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Article

Growth and Genome Features of Non-O1/O139 Vibrio cholerae Isolated from Three Species of Common Freshwater Fish

by
Xinchi Qin
1,2,
Lianzhi Yang
1,2,
Yingwei Xu
1,2,
Lu Xie
3,
Yongjie Wang
1,2 and
Lanming Chen
1,2,*
1
Key Laboratory of Quality and Safety Risk Assessment for Aquatic Products on Storage and Preservation (Shanghai), Ministry of Agriculture and Rural Affairs of China, Shanghai 201306, China
2
College of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, China
3
Shanghai-MOST Key Laboratory of Health and Disease Genomics, Institute for Genome and Bioinformatics, Shanghai Institute for Biomedical and Pharmaceutical Technologies, Shanghai 200032, China
*
Author to whom correspondence should be addressed.
Diversity 2024, 16(5), 268; https://doi.org/10.3390/d16050268
Submission received: 16 March 2024 / Revised: 26 April 2024 / Accepted: 27 April 2024 / Published: 30 April 2024
(This article belongs to the Special Issue Diversity, Occurrence and Distribution of Foodborne Pathogens)
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Abstract

:
Vibrio cholerae is the etiological agent of cholera in humans. The bacterium is frequently detected in aquatic products worldwide. However, the current literature on the genome evolution of V. cholerae of aquatic animal origins is limited. Here, we firstly characterized the growth and genome features of V. cholerae isolates with different resistance phenotypes from three species of common freshwater fish. The results revealed that the non-O1/O139 V. cholerae isolates (n = 4) were halophilic and grew optimally at 2% NaCl and pH 8.0. Their draft genome sequences were 3.89 Mb–4.15 Mb with an average GC content of 47.35–47.63%. Approximately 3366–3561 genes were predicted to encode proteins, but 14.9–17.3% of them were of an unknown function. A number of strain-specific genes (n = 221–311) were found in the four V. cholerae isolates, 3 of which belonged to none of any of the known sequence types (STs). Several putative mobile genetic elements (MGEs) existed in the V. cholerae isolates, including genomic islands (n = 4–9), prophages (n = 0–3), integrons (n = 1–1), and insertion sequences (n = 0–3). Notably, CRISPR-Cas system arrays (n = 2–10) were found in the V. cholerae genomes, whereby the potential immunity defense system could be active. Comparative genomic analyses also revealed many putative virulence-associated genes (n = 106–122) and antibiotic resistance-related genes (n = 6–9). Overall, the results of this study demonstrate the bacterial broader-spectrum growth traits and fill prior gaps in the genomes of V. cholerae originating from freshwater fish.

1. Introduction

Vibrio cholerae is the etiological agent of cholera, a highly contagious diarrhea disease that affects millions of people worldwide each year [1]. Currently, outbreaks of cholera still occur, especially in regions where access to safe drinking water is limited [1]. For instance, in Mozambique, in the most recent outbreak, the first case of cholera was reported to the World Health Organization (WHO) from the Lago district in Niassa Province on 14 September 2022. Until 19 February 2023, a total of 5237 suspected cases and 37 deaths have been reported from 29 districts across six of the eleven Provinces in Mozambique (https://www.who.int/emergencies/disease-outbreak-news, accessed on 2 June 2023). In addition, the consumption of fish products contaminated by V. cholerae has also been linked to cholera outbreaks [2]. The cholerae toxin (CT) and toxin-coregulated pilus (TCP) are the primary virulence factors present in epidemic V. cholerae strains of serotypes O1 and O139 [3].
Antibiotic treatment can effectively shorten the duration of diarrhea and limit the spread of cholera. Nevertheless, the intensive use of antibiotics in health and agriculture sectors has exacerbated the resistance crisis. The emergence of multidrug-resistant (MDR) pathogens is a global health issue, which leads to higher morbidity and mortality and more healthcare costs [4]. The MDR V. cholerae strains have emerged and rapidly spread worldwide, particularly in Africa in recent years [5,6]. For example, Taviani et al. [5] isolated V. cholerae strains (n = 70) from drinking water samples (n = 91) collected in Moamba, Mozambique, in 2008. They found that all the isolates were resistant to Ampicillin (AMP); 51% and 13% of the isolates were also resistant to streptomycin (STP) and gentamicin (GM); and 13% of the isolates exhibited MDR profiles, showing resistance to at least three antibiotics. Abioye et al. [6] isolated V. cholerae strains (n = 34) from some seafood in the Eastern Cape Province, South Africa, in 2022, and found that 65.71% of the strains had MDR phenotypes. Recently, Schmidt et al. [7] reported the antibiotic resistance of 24 environmental V. cholerae non-O1/O139 strains collected in Germany and other European countries between 2011 and 2021. They found that 42% of the isolates (n = 24) were resistant to meropenem (MEM), 16.7% to tetracycline (TET) and doxycycline (DOX), 12.5% to trimethoprimand, and 8% to piperacillin-tazobactam (TZP).
Resistance genes (RGs) may mainly be spread through horizontal gene transfer (HGT), which is arguably the most significant driving force of bacterial evolution [8]. This transfer could occur specifically through mobile genomic elements (MGEs), such as integrons (Ins), prophages, genomic islands (GIs), and insertion sequences (ISs) [9]. HGT significantly contributes to microbial adaptation to the environments and to the hosts [10]. In the past decades, sequencing technology has been rapidly advancing, e.g., single-molecule real-time sequencing, Oxford nanopore sequencing, single-cell sequencing, and multi-omics sequencing [11,12]. This facilitates the deciphering of the molecule mechanism underlying the MDR and pathogenesis of V. cholerae at the whole-genome level. A total of 1708 V. cholerae isolates have been sequenced so far (GenBank database, https://www.ncbi.nlm.nih.gov/, accessed on 18 June 2023). Of these, complete genome sequences of 112 V. cholerae isolates are available in genome databases. However, the current literature on genome features of V. cholerae strains in aquatic animals is limited, particularly from freshwater fish.
In our prior studies, we isolated and identified a number of V. cholerae strains from many species of aquatic animals and water environments [2,13,14,15]. In follow up to our previous work, in this study, we further investigated the growth and genome features of the V. cholerae isolates, which displayed different resistance phenotypes and originated from three species of common freshwater fish, including Aristichthys nobilis, Ctenopharyngodon idellus, and Parabramis pekinensis. The prevalence of V. cholerae in these fish species has been reported in our previous study [2]. The objectives of this study were the following: (1) to examine the growth of V. cholerae isolates (n = 4) under varying pH and NaCl circumstances; (2) to sequence whole genomes of the V. cholerae isolates and identify MGEs and virulence- and resistance-related genes; and (3) to investigate the phylogenetic relatedness of the V. cholerae isolates.

2. Materials and Methods

2.1. V. cholerae Isolates and Culture Conditions

V. cholerae 7-6-5 and V. cholerae L10-48, V. cholerae L1-1, and V. cholerae B5-86 isolates were isolated from A. nobilis, C. idellus, and P. pekinensis, respectively (Table S1), which were sampled in fish markets in Shanghai, China, in 2017–2019 [2,13]. The V. cholerae isolates were routinely incubated in Trypticase Soy Broth (TSB) (3% NaCl, pH 8.5) at 37 °C with shaking at 180 rpm [2,13,14,15], unless otherwise specified.

2.2. Growth of V. cholerae Isolates under Different pH and NaCI Conditions

The TSB was adjusted to different pH values (6.0, 6.5, 7.0, 7.5, 8.0, and 8.5) and NaCl concentrations (0.5%, 1%, 2%, 3%, 4%, and 5%), respectively [16]. Growth curves of V. cholerae isolates under the different pH (6.0–8.5) and NaCl concentrations (0.5–5%) were determined individually at 37 °C for 24 h using Multimode Microplate Reader (BioTek Instruments, Winooski, VT, USA).

2.3. Genome Sequencing, Assembly, and Annotation

The V. cholerae strains were cultured in the TSB (2% NaCl, pH 8.0) until reaching the mid-logarithmic growth phase (mid-LGP) at 37 °C. The bacterial cells were collected at 2700× g for 10 min at 4 °C. The genomic DNA was extracted using the TIANamp Bacteria DNA Kit (Tiangen Biochemical Technology Co., Ltd., Beijing, China) and analyzed [9,17,18,19].
The 16S rRNA gene of V. cholerae strains was individually amplified by the PCR assay, and the PCR products were purified, sequenced, and analyzed [9,17,18,19].
Genome sequences of the V. cholerae isolates were determined by Shanghai Majorbio Bio-Pharm Technology Co., Ltd. in Shanghai, China, using the Illumina HiSeq × 10 platform (Illumina, San Diego, CA, USA). The average length of sequencing reads was 150 bp. Sequence quality control and assembly were performed using SOAPdenovo software (version 2.04) [20]. The coding sequences (CDSs), rRNA genes, and tRNA genes were predicted using Glimmer software (version 3.02) [21], Barrnap tool (https://github.com/tseemann/barrnap, accessed on 11 April 2021), and tRNAscan-SE (version 2.0) software [22], respectively.
Each gene in V. cholerae genomes was functionally assigned against the non-redundant protein database of the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov, accessed on 16 June 2023) and classified by assigning a Clusters of Orthologous Groups (COGs) number with 80% identity and 90% coverage at E ≤ 1 × 10−5. If a CDS did not hit any known function, it was labeled as function unknown. The Virulence Factor Database (http://www.mgc.ac.cn/VFs, accessed on 16 June 2023), Antibiotic Resistance Gene Database (http://arpcard.Mcmaster.ca, accessed on 16 June 2023), and BacMET database [23] were used to predict virulence-, antibiotic-, and heavy metal resistance-related genes, respectively.

2.4. Comparative Genome Analysis

GIs in each of the V. cholerae genomes were predicted using IslandPath-DIMOB and IslandViewer software (version 1.0) [24], while Prophages, Ins, and ISs were predicted using Phage_Finder software (version 2.3.0) [25], Integron_Finder software (version 2.0) [26], and Isfinder software (https://www-is.biotoul.fr/search.php, accessed on 20 May 2023) [27], respectively. The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas arrays were predicted using Minced software (version 3) and CRISPRtyper (https://cctyper.crispr.dk/#/submit, accessed on 11 April 2021) [28].
Orthologous genes in each of V. cholerae genomes were predicted using OrthoMCL software (version 14-137) [29], with their encoding proteins having at least more than 60% amino acid identity and 80% sequence coverage. Proteins with less than 30% identity or no matches were classified as strain-specific genes at a significance level of E ≤ 1 × 10−5 [9,17,18,19].

2.5. Multilocus Sequence Typing (MLST) Analysis

The MLST analysis of each of the V. cholerae isolates was performed against the PubMLST database, on the basis of the conserved gyrB, mdh, pntA, metE, pyrC, adk, and purM genes in V. cholerae [30].

2.6. Phylogenetic Tree Construction

The phylogenetic tree of 71 V. cholerae isolates was constructed using the maximum likelihood approach [9,17,18,19]. The cutoff threshold for bootstrap values was set above 50%. Complete genome sequences of the 69 V. cholerae isolates were downloaded from the GenBank database (Table S2).

2.7. Antibiotic and Heavy Metal Resistance Assays

Antibiotic resistance of V. cholerae isolates was examined using the standard disco diffusion method approved by the Clinical and Laboratory Standards Institute (CLSI, M100-S23, 2018), Malvern, PA, USA. The heavy metal tolerance of V. cholerae isolates was also examined following the broth dilution testing (microdilution, CLSI) [2,13,14,15]. The same chemicals and quality control strains were used as described in our recent reports [2,13,14,15].
The SPSS software (version 22, IBM, Armonk, NY, USA) was utilized to analyze the collected data. All tests were performed in triplicate.

3. Results and Discussion

3.1. Growth Profiles of the Four V. cholerae Isolates under Different pH and NaCI Conditions

V. cholerae 7-6-5 and L1-1, L10-48, and B5-86 isolates were isolated from A. nobilis, C. idellus, and P. pekinensis, respectively (Table S1). The strains were confirmed by 16S rRNA gene sequencing and analysis (Table S1).
Human acidic stomach pH is close to 2.0, and reaches 6.0 after eating food, while intestinal pH is close to 8.0. This challenges the survival of V. cholerae, which forms colonies in the intestinal tract and causes severe diarrhea disease and even death [3]. In our prior study, we observed that the growth of V. cholerae isolates was highly inhibited below pH 6.0. Therefore, we examined the growth traits of the four V. cholerae isolates of aquatic animal origins under different pH conditions (pH 6.0–8.5). As shown in Figure 1A–D, different growth profiles were observed among the V. cholerae isolates incubated in the TSB (3% NaCl) at 37 °C. For instance, the acidic condition (pH 6.0) slightly repressed the growth of V. cholerae 7-6-5. This isolate was capable of growing vigorously in a broader spectrum of pH conditions (pH 6.5–8.5) but showed the maximum biomass with an OD600 value of 1.332 at pH 8.0 (Figure 1A). Conversely, the V. cholerae L1-1 isolate was the most sensible to the acidic conditions among the test V. cholerae strains, as the growth of V. cholerae L1-1 was obviously inhibited at pH 6.0–7.5 (Figure 1C).
Although the four V. cholerae isolates were isolated from A. nobilis, C. idellus, and P. pekinensis, which were aquacultured in a freshwater environment, distinct growth profiles of the V. cholerae strains were observed under different NaCl concentrations (0.5–5%), when incubated in the TSB (pH 8.0) at 37 °C (Figure 2A–D). For example, V. cholerae 7-6-5 appeared to be the most adaptable to the lower NaCl concentrations (0.5–1.0%), under which this isolate was able to reach the maximum biomass with OD600 values of 0.725–0.796 (Figure 2A). Conversely, the growth of V. cholerae L10-48, L1-1, and B5-86 isolates was inhibited with 0.5–1.0% of NaCl (Figure 2B–D). All the strains grew well at 2–5% of NaCl, but the maximum biomass was at 2% NaCI, which was lower than the routine culture condition (3% NaCl) for V. cholerae strains [2].
Taken together, these results indicated that the four V. cholerae isolates that originated from the common freshwater fish were halophilic and grew optimally at pH 8.0 and 2% NaCl. V. cholerae 7-6-5 was the most fit to the broader spectrum of pH (6.5–8.5) and NaCl (0.5–3.0%) circumstances among the test isolates.

3.2. Genome Features of the Four V. cholerae Isolates from the Common Freshwater Fish

In this study, the genome sequences of the four V. cholerae isolates were determined using the Illumina HiSeq × 10 sequencing platform. Approximately 61,043–102,056 clean and high-quality sequencing reads were obtained, and the sequence assembly yielded 49–175 scaffolds with an average sequencing depth of 288.0–fold to 321.14–fold. The assembled genome sizes of the four V. cholerae isolates were 3.89 Mb–4.15 Mb of two chromosomes, with an average GC content of 47.35–47.63% (Table 1, Figure 3), consistent with the other V. cholerae strains. For example, the reference genome of V. cholerae N16961 was 4.03 Mb with an average 47.3% G + C content [31]. In this study, approximately 3486–3659 genes were predicted to encode proteins, but 14.4–16.8% of which were of an unknown function that accounted for the largest proportion among the 24 catalogs in the COG database (Figure S1).
Comparative genomics analysis also revealed several MGEs in the four V. cholerae genomes, including the GIs (n = 4–9), prophage gene clusters (n = 0–3), Ins (n = 1–1), and ISs (n = 0–3), suggesting the potential HGT facilitated by the MGEs.
Additionally, the distribution of sequencing depth displayed a typical Poisson distribution (Figure S2), suggesting a lower proportion of repetitive DNA in the four V. cholerae genomes. On the other hand, a few repeats were observed at the end of scaffolds (n = 3–7, <1.3 Kb), suggesting that the un-assembled gaps between scaffolds were repetitive DNA (Table S3).
The draft genomes of the V. cholerae 7-6-5, L10-48, L1-1, and B5-86 isolates were deposited in the GenBank database under the bioSample accession numbers SAMN37882014, SAMN37882015, SAMN37882016, and SAMN37882017, respectively.

3.3. Putative MGEs in the Four V. cholerae Isolates from the Common Freshwater Fish

3.3.1. GIs

The GIs carry large foreign DNA fragments (~200 Kb) that facilitate the bacterial survival in the hosts and in the environments [17]. In this study, several GIs (n = 4–9) were identified in the four V. cholerae genomes (Figure S3, Table S4), among which V. cholerae L10-48 derived from C. idellus had the maximum number of GIs (n = 9, GIs 1–9). Nevertheless, the number of GIs was less than those (n = 11) identified in the genome of V. cholerae N16961 of serotype O1.
A total of 331 genes were predicted in the 23 GIs (1302 bp–37,045 bp) identified in the four V. cholerae genomes, which endowed the bacterium with additional biological functions, e.g., resistance, and substance metabolism. For instance, antibiotic resistance-related genes were found in some GIs, e.g., GI 3 (5770 bp) in V. cholerae 7-6-5 and GI 3 (12,834 bp) in V. cholerae B5-86. Phages-related genes were also found in some GIs in the V. cholerae genomes, e.g., GI 2 (8745 bp) in V. cholerae L10-48, GI 3 (5552 bp) in V. cholerae L1-1, and GI 2 (27,273 bp) in V. cholerae B5-86.

3.3.2. Prophages

Phages dominate the aquatic ecosystems on the Earth [32]. To the best of our knowledge, the current literature on prophages in V. cholerae genomes is rare.
In this study, six prophage gene clusters (9538 bp–42,593 bp) were identified in the V. cholerae L1-1, L10-48, and B5-86 genomes (n = 1–3), whereas none were found in V. cholerae 7-6-5. A total of 248 genes were predicted in the six prophages, which encoded phage structure proteins, e.g., the phage holin family proteins; phage tail, sheath, and assembly proteins; phage baseplate assembly proteins; and phage small head proteins. Notably, approximately 53.6% of the prophage genes coded for unknown proteins (Figure S4, Table S5).
The V. cholerae L1-1 genome contained three prophage gene clusters, which showed sequence similarity to Escherichia_phage_lys12581Vzw (38,198 bp, NCBI accession number: NC_049917), Vibrio_phage_VHML (42,593 bp, NCBI accession number: NC_004456), and Vibrio_phage_VCY_phi (9538 bp, NCBI accession number: NC_016162). V. cholerae L10-48 harbored two prophage gene clusters, showing sequence similarity to Burkholderia_cenocepacia_phage_BcepMa (24,437 bp, NCBI accession number: NC_005882) and Escherichia_phage_Arg0145 (42,272 bp, NCBI accession number: NC_049918). Additionally, only one Escherichia_converting_phage_Stx2a_F451 (40,712 bp, NCBI accession number: NC_049924) homologue was found in V. cholerae B5-86. Of note, the six identified prophages in the V. cholerae genomes were derived from three different genera, including Burkholderia cenocepacia, Escherichia spp., and Vibrio spp., indicating the possible phage transmission across these genera boundaries.
Additionally, one Vibrio_phage_CTX (13,121 bp, NCBI accession number: NC_015209) was identified in the V. cholerae N16961 genome, which encoded the CT and TCP, consistent with the previous report [31].

3.3.3. Ins

Mobile Ins were widespread in environments heavily influenced by human activity, with prolonged exposure to detergents, antibiotics, and heavy metals [33]. Ins are grouped into Type I, Type II, Type III, and super integrons, based on integrase genes (intI1, intI2, intI3, and intI4) [34].
In this study, each of the four V. cholerae genomes contained one complete In (2759–13,159 bp) (Figure S5, Table S6). For example, the identified In in the V. cholerae 7-6-5 genome encoded the IntI4 (Vc 7_6_5_1252) and QnrVC family quinolone resistance pentapeptide repeat protein (Vc 7_6_5_1250), suggesting that it was a super In. Notably, a similar super In encoding the quinolone resistance protein was also found in the V. cholerae L1-1 and B5-86 genomes. The former also coded for the DUF4144 domain-containing proteins (Vc L1_1_2144), while the latter encoded the GNAT family N-acetyltransferase (Vc B5_86_2255) as well. One super In was found in the V. cholerae L10-48 genome, which encoded the IntI4, HNH endonuclease, and IS30 family transposase (Vc L10_48_2217, Vc L10_48_2215, and Vc L10_48_2214). Additionally, no Ins were identified in the V. cholerae N16961 genome.

3.3.4. ISs

ISs belonging to the IS3 and IS5 families play an important role in the evolution of antibiotic resistance and virulence in Gram-negative bacteria [35]. In this study, only a few ISs (n = 1–3, 620 bp–1551 bp) were identified in the V. cholerae L10-48, L1-1, and B5-86 genomes, whereas none were found in V. cholerae 7-6-5 (Table S7).
For instance, the V. cholerae L10-48 genome contained three ISs (IS001–IS003). The IS001 (1238 bp) coded for the ISAs1 family transposase (Vc L10_48_1267); IS002 (1045 bp) for the IS30 family transposase (Vc L10_48_2214); and IS003 (961 bp) for the IS5 family transposase (Vc L10_48_1263). However, none of the ISs carried antibiotic resistance- or virulence-related genes in the three V. cholerae genomes.

3.4. CRISPR-Cas System Arrays

The CRISPR-Cas system provides the host with an adaptive and hereditary immunity against exogenous nucleic acids [36]. In this study, the CRISPR-Cas system arrays (n = 2–10, 74 bp–4468 bp) were identified in all the four V. cholerae genomes (Figure S6).
For example, the V. cholerae L10-48 genome had the maximum number of CRISPR-Cas arrays (n = 10). Of these, the CRISPR 4 was the longest in size (2834 bp), with the maximum number of repetitive sequences (n = 47). In contrast, the V. cholerae B5-86 genome had the fewest CRISPR-Cas arrays (n = 2).
Remarkably, the genes encoding CRISPR-associated Cas proteins were identified in the V. cholerae L10-48 (n = 5), 7-6-5 (n = 3), and L1-1 (n = 1) genomes, whereas no such gene was found in V. cholerae B5-86. For instance, the V. cholerae L10-48 genome contained the casA (Vc L10_48_3096), casB (Vc L10_48_3095), casC (Vc L10_48_3093), casD (Vc L10_48_3092), and casE (Vc L10_48_3094) genes, while V. cholerae 7-6-5 contained three copies of the cas6f (Vc 7_6_5_3049, Vc 7_6_5_2545, and Vc 7_6_5_0501) genes. Only one cas6f (Vc L1_1_0413) gene was found in V. cholerae L1-1 (Table S8). The Cas is an endonuclease that can cleave foreign DNA [37]. These results suggested the potential active immunity defense systems in the V. cholerae isolates, which may have led to the fewer MGEs in the isolates. In contrast, in our recent study, we found many more MGEs present in Klebsiella oxytoca strains (n = 8) isolated from eight species of aquatic animals, particularly GIs (n = 105) and prophages (n = 24) [9]. Of note, although the CRISPR-Cas arrays were identified, the cas gene was absent from the K. oxytoca genomes, suggesting their inactive bacterial CRISPR-Cas systems [9]. Most recently, Wang et al. have successfully applied the Cas9-natural excision (NE) method to remove four representative MGEs, including plasmids, prophages, and GIs, from Vibrio strains [38], which provided the experimental evidence for the possible correlation between the Cas protein and MGEs. Interestingly, the cas gene appeared to be absent from V. cholerae N16961 as well.

3.5. Putative Virulence-Associated Genes in the Four V. cholerae Genomes

The genes encoding the CT and TCP were absent from the four V. cholerae genomes. The non-epidemic V. cholerae strains without the CT and TCP, referred to as non-O1/O139, can cause sporadic episodes of diarrhea and gastrointestinal infection [39,40]. Nevertheless, the Zonula occludens toxin gene (zot) was found in V. cholerae L1-1 and B5-86. Zot can increase the permeability of the small intestinal mucosa [41].
In addition, the genomes of V. cholerae 7-6-5, L1-1, L10-48, and B5-86 isolates contained the genes encoding the Type VI secretion system (T6SS) [42], the latter two of which also encoded the Type III secretion system (T3SS) [43]. The four V. cholerae genomes also contained the genes encoding the virulence-related flagella, mannose hemagglutinin pili, and autoinducers (e.g., autoinducer-2, AI-2; and cholerae autoinducer-1, CAI-1) required for the biofilm formation in V. cholerae. The co-existence of the other virulence-related genes was found in the V. cholerae genomes as well (Table S9). For example, the V. cholerae 7-6-5 genome encoded the cell surface-expressed elongation factor EF-Tu (Vc 7_6_5_2186, Vc 7_6_5_2235, Vc 7_6_5_3026, and Vc 7_6_5_3364) [44], and lipid mediator receptors connected lipo-oligosaccharides (LOSs) (Vc 7_6_5_0960) [45]. The LOSs of Histophilus somni may act as an adhesin and an endotoxin that signals through toll-like receptor 4 and NF-κB to cause inflammation in the host [45]. The RTX toxin genes (Vc 7_6_5_2268; Vc 7_6_5_2265; and Vc 7_6_5_2266) were also identified in V. cholerae 7-6-5, which was previously characterized by its ability to round human laryngeal epithelial (HEp-2) cells [46]. Two genes (Vc 7_6_5_2990; Vc 7_6_5_2991) involved in the V. cholerae cytolysin (VCC) were found in V. cholerae 7-6-5, which was among disparate pore-forming toxins [47].
Of note, V. cholerae B5-86 originating from P. pekinensis had the maximum number of virulence-related genes (n = 122), whereas V. cholerae L1-1 from C. idellus had the fewest ones (n = 106). These results suggested the potential risk of consuming the freshwater fish contaminated by these V. cholerae strains. In future research, the potential virulence of these V. cholerae strains should be further investigated at cell and animal mode levels.

3.6. Resistance-Associated Genes in the Four V. cholerae Genomes and Their Resistance Phenotypes

AMP is widely applied to treat foodborne bacterial infections. However, the treatment efficiency on the Vibrio infection has increasingly become ineffective, possibly due to the crp gene [48]. In this study, antibiotic resistance-related genes (n = 6–9), e.g., crp, VarG, catB9, CARB-7, QnrVC4, and almG, were identified in the four V. cholerae genomes (Table 2).
For example, the four V. cholerae genomes all carried the crp and AlmG genes. The former encoded a crp regulator in Escherichia coli by repressing the expression of the MdtEF multidrug efflux pump [48]. AlmG is a glycyltransferase responsible for the polymyxin resistance in pandemic V. cholerae [49]. V. cholerae 7-6-5 also contained the QnrVC4 and VarG genes. The former is an In-mediated quinolone resistance protein in Aeromonas punctata [50]. Lin et al. [51] reported that VarG of V. cholerae N16961 has β-lactamase activity against penicillins, cephalosporins and carbapenems. In this study, we found that V. cholerae L10-48 also contained the catB9 and CARB-7 genes responsible for the chloramphenicol and ampicillin resistance (CARB-7), respectively [52,53].
Table 2. The antimicrobial resistance-associated genes identified in the four V. cholerae genomes.
Table 2. The antimicrobial resistance-associated genes identified in the four V. cholerae genomes.
Antibiotic AgentResistance-Related GeneV. cholerae IsolateReference
Fluoroquinolonecrp7-6-5, L1-1, L10-48, B5-86[48]
QnrVC47-6-5, L1-1, B5-86[50]
ChloramphenicolcatB9L10-48[52]
CarbapenemVarG7-6-5, B5-86[51]
PolymyxinAlmG, ugd7-6-5, L1-1, L10-48, B5-86[49,54]
beta-LactamCARB-7L10-48[53]
NitroimidazolemsbA7-6-5, L1-1, L10-48, B5-86[55]
StreptograminvatF7-6-5, L1-1, L10-48, B5-86[56]
To verify the in silico predicted resistance genes, we examined the resistance phenotypes of the V. cholerae isolates experimentally, and the results are shown in Table S1. For example, V. cholerae 7-6-5 was resistant to antibiotics moxifloxacin (MFX) and rifampicin (RIF), as well as heavy metals Hg, Ni, Pb, and Zn; V. cholerae L10-48 was only resistant to AMP and STR; V. cholerae L1-1 solely tolerated Hg and Pb; and V. cholerae B5-86 was resistant to STR, Hg, Pb, and Zn (Table S1).
Based on the BacMET database, no heavy metal tolerance-related genes were identified in the V. cholerae genomes, which was not consistent with the heavy metal tolerance phenotypes of V. cholerae 7-6-5, L1-1, and B5-86. We could not rule out the possibility that certain MDR efflux pumps are involved in the pumping out of heavy metals as well [57].

3.7. Strain-Specific Genes of the Four V. cholerae Isolates from the Common Freshwater Fish

A number of strain-specific genes (n = 221–311) were identified in the four V. cholerae isolates (Figure 4). V. cholerae 7-6-5 from A. nobilis harbored the highest number of strain-specific genes (n = 311), whereas V. cholerae B5-86 from P. pekinensis had the relative fewer ones (n = 221). Additionally, 2922 core genes were found in the four V. cholerae strains, which accounted for 65.6% of the pan genes (n = 4457) that were conserved in all the analyzed genomes. In addition, the genes of plasmid origins were also found in the four V. cholerae genomes. In future research, it will be interesting to sequence the plasmids extracted from the V. cholerae strains and decipher their function and evolution.

3.8. MLST of the Four V. cholerae Isolates from the Common Freshwater Fish

In this study, the MLST analysis against the PubMLST database revealed that V. cholerae L10-48 belonged to the ST type of 884. Remarkably, V. cholerae 7-6-5, L1-1, and B5-86 strains were novel ST types, matching none of any known STs.

3.9. Phylogenetic Relatedness of the Four V. cholerae Isolates from the Common Freshwater Fish

To further investigate the phylogenetic relatedness of the four V. cholerae isolates originating in common freshwater fish, we constructed a phylogenetic tree together with the other 67 V. cholerae genomes (Figure 5). Among the 67 V. cholerae strains of serotypes O1/O139, and non O1/O139, 32 were isolated from humans, 12 from the environment, 11 from shrimp, 9 from pork, 2 from chicken, and 1 from crab samples between 1962 and 2019 in Asia, Africa, and the USA. This analysis revealed four distinct clusters, designated as Clusters A to D (Table S2).
V. cholerae 7-6-5 (SRA submission no: SAMN37882014) isolated from A. nobilis was classified into Cluster A and showed the closest evolutionary distance to V. cholerae Vc401 (GenBank accession no: GCA_0016456995.1) isolated from shrimp in 2015 in China. V. cholerae L10-48 (SRA submission no: SAMN37882015) isolated from A. nobilis was also grouped into Cluster A, but it had the closest distance to V. cholerae Vc306 (GenBank assembly accession no: GCA_016456645.1) isolated from pork in 2015 in China. Interestingly, V. cholerae B5-86 (SRA submission no: SAMN37882017) isolated from P. pekinensis was classified into Cluster D, phylogenetically closest to V. cholerae BD18 (GenBank assembly accession no: GCA_03348365.1) isolated from the environment in 2013 in Bangladesh. Additionally, the evolutionary distance between V. cholerae L1-1 (SRA submission no: SAMN37882016) isolated from C. idellus and V. cholerae BD06 (GenBank assembly accession no: GCA_003348715.1) isolated from the environment in 2013 in Bangladesh was the closest, both of which were classified into Cluster B. Notably, the majority (n = 27) of O1/O139 V. cholerae isolates from human clinical samples fell into Cluster B.
These results indicated the genome diversity of V. cholerae isolates from the common freshwater fish and the possible transmission of V. cholerae across the national geographic (e.g., Bangladesh and China) and animal species (e.g., fish, shrimp, and pork) boundaries.
The freshwater fish had been tested for the presence of V. cholerae in three surveys of our previous research, by which a number of V. cholerae strains were isolated and identified [2,13,14]. Of these, enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR)-based fingerprinting of the V. cholerae isolates (n = 400, 100 from each of four species, e.g., A. nobilis, C. idellus, and P. pekinensis) revealed 328 ERIC-genotypes, which demonstrated a large degree of genomic variation among the isolates [2]. The results in this study provided genome-level evidence of V. cholerae’s diversity in freshwater fish.

4. Conclusions

We previously reported that V. cholerae was found in freshwater fish such as A. nobilis, C. idellus, and P. pekinensis. In this study, we firstly characterized the growth and genome features of V. cholerae isolates with different resistance phenotypes from commonly consumed freshwater fish. The results revealed that the non-O1/O139 V. cholerae isolates (n = 4) were halophilic and grew optimally at 2% NaCl and pH 8.0 in the TSB medium at 37 °C. V. cholerae 7-6-5 from A. nobilis had the best fit to the broader spectrum of pH (6.5–8.5) and NaCl (0.5–3.0%) conditions among the isolates.
The draft genome sequences of the four V. cholerae isolates were 3.89 Mb–4.15 Mb with an average GC content of 47.35–47.63%. Approximately 3366–3561 genes were predicted to encode proteins, but 14.9–17.3% of them were of an unknown function. A number of strain-specific genes (n = 221–311) were found in the four V. cholerae isolates, 3 of which belonged to none of any of the known STs. Several putative MGEs existed in the V. cholerae isolates, including GIs (n = 4–9), prophages (n = 0–3), Ins (n = 1–1), and ISs (n = 0–3). Notably, CRISPR-Cas system arrays (n = 2–10) were found in the V. cholerae genomes, whereby the potential immunity defense system could be active. Comparative genomic analyses also revealed many putative virulence-associated genes (n = 106–122) and antibiotic resistance-related genes (n = 4–5), suggesting a potential risk of consuming the aquatic products contaminated by these V. cholerae isolates.
Taken together, the results of this study demonstrated the bacterial broader-spectrum growth traits and fill prior gaps of knowledge in the genomes of V. cholerae from freshwater fish. This study should be useful for the future investigation of the evolution of the waterborne pathogen worldwide.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/d16050268/s1. Table S1: The phenotypes of the four V. cholerae isolates used in this study; Table S2: The seventy-one V. cholerae strains with genome sequences used in the phylogenetic tree; Table S3: The identified repeats in the four V. cholerae genomes; Table S4: The identified GIs in the four V. cholerae genomes; Table S5: The identified prophage gene clusters in the four V. cholerae genomes; Table S6: The identified Ins in the four V. cholerae genomes; Table S7: The identified ISs in the four V. cholerae genomes; Table S8: The identified CRISPR-Cas arrays in the four V. cholerae genomes; Table S9: The potential virulence factor-encoding genes identified in the four V. cholerae genomes [58,59,60,61,62,63,64]; Figure S1: The gene function of the GIs identified in the four V. cholerae genomes. Different colors refer to COG classification to mark gene function and genes not annotated to the COG database are displayed in grey. (A–D): the V. cholerae 7-6-5, L10-48, L1-1, and B5-86 genomes, respectively; Figure S2: The k-mer analysis for the four V. cholerae subread data based on the number of unique 17-mers. (A–D): the V. cholerae 7-6-5, L10-48, L1-1, and B5-86 genomes, respectively; Figure S3: Gene organizations of the GIs identified in the four V. cholerae genomes. Different colors refer to COG classification in Figure S1; Figure S4: The structure diagram of the prophage gene clusters identified in the four V. cholerae genomes; Figure S5: The structure diagram of the Ins identified in the four V. cholerae genomes; Figure S6: The structural features of the CRISPR arrays identified in the four V. cholerae genomes. The repeat sequences are shown by the rectangle of different colors and the spacer regions are represented by rhombuses of different colors.

Author Contributions

X.Q.: investigation, data curation, and writing—original draft preparation; L.Y.: investigation; Y.X.: data analysis; L.X. and Y.W.: supervision and discussion; L.C.: funding acquisition, conceptualization, and writing—review and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by Science and Technology Commission of Shanghai Municipality, grant number 17050502200.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

Draft genome sequences of the four V. cholerae isolates were deposited in the GenBank database under the bioSample accession numbers SAMN37882014, SAMN37882015, SAMN37882016, and SAMN37882017, respectively.

Acknowledgments

The authors are grateful to Huiqiong Guan and Suo Sun at Shanghai Ocean University for their help in the data analyses.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Montero, D.A.; Vidal, R.M.; Velasco, J.; George, S.; Lucero, Y.; Gómez, L.A.; Carreño, L.J.; García-Betancourt, R.; O’Ryan, M. Vibrio cholerae, classification, pathogenesis, immune response, and trends in vaccine development. Front. Med. 2023, 10, 1155751. [Google Scholar] [CrossRef] [PubMed]
  2. Xu, M.; Wu, J.; Chen, L. Virulence, antimicrobial and heavy metal tolerance, and genetic diversity of Vibrio cholerae recovered from commonly consumed freshwater fish. Environ. Sci. Pollut. Res. Int. 2019, 26, 27338–27352. [Google Scholar] [CrossRef] [PubMed]
  3. Waldor, M.K.; Mekalanos, J.J. Lysogenic conversion by a filamentous phage encoding cholera toxin. Science 1996, 272, 1910–1914. [Google Scholar] [CrossRef] [PubMed]
  4. Almansour, A.M.; Alhadlaq, M.A.; Alzahrani, K.O.; Mukhtar, L.E.; Alharbi, A.L.; Alajel, S.M. The silent threat: Antimicrobial-resistant pathogens in food-producing animals and their impact on public health. Microorganisms 2023, 11, 2127. [Google Scholar] [CrossRef]
  5. Taviani, E.; van den Berg, H.; Nhassengo, F.; Nguluve, E.; Paulo, J.; Pedro, O.; Ferrero, G. Occurrence of waterborne pathogens and antibiotic resistance in water supply systems in a small town in Mozambique. BMC Microbiol. 2022, 22, 243. [Google Scholar] [CrossRef] [PubMed]
  6. Abioye, O.E.; Nontongana, N.; Osunla, C.A.; Okoh, A.I. Antibiotic resistance and virulence genes profiling of Vibrio cholerae and Vibrio mimicus isolates from some seafood collected at the aquatic environment and wet markets in Eastern Cape Province, South Africa. PLoS ONE 2023, 18, e0290356. [Google Scholar] [CrossRef] [PubMed]
  7. Schmidt, K.; Scholz, H.C.; Appelt, S.; Michel, J.; Jacob, D.; Dupke, S. Virulence and resistance patterns of Vibrio cholerae non-O1/non-O139 acquired in Germany and other European countries. Front. Microbiol. 2023, 14, 1282135. [Google Scholar] [CrossRef] [PubMed]
  8. Arnold, B.J.; Huang, I.T.; Hanage, W.P. Horizontal gene transfer and adaptive evolution in bacteria. Nat. Rev. Microbiol. 2022, 20, 206–218. [Google Scholar] [CrossRef] [PubMed]
  9. Sun, S.; Gu, T.; Ou, Y.; Wang, Y.; Xie, L.; Chen, L. Environmental compatibility and genome flexibility of Klebsiella oxytoca isolated from eight species of aquatic animals. Diversity 2024, 16, 30. [Google Scholar] [CrossRef]
  10. Haudiquet, M.; Sousa, J.M.; Touchon, M.; Rocha, E.P.C. Selfish, promiscuous and sometimes useful: How mobile genetic elements drive horizontal gene transfer in microbial populations. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2022, 377, 20210234. [Google Scholar] [CrossRef]
  11. Dorado, G.; Gálvez, S.; Rosales, T.E.; Vásquez, V.F.; Hernández, P. Analyzing modern biomolecules: The revolution of nucleic-acid sequencing—Review. Biomolecules 2021, 11, 1111. [Google Scholar] [CrossRef] [PubMed]
  12. Evrony, G.D.; Hinch, A.G.; Luo, C. Applications of single-cell DNA sequencing. Annu. Rev. Genom. Hum. G. 2021, 22, 171–197. [Google Scholar] [CrossRef] [PubMed]
  13. Chen, D.; Li, X.; Ni, L.; Xu, D.; Xu, Y.; Ding, Y.; Xie, L.; Chen, L. First experimental evidence for the presence of potentially toxic Vibrio cholerae in snails, and virulence, cross-resistance and genetic diversity of the bacterium in 36 species of aquatic food animals. Antibiotics 2021, 10, 412. [Google Scholar] [CrossRef] [PubMed]
  14. Fu, H.; Yu, P.; Liang, W.; Kan, B.; Peng, X.; Chen, L. Virulence, resistance, and genomic fingerprint traits of Vibrio cholerae isolated from 12 species of aquatic products in Shanghai, China. Microb. Drug. Resist. 2020, 26, 1526–1539. [Google Scholar] [CrossRef] [PubMed]
  15. Song, Y.; Yu, P.; Li, B.; Pan, Y.; Zhang, X.; Cong, J.; Zhao, Y.; Wang, H.; Chen, L. The mosaic accessory gene structures of the SXT/R391-like integrative and conjugative elements derived from Vibrio spp. isolated from aquatic products and environment in the Yangtze River Estuary, China. BMC Microbiol. 2013, 13, 214. [Google Scholar] [CrossRef]
  16. Miller, C.J.; Drasar, B.S.; Feachem, R.G. Response of toxigenic Vibrio cholerae 01 to physico-chemical stresses in aquatic environments. J. Hygiene. 1984, 93, 475–495. [Google Scholar] [CrossRef] [PubMed]
  17. Guan, H.; Xie, L.; Chen, L. Survival and genome evolution signatures of Klebsiella pneumoniae isolates originated in seven species of aquatic animals. Diversity 2023, 15, 527. [Google Scholar] [CrossRef]
  18. Xu, D.; Peng, X.; Xie, L.; Chen, L. Survival and genome diversity of Vibrio parahaemolyticus isolated from edible aquatic animals. Diversity 2022, 14, 350. [Google Scholar] [CrossRef]
  19. Yu, P.; Yang, L.; Wang, J.; Su, C.; Qin, S.; Zeng, C.; Chen, L. Genomic and transcriptomic analysis reveal multiple strategies for the cadmium tolerance in Vibrio parahaemolyticus N10-18 isolated from aquatic animal Ostrea Gigas Thunberg. Foods 2022, 11, 3777. [Google Scholar] [CrossRef] [PubMed]
  20. Luo, R.; Liu, B.; Xie, Y.; Li, Z.; Huang, W.; Yuan, J.; He, G.; Chen, Y.; Pan, Q.; Liu, Y.; et al. SOAPdenovo2: An empirically improved memory-efficient short-read de novo assembler. GigaScience 2012, 1, 18. [Google Scholar] [CrossRef]
  21. Delcher, A.L.; Bratke, K.A.; Powers, E.C.; Salzberg, S.L. Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics 2007, 23, 673–679. [Google Scholar] [CrossRef] [PubMed]
  22. Chan, P.P.; Lowe, T.M. tRNAscan-SE: Searching for tRNA genes in genomic sequences. Methods Mol. Biol. 2019, 1962, 1–14. [Google Scholar] [CrossRef]
  23. Pal, C.; Bengtsson-Palme, J.; Rensing, C.; Kristiansson, E.; Larsson, D.G.J. BacMet: Antibacterial biocide and metal resistance genes database. Nucleic. Acids. Res. 2014, 42, D737–D743. [Google Scholar] [CrossRef] [PubMed]
  24. Bertelli, C.; Fiona, S.L. Improved genomic island predictions with IslandPath-DIMOB. Bioinformatics 2018, 34, 2161–2167. [Google Scholar] [CrossRef]
  25. Fouts, D.E. Phage_Finder: Automated identification and classification of prophage regions in complete bacterial genome sequences. Nucleic. Acids. Res. 2006, 34, 5839–5851. [Google Scholar] [CrossRef]
  26. Cury, J.; Jové, T.; Touchon, M.; Néron, B.; Rocha, E.P. Identification and analysis of integrons and cassette arrays in bacterial genomes. Nucleic. Acids. Res. 2016, 44, 4539–4550. [Google Scholar] [CrossRef]
  27. Siguier, P.; Perochon, J.; Lestrade, L.; Mahillon, J.; Chandler, M. ISfinder: The reference centre for bacterial insertion sequences. Nucleic. Acids. Res. 2006, 34, D32–D36. [Google Scholar] [CrossRef]
  28. Bland, C.; Ramsey, T.L.; Sabree, F.; Lowe, M.; Brown, K.; Kyrpides, N.C.; Hugenholtz, P. CRISPR recognition tool (CRT): A tool for automatic detection of clustered regularly interspaced palindromic repeats. BMC Bioinform. 2007, 8, 209. [Google Scholar] [CrossRef] [PubMed]
  29. Li, L.; Stoeckert, C.J., Jr.; Roos, D.S. OrthoMCL: Identification of ortholog groups for eukaryotic genomes. Genome. Res. 2003, 13, 2178–2189. [Google Scholar] [CrossRef]
  30. Jolley, K.A.; Bray, J.E.; Maiden, M.C.J. Open-access bacterial population genomics: BIGSdb software, the PubMLST.org website and their applications. Wellcome. Open. Res. 2018, 3, 124. [Google Scholar] [CrossRef]
  31. Heidelberg, J.F.; Eisen, J.A.; Nelson, W.C.; Clayton, R.A.; Gwinn, M.L.; Dodson, R.J.; Haft, D.H.; Hickey, E.K.; Peterson, J.D.; Umayam, L.; et al. DNA sequence of both chromosomes of the cholera pathogen Vibrio cholerae. Nature 2000, 406, 477–483. [Google Scholar] [CrossRef] [PubMed]
  32. McKerral, J.C.; Papudeshi, B.; Inglis, L.K.; Roach, M.J.; Decewicz, P.; McNair, K.; Luque, A.; Dinsdale, E.A.; Edwards, R.A. The promise and pitfalls of prophages. bioRxiv 2023. bioRxiv: 537752. [Google Scholar] [CrossRef] [PubMed]
  33. An, X.L.; Chen, Q.L.; Zhu, D.; Zhu, Y.G.; Gillings, M.R.; Su, J.Q. Impact of wastewater treatment on the prevalence of integrons and the genetic diversity of integron gene gassettes. Appl. Environ. Microb. 2018, 84, e02766-17. [Google Scholar] [CrossRef] [PubMed]
  34. Sabbagh, P.; Rajabnia, M.; Maali, A.; Ferdosi-Shahandashti, E. Integron and its role in antimicrobial resistance: A literature review on some bacterial pathogens. Iran. J. Basic. Med. Sci. 2021, 24, 136–142. [Google Scholar] [CrossRef] [PubMed]
  35. Gonçalves, O.S.; Campos, K.F.; Assis, J.C.S.; Fernandes, A.S.; Souza, T.S.; Rodrigues, L.G.C.; Queiroz, M.V.; Santana, M.F. Transposable elements contribute to the genome plasticity of Ralstonia solanacearum species complex. Microb. Genom. 2020, 6, e000374. [Google Scholar] [CrossRef]
  36. Zakrzewska, M.; Burmistrz, M. Mechanisms regulating the CRISPR-Cas systems. Front. Microbiol. 2023, 14, 1060337. [Google Scholar] [CrossRef] [PubMed]
  37. Deveau, H.; Garneau, J.E.; Moineau, S. CRISPR/Cas system and its role in phage-bacteria interactions. Annu. Rev. Microbiol. 2010, 64, 475–493. [Google Scholar] [CrossRef] [PubMed]
  38. Wang, P.; Du, X.; Zhao, Y.; Wang, W.; Cai, T.; Tang, K.; Wang, X. Combining CRISPR/Cas9 and natural excision for the precise and complete removal of mobile genetic elements in bacteria. Appl. Environ. Microbiol. 2024, 90, e0009524. [Google Scholar] [CrossRef]
  39. Austin, B. Vibrios as causal agents of zoonoses. Vet. Microbiol. 2010, 140, 310–317. [Google Scholar] [CrossRef]
  40. Ceccarelli, D.; Chen, A.; Hasan, N.A.; Rashed, S.M.; Huq, A.; Colwell, R.R. Non-O1/non-O139 Vibrio cholerae carrying multiple virulence factors and V. cholerae O1 in the Chesapeake Bay, Maryland. Appl. Environ. Microbiol. 2015, 81, 1909–1918. [Google Scholar] [CrossRef]
  41. Lee, A.; White, N.; Walle, C.F. The intestinal zonula occludens toxin (ZOT) receptor recognises non-native ZOT conformers and localises to the intercellular contacts. FEBS Lett. 2003, 555, 638–642. [Google Scholar] [CrossRef] [PubMed]
  42. Dong, T.G.; Ho, B.T.; Yoder-Himes, D.R.; Mekalanos, J.J. Identification of T6SS-dependent effector and immunity proteins by Tn-seq in Vibrio cholerae. Proc. Natl. Acad. Sci. USA 2013, 110, 2623–2628. [Google Scholar] [CrossRef] [PubMed]
  43. Kodama, T.; Rokuda, M.; Park, K.S.; Cantarelli, V.V.; Matsuda, S.; Iida, T.; Honda, T. Identification and characterization of VopT, a novel ADP-ribosyltransferase effector protein secreted via the Vibrio parahaemolyticus type III secretion system 2. Cell. Microbiol. 2007, 9, 2598–2609. [Google Scholar] [CrossRef] [PubMed]
  44. Sun, Y.; Wang, X.; Li, J.; Xue, F.; Tang, F.; Dai, J. Extraintestinal pathogenic Escherichia coli utilizes the surface-expressed elongation factor Tu to bind and acquire iron from holo-transferrin. Virulence 2022, 13, 698–713. [Google Scholar] [CrossRef] [PubMed]
  45. Inzana, T.J. The many facets of lipooligosaccharide as a virulence factor for Histophilus somni. Curr. Top. Microbiol. Immunol. 2016, 396, 131–148. [Google Scholar] [PubMed]
  46. Sheahan, K.L.; Cordero, C.L.; Satchell, K.J. Identification of a domain within the multifunctional Vibrio cholerae RTX toxin that covalently cross-links actin. Proc. Natl. Acad. Sci. USA 2004, 101, 9798–9803. [Google Scholar] [CrossRef] [PubMed]
  47. De, S.; Olson, R. Crystal structure of the Vibrio cholerae cytolysin heptamer reveals common features among disparate pore-forming toxins. Proc. Natl. Acad. Sci. USA 2011, 108, 7385–7390. [Google Scholar] [CrossRef]
  48. Nishino, K.; Senda, Y.; Yamaguchi, A. CRP regulator modulates multidrug resistance of Escherichia coli by repressing the mdtEF multidrug efflux genes. J. Antibiot. 2008, 61, 120–127. [Google Scholar] [CrossRef] [PubMed]
  49. Henderson, J.C.; Herrera, C.M.; Trent, M.S. AlmG, responsible for polymyxin resistance in pandemic Vibrio cholerae, is a glycyltransferase distantly related to lipid A late acyltransferases. J. Biol. Chem. 2017, 292, 21205–21215. [Google Scholar] [CrossRef]
  50. Tacão, M.; Moura, A.; Correia, A.; Henriques, I. Co-resistance to different classes of antibiotics among ESBL-producers from aquatic systems. Water Res. 2014, 48, 100–107. [Google Scholar] [CrossRef]
  51. Lin, H.V.; Massam-Wu, T.; Lin, C.P.; Wang, Y.A.; Shen, Y.; Lu, W.; Hsu, P.; Chen, Y.; Borges-Walmsley, M.I.; Walmsley, A.R. The Vibrio cholerae var regulon encodes a metallo-β-lactamase and an antibiotic efflux pump, which are regulated by VarR, a LysR-type transcription factor. PLoS ONE 2017, 12, e0184255. [Google Scholar] [CrossRef] [PubMed]
  52. Lepuschitz, S.; Baron, S.; Larvor, E.; Granier, S.A.; Pretzer, C.; Mach, R.L.; Farnleitner, A.H.; Ruppitsch, W.; Pleininger, S.; Indra, A.; et al. Phenotypic and genotypic antimicrobial resistance traits of Vibrio cholerae non-O1/non-O139 isolated from a large Austrian lake frequently associated with cases of human infection. Front. Microbiol. 2019, 10, 2600. [Google Scholar] [CrossRef]
  53. Melano, R.; Petroni, A.; Garutti, A.; Saka, H.A.; Mange, L.; Pasterán, F.; Rapoport, M.; Rossi, A.; Galas, M. New carbenicillin-hydrolyzing β-lactamase (CARB-7) from Vibrio cholerae non-O1, non-O139 strains encoded by the VCR region of the V. cholerae genome. Antimicrob. Agents Chemother. 2002, 46, 2162–2168. [Google Scholar] [CrossRef] [PubMed]
  54. Lacour, S.; Bechet, E.; Cozzone, A.J.; Mijakovic, I.; Grangeasse, C. Tyrosine phosphorylation of the UDP-glucose dehydrogenase of Escherichia coli is at the crossroads of colanic acid synthesis and polymyxin resistance. PLoS ONE 2008, 3, e3053. [Google Scholar] [CrossRef] [PubMed]
  55. Bonifer, C.; Glaubitz, C. MsbA: An ABC transporter paradigm. Biochem. Soc. Trans. 2021, 49, 2917–2927. [Google Scholar] [CrossRef] [PubMed]
  56. Siddi, G.; Piras, F.; Meloni, M.P.; Gymoese, P.; Torpdahl, M.; Fredriksson-Ahomaa, M.; Migoni, M.; Cabras, D.; Cuccu, M.; De Santis, E.P.L.; et al. Hunted wild boars in sardinia: Prevalence, antimicrobial resistance and genomic analysis of Salmonella and Yersinia enterocolitica. Foods 2023, 13, 65. [Google Scholar] [CrossRef] [PubMed]
  57. Nguyen, T.H.T.; Nguyen, H.D.; Le, M.H.; Nguyen, T.T.H.; Nguyen, T.D.; Nguyen, D.L.; Nguyen, Q.H.; Nguyen, T.K.O.; Michalet, S.; Dijoux-Franca, M.; et al. Efflux pump inhibitors in controlling antibiotic resistance: Outlook under a heavy metal contamination context. Molecules 2023, 28, 2912. [Google Scholar] [CrossRef] [PubMed]
  58. Syed, K.A.; Beyhan, S.; Correa, N.; Queen, J.; Liu, J.; Peng, F.; Satchell, K.J.F. Yildiz, F.; Klose, K.E. The Vibrio cholerae flagellar regulatory hierarchy controls expression of virulence factors. J. Bacteriol. 2009, 191, 6555–6570. [Google Scholar] [CrossRef] [PubMed]
  59. Utada, A.S.; Bennett, R.R.; Fong, J.C.N.; Gibiansky, M.L.; Yildiz, F.H.; Golestanian, R.; Wong, G.C.L. Vibrio cholerae use pili and flagella synergistically to effect motility switching and conditional surface attachment. Nat. Commun. 2014, 5, 4913. [Google Scholar] [CrossRef]
  60. Chen, X.; Schauder, S.; Potier, N.; Dorsselaer, A.V.; Pelczer, I.; Bassler, B.L.; Hughson, F.M. Structural identification of a bacterial quorum-sensing signal containing boron. Nature. 2002, 415, 545–549. [Google Scholar] [CrossRef]
  61. Chen, L.; Yang, J.; Yu, J.; Yao, Z.; Sun, L.; Shen, Y.; Jin, Q. VFDB: A reference database for bacterial virulence factors. Nucleic. Acids. Res. 2005, 33, D325–D328. [Google Scholar] [CrossRef] [PubMed]
  62. Lee, N.Y.; Lee, H.Y.; Lee, K.H.; Han, S.H.; Park, S.J. Vibrio vulnificus IlpA induces MAPK-mediated cytokine production via TLR1/2 activation in THP-1 cells, a human monocytic cell line. Mol. Immunol. 2011, 49, 143–154. [Google Scholar] [CrossRef] [PubMed]
  63. Fullner, K.J.; Mekalanos, J.J. In vivo covalent cross-linking of cellular actin by the Vibrio cholerae RTX toxin. EMBO J. 2000, 19, 5315–5323. [Google Scholar] [CrossRef]
  64. Higgins, D.A.; Pomianek, M.E.; Kraml, C.M.; Taylor, R.K.; Semmelhack, M.F.; Bassler, B.L. The major Vibrio cholerae autoinducer and its role in virulence factor production. Nature 2007, 450, 883–886. [Google Scholar] [CrossRef] [PubMed]
Diversity 16 00268 g001
Figure 1. Growth profiles of the four V. cholerae isolates under different pH conditions. The isolates were incubated in the TSB (3% NaCl) at 37 °C. (A–D): V. cholerae 7-6-5, L10-48, L1-1, and B5-86 isolates, respectively.
Figure 1. Growth profiles of the four V. cholerae isolates under different pH conditions. The isolates were incubated in the TSB (3% NaCl) at 37 °C. (A–D): V. cholerae 7-6-5, L10-48, L1-1, and B5-86 isolates, respectively.
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Figure 2. Growth profiles of the four V. cholerae isolates under different concentrations of NaCl. The isolates were incubated in the TSB (pH 8.0) at 37 °C. (A–D): V. cholerae 7-6-5, L10-48, L1-1, and B5-86 isolates, respectively.
Figure 2. Growth profiles of the four V. cholerae isolates under different concentrations of NaCl. The isolates were incubated in the TSB (pH 8.0) at 37 °C. (A–D): V. cholerae 7-6-5, L10-48, L1-1, and B5-86 isolates, respectively.
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Figure 3. Genome circle maps of the four V. cholerae isolates from common freshwater fish. (A,B): represent the larger and smaller chromosomes, respectively. Circles from the inwards to outside: the GC content, GC-skew, the reference genome of V. cholerae N16961, and V. cholerae 7-6-5, L10-48, L1-1, and B5-86 genomes, respectively. The eighth and ninth circles (in red) represent CDSs on the positive and negative chains (inward and outward parts), respectively.
Figure 3. Genome circle maps of the four V. cholerae isolates from common freshwater fish. (A,B): represent the larger and smaller chromosomes, respectively. Circles from the inwards to outside: the GC content, GC-skew, the reference genome of V. cholerae N16961, and V. cholerae 7-6-5, L10-48, L1-1, and B5-86 genomes, respectively. The eighth and ninth circles (in red) represent CDSs on the positive and negative chains (inward and outward parts), respectively.
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Figure 4. The Venn diagram shows the pan genes of the four V. cholerae strains from the freshwater fish. The central region represents the number of core genes, and each petal displays the number of specific genes for each strain.
Figure 4. The Venn diagram shows the pan genes of the four V. cholerae strains from the freshwater fish. The central region represents the number of core genes, and each petal displays the number of specific genes for each strain.
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Figure 5. The phylogenetic tree showing the relationship of the 71 V. cholerae genomes. The genome sequences of the four V. cholerae isolates determined in this study were marked with red dots. The second to the sixth circles represent the host, collected date, serotypes, STs, and location, respectively.
Figure 5. The phylogenetic tree showing the relationship of the 71 V. cholerae genomes. The genome sequences of the four V. cholerae isolates determined in this study were marked with red dots. The second to the sixth circles represent the host, collected date, serotypes, STs, and location, respectively.
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Table 1. Genome features of the four V. cholerae isolates from common freshwater fish.
Table 1. Genome features of the four V. cholerae isolates from common freshwater fish.
Genome FeatureV. cholerae Isolate
7-6-5L10-48L1-1B5-86
Genome size (bp)3,886,2084,145,5664,032,2203,942,938
G + C (%)47.4447.3547.5847.63
DNA Scaffold175807949
Predicted gene3500366335703487
Protein coding gene3404 356134763366
RNA gene9610294121
Genes assigned to COG2981304630102985
Genes with unknown function519617560502
GIs4964
Prophage gene cluster0231
Ins1111
ISs0312
CRISPR-Cas array81062
BioSample accession no.SAMN37882014SAMN37882015SAMN37882016SAMN37882017
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Qin, X.; Yang, L.; Xu, Y.; Xie, L.; Wang, Y.; Chen, L. Growth and Genome Features of Non-O1/O139 Vibrio cholerae Isolated from Three Species of Common Freshwater Fish. Diversity 2024, 16, 268. https://doi.org/10.3390/d16050268

AMA Style

Qin X, Yang L, Xu Y, Xie L, Wang Y, Chen L. Growth and Genome Features of Non-O1/O139 Vibrio cholerae Isolated from Three Species of Common Freshwater Fish. Diversity. 2024; 16(5):268. https://doi.org/10.3390/d16050268

Chicago/Turabian Style

Qin, Xinchi, Lianzhi Yang, Yingwei Xu, Lu Xie, Yongjie Wang, and Lanming Chen. 2024. "Growth and Genome Features of Non-O1/O139 Vibrio cholerae Isolated from Three Species of Common Freshwater Fish" Diversity 16, no. 5: 268. https://doi.org/10.3390/d16050268

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