors, found in the genomes of many Vibrio species, indicates the ability of these vibrios to be potential pathogens, as well as the important role of these factors in their ecology [23].

Important factors in the pathogenicity of NAG vibrios are hemolysin HlyA and cytolysin/cytokin MARTX [8, 24, 25]. A complete hlyA gene, corresponding in structure to the locus of the reference strain V. cholerae N16961 O1 biovar El Tor, was found in the genome of 10 NAG vibrios and in strain V. paracholerae No. 5 2207. In the remaining isolates, this gene contains mutations, and its identity varies from 94.5 to 99.0%. The presence of the hlyA gene correlated with the production of β-hemolysin by all strains, with erythrocyte hemolysis zones ranging from 1 to 8 mm. The rtxA gene, which directly encodes the MARTX toxin, is present in 10 NAG vibrios, but this gene has mutations in all of them. At the same time, other loci were identified in the genome of all strains (Table 2): rtxC (encodes acyltransferase) and vc1447 (encodes a transporter). Three NAG vibrios have an intact rtxC gene sequence, while the remaining strains of V. cholerae and V. paracholerae have a gene that is 98.6–99.6% identical (Table 2).

It is worth noting that four strains contain the cas3 gene, which is part of the CRISPR-cas system—a specific adaptive immune system in bacteria that protects them from the penetration of foreign DNA (phages, plasmids). The cas3 gene encodes a nuclease that fragments foreign DNA [26]. Further analysis has established that the CRISPR-cas system identified in the strains belongs to class I (subtypes I-F, I-C) and III (subtype III-B). The V. cholerae non-O1/non-O139 strain No. 1 2207 has two systems of subtypes III-B and I-F, whose CRISPR cassettes include 33 and 6 spacers, respectively. V. cholerae non-O1/non-O139 No. 2 2207 and No. 4 0907 have an I-F system with 34 and 13 spacers, respectively. The V. paracholerae strain No. 5-2 0207 contains a CRISPR-cas system of subtypes I-C (35 spacers) and I-F (58 spacers) (Fig. 4).

 

Fig. 4. Structure of the CRISPR-cas 1 system of V. cholerae non-O1/non-O139 and V. paracholerae strains.

Shown are subtype III-B V. cholerae No. 1 2207 and subtypes I-F, I-C of V. paracholerae strain No. 5-2 0207.

 

Spacers are sections of foreign DNA homologous to phages and plasmids with which bacteria have previously come into contact. The presence of spacers specific to lytic cholera phages protects cells from subsequent infections. Analysis of the spacers revealed their correspondence to the protospacer DNA sequences of various cholera phages: ICP1, JSF14, JSF1, JSF5, JSF6, JSF2, JSF13, JSF4, VMJ710, JSF17, Rostov M3, CP-T1, vB_VchM-138, vB_VchM_VP-3213, 24, Х29, phi 2, Rostov 7, VPUSM 8, Ch457, VcP032, V86, K139, vB_VchM_Kuja, Kappa, K139, as well as V. cholerae pE7G and pSA7G2 plasmids. Furthermore, spacers homologous to V. vulnificus 48/10 and Salinivibrio costicola GSL5 plasmids were detected. The data obtained indicate that, living in an aquatic environment, NAG vibrios and V. paracholerae are exposed to various cholera phages. Given that the analyzed strains were isolated from the external environment, it was expected that all of them would have a DNA segment encoding the biosynthesis of mannose-sensitive hemagglutination pilus (MSHA), which facilitate the attachment of vibrios to biotic and abiotic surfaces during biofilm formation (secretory operon — mshHIJKLMNIJ, structural operon — mshBACDOPQ). As is well known, biofilm protects vibrios from adverse factors and increases their survival in the environment [27]. However, the mshA gene, which encodes the major subunit of MSHA pilus, was found only in four strains of NAG vibrios, and its sequence was highly homologous to the prototype (99.3–99.4% identity). At the same time, all strains have the mshD gene, which is responsible for the biosynthesis of the minor subunit of MSHA pilus (Table 1). It is possible that the strains under study produce MSHA pilus, but further research is needed to verify this assumption.

Identification of genetic differences characteristic of V. paracholerae strains

Genetic analysis showed that V. paracholerae did not differ from NAG vibrios in terms of the presence and structure of genes commonly found in V. cholerae isolated from the external environment (Table 1). In particular, the V. paracholerae genome contains the ompW gene, which encodes an outer membrane protein specific to V. cholerae. This gene is used in foreign PCR test systems to detect V. cholerae strains [28]. The V. paracholerae genome also contains the tоxR/toxS, hlyA, rtx, T6SS and T3SS genes, as well as other genes found in V. cholerae non-O1/non-O139 [8, 10, 24]. However, according to the literature, V. paracholerae has a number of genetic features that distinguish it from V. cholerae [20]. Thus, the genomic markers that differentiate V. cholerae and V. paracholerae are two loci of the Tor operon (vc1692–1694 and vc1719–1720) and the genes of the glutathione-regulated potassium pump (vc2606, vc2607), which are present in V. cholerae and not detected in V. paracholerae [29]. The five strains we studied also lack all of these genes (Table 2).

Thus, the data obtained confirm that the isolated strains No. 5-2 0207, No. 5 2207, No. 6 2207, No. 5 3007, and No. 3 1808 belong to the species V. paracholerae.

Discussion

Analysis of V. cholerae non-O1/non-O139 and V. paracholerae strains isolated from the Volga River near Saratov showed that these strains do not contain a number of mobile genetic elements with virulence and pandemic genes. At the same time, loci encoding additional virulence and adaptation factors, including hlyA responsible for hemolysin biosynthesis, were identified in their genome. In 52.4% of strains, the structure of this locus corresponds to the toxigenic reference strain V. cholerae N16961 O1 El Tor. Hemolysin HlyA, which has cytotoxic activity and the ability to damage intestinal epithelial cells, is an important factor in the pathogenicity of V. cholerae non-O1/non-O139 strains [8, 24, 25]. The loci responsible for the biosynthesis of the pore-forming toxin MARTX, which destroys the actin cytoskeleton of host cells, and T6SS genes have also been identified. It has been experimentally proven that NAG vibrios expressing T6SS effector proteins and producing hemolysin suppressed the growth of the toxigenic strain V. cholerae N16961 O1 El Tor when co-cultured in an aqueous medium [25].

Three strains of NAG vibrios and one isolate of V. paracholerae containing a class 1 CRISPR-cas system were identified. The presence of the cas gene encoding nuclease and a significant number of spacers (from 6 to 58) prove that this system is functional in these strains. Furthermore, spacers corresponding to the genetic material of various types of ICP1phage (ICP1_2001, ICP1_2004, ICP1_2005, ICP1_2006, ICP1_2006_E, ICP1_2006_D, ICP1_2011, ICP1_2011_A, ICP1_2012, ICP1_2017_F_Mathbaria). This phage is the most widespread in the endemic territory [30]. Based on the information obtained, it can be assumed that V. cholerae non-O1/non-O139 strains brought from the endemic territory may circulate in the Volga River near Saratov. Earlier, D.Y. Kang et al., studying V. cholerae non-O1/non-O139 strains isolated from open water bodies in northern Cameroon, showed their close relationship with isolates circulating in Kenya, Argentina, Haiti, suggesting that these vibrios may spread not only between countries but also between continents [31].

Given that V. paracholerae strains were first detected by us in Russia, further detailed phenotypic and molecular genetic studies of representatives of this species are necessary. There is no doubt that V. paracholerae circulates in other regions of our country and is isolated under the guise of V. cholerae non-O1/non-O139. The presence/absence of specific genes in V. paracholerae [20] indicates the possibility of creating diagnostic test systems for their detection and differentiation from V. cholerae non-O1/non-O139. It is worth noting that among the 38 nucleotide sequences of complete genomes of V. paracholerae strains deposited in NCBI GenBank with an established source of isolation, 10 (26%) isolates were isolated from the stool of patients. There is a strong possibility that V. paracholerae strains circulating in the Volga River water are also capable of causing acute intestinal infection, but additional research is needed to confirm this assumption.

Conclusion

Not only V. cholerae non-O1/non-O139 strains, but also V. paracholerae strains circulate in the Volga River near Saratov. Both groups of strains have similar biochemical properties and genetic structure. Their genomes lack pathogenicity genes and a number of pandemic genes, but loci for additional toxins have been identified, the presence of which is characteristic of representatives of NAG vibrios isolated from open water bodies in other regions of our country. Further research is needed on the comparative study of the phenotypic and molecular-genetic properties of V. paracholerae strains.

About the authors

Svetlana P. Zadnova

Research Anti-Plague Institute «Microbe»

Email: svetlanazadnova@mail.ru
ORCID iD: 0000-0003-4366-0562

Dr. Sci. (Biol.), Head, Laboratory of pathogenic vibrios, Microbiology department

Russian Federation, Saratov

Nadezhda B. Cheldyshova

Research Anti-Plague Institute «Microbe»

Email: rusrapi@microbe.ru
ORCID iD: 0000-0001-5759-3765

Cand. Sci. (Med.), senior researcher, Laboratory of pathogenic vibrios, Microbiology department

Russian Federation, Saratov

Daria L. Kusmartseva

Research Anti-Plague Institute «Microbe»

Email: rusrapi@microbe.ru
ORCID iD: 0009-0002-3235-7357

junior researcher, Laboratory of pathogenic vibrios, Microbiology department

Russian Federation, Saratov

Darya A. Rybalchenko

Research Anti-Plague Institute «Microbe»

Email: arashis@mail.ru
ORCID iD: 0000-0002-3117-8229

researcher, Laboratory of pathogenic vibrios, Microbiology department

Russian Federation, Saratov

Danil A. Sergutin

Research Anti-Plague Institute «Microbe»

Author for correspondence.
Email: sergutin322@mail.ru
ORCID iD: 0009-0003-7525-9682

junior researcher, Laboratory of pathogenic vibrios

Russian Federation, Saratov

Andrey V. Boyko

Research Anti-Plague Institute «Microbe»

Email: rusrapi@microbe.ru
ORCID iD: 0000-0001-9576-4959

Dr. Sci. (Med), leading researcher, Department of diagnostics of infectious diseases

Russian Federation, Saratov

Andrey V. Kazantsev

Research Anti-Plague Institute «Microbe»

Email: rusrapi@microbe.ru
ORCID iD: 0000-0003-1790-0411

researcher, Department of diagnostics of infectious diseases

Russian Federation, Saratov

Oksana A. Koreshkova

Research Anti-Plague Institute «Microbe»

Email: rusrapi@microbe.ru
ORCID iD: 0009-0008-2029-0646

junior researcher, Department of infectious disease diagnostics

Russian Federation, Saratov

Andrey V. Fedorov

Research Anti-Plague Institute «Microbe»

Email: rusrapi@microbe.ru
ORCID iD: 0000-0001-7190-4427

junior researcher, Laboratory of genomic and proteomic analysis

Russian Federation, Saratov

Yaroslav M. Krasnov

Research Anti-Plague Institute «Microbe»

Email: rusrapi@microbe.ru
ORCID iD: 0000-0002-4909-2394

Cand. Sci. (Chem.), Head, Laboratory of Genomic and proteomic analysis

Russian Federation, Saratov

Svetlana A. Portenko

Research Anti-Plague Institute «Microbe»

Email: rusrapi@microbe.ru
ORCID iD: 0000-0002-8334-9173

Cand. Sci. (Biol.), Head, Department of infectious disease diagnostics

Russian Federation, Saratov

Svetlana A. Shcherbakova

Research Anti-Plague Institute «Microbe»

Email: rusrapi@microbe.ru
ORCID iD: 0000-0003-1143-4069

D. Sci. (Biol.), Deputy director

Russian Federation, Saratov

References

Supplementary files