Research Progress on Non-O1 and non-O139 Vibrio cholerae in Aquatic Animals and Its Public Health Significance

1. Introduction

Vibrio cholerae is a gram-negative, curved bacterium widely distributed in aquatic environments. Among its serotypes, O1 and O139 are recognized as the primary pathogens responsible for cholera outbreaks.^1,2 However, in recent years, non-O1/non-O139 V. cholerae strains have gained increasing importance in aquaculture, attracting widespread attention from researchers.^3–5 Although non-O1/non-O139 V. cholerae strains do not cause typical cholera, they have been confirmed to cause human diseases such as diarrhea, wound infections, and septicemia.^6–9 In aquaculture, these strains are considered significant pathogens, capable of causing diseases in various aquatic animals and resulting in substantial economic losses.^10–12 Furthermore, non-O1/non-O139 V. cholerae may serve as a reservoir of virulence genes in the environment, potentially influencing the evolution of O1 and O139 V. cholerae through horizontal gene transfer.¹³

Aquatic animals play a crucial role in the ecology and epidemiology of non-O1/non-O139 V. cholerae as important hosts and vectors.¹⁴ With the rapid global development of aquaculture, the interactions between aquatic animals and non-O1/non-O139 V. cholerae have become increasingly complex. This not only affects the sustainable development of aquaculture but may also pose potential threats to public health safety. In recent years, significant progress has been made in the study of non-O1/non-O139 V. cholerae, thanks to advances in molecular biology techniques and omics methods. However, many mysteries remain regarding the distribution, pathogenic mechanisms, antimicrobial resistance, and ecological characteristics of these strains in aquatic animals.

This article aims to review the latest advances in research on non-O1/non-O139 V. cholerae in aquatic animals, including their classification and identification, epidemiological characteristics, virulence factors, impact on aquatic animals, antimicrobial resistance, prevention and control measures, and public health significance. By systematically examining existing research findings, we hope to provide a reference for a deeper understanding of the biological properties of non-O1/non-O139 V. cholerae and offer scientific basis for disease prevention and control in aquaculture, as well as food safety management.

2. Classification and Identification of non-O1/non-O139 V. cholerae

The classification and identification of non-O1/non-O139 V. cholerae form the foundation for understanding their biological properties and epidemiological characteristics. As research deepens and technology advances, this field continues to evolve, providing new insights into the classification system of V. cholerae.

2.1. Serological Classification

Traditionally, V. cholerae classification has been primarily based on the serological characteristics of the O antigen.¹⁵ Currently, over 200 O serogroups of V. cholerae are known, with non-O1/non-O139 types comprising the vast majority.¹⁶ Serological typing methods are simple to perform and relatively low-cost, remaining widely used for initial screening of clinical and environmental samples. However, this method has limitations such as cross-reactivity and subjective judgment, making it challenging to meet the needs for precise classification.¹⁷

2.2. Molecular Biological Identification Methods

The application of molecular biology techniques has greatly improved the accuracy and efficiency of non-O1/non-O139 V. cholerae identification. Multiplex PCR techniques are widely used to detect specific genes, such as the rfb gene cluster (encoding O antigen) and the toxR gene (species-specific regulatory gene).^1,18 16S rRNA gene sequencing is another commonly used molecular identification method that can provide accurate identification at the genus level.¹⁹ Additionally, Multi-Locus Sequence Typing (MLST) technology, which analyzes sequence variations in multiple housekeeping genes,²⁰ has provided a powerful tool for typing and evolutionary studies of V. cholerae.^13,21,22

2.3. Emerging Classification and Identification Technologies

In recent years, emerging technologies have brought new opportunities for the classification and identification of non-O1/non-O139 V. cholerae. The application of Whole Genome Sequencing (WGS) technology has enabled researchers to comprehensively analyze the genomic characteristics of strains, not only allowing for precise classification but also revealing evolutionary relationships between strains.^23–25 Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) technology is gradually becoming an important tool for bacterial identification due to its rapid, accurate, and economical features.^26,27 Furthermore, real-time genomic analysis technology based on nanopore sequencing has shown potential in rapid identification and typing.²⁸

Although these new technologies have brought significant advances in the classification and identification of non-O1/non-O139 V. cholerae, some challenges remain in practical applications. For example, issues such as standardization and sharing of data between different laboratories, identification in complex environmental samples, and classification of new variant strains still require further research.

In conclusion, the classification and identification methods for non-O1/non-O139 V. cholerae are transitioning from traditional phenotypic analysis to genome-based precise typing. This trend not only improves the accuracy of classification but also provides new perspectives for in-depth understanding of the evolution and adaptability of non-O1/non-O139 V. cholerae. In the future, with continuous technological advancements and database improvements, we hope to establish a more comprehensive and precise classification system for non-O1/non-O139 V. cholerae, providing more reliable scientific basis for disease prevention and control in aquaculture and public health safety.

3. Epidemiology of non-O1/non-O139 V. cholerae in Aquatic Animals

Epidemiological studies of non-O1/non-O139 V. cholerae in aquatic animals are crucial for understanding their ecological distribution, transmission dynamics, and potential public health risks. In recent years, with advances in monitoring techniques and expanded research scope, we have gained a deeper understanding of the epidemiological characteristics of these pathogens in aquaculture environments.

3.1. Geographical Distribution

Non-O1/non-O139 V. cholerae are widely distributed in aquaculture environments globally.^29,30 Studies have shown that these strains can be isolated from freshwater, saltwater, and brackish water aquaculture systems across all continents.^29,31 In China, India, and Southeast Asian countries, the detection rates of non-O1/non-O139 V. cholerae are relatively high due to intensive aquaculture activities.^{12,30,32–34} Coastal waters and farms in Europe and the Americas have also reported the presence of these strains.^5,35–37 Notably, strains from different geographical regions may exhibit genetic diversity, reflecting the results of local adaptive evolution .³⁸

3.2. Host Range

Non-O1/non-O139 V. cholerae demonstrates a wide range of aquatic animal hosts, spanning multiple ecological niches. These hosts include economically important fish species such as carp (Cyprinus carpio),^39,40 Zebrafish (Danio rerio),⁴¹ tilapia (Oreochromis niloticus),⁴² bluegill sunfish (Lepomis macrochirus)⁴³ and pink-tailed chalceus (Chalceus macrolepidotus)⁴⁴; crustaceans like giant freshwater prawn (Macrobrachium rosenbergii)^10,45 and red swamp crayfish (Procambarus clarkia)⁴⁶; and mollusks such as oysters (Crassostrea gigas)^47,48 and mussels (Mytilus edulis).⁴⁹ Furthermore, amphibians like the American bullfrog (Rana catesbeiana),⁴³ aquatic mammals such as dolphins (Tursiops truncatus)⁵⁰ have also been identified as hosts. This diverse host range not only reflects the ecological adaptability of non-O1/non-O139 V. cholerae but also highlights its potential significance in aquaculture and aquatic ecosystem health. These findings provide a foundation for further research into the ecology, evolution, and potential health impacts of these strains.

3.3. Seasonal Variations

The prevalence of non-O1/non-O139 V. cholerae in the environment exhibits distinct seasonal patterns, influenced by various environmental factors and displaying differentiated characteristics across geographical regions. In temperate areas, such as Southern California and Mediterranean coasts, the highest detection rates are typically observed during summer and early autumn, primarily associated with rising water temperatures.^51,52 Water temperature is considered one of the key factors affecting the survival and growth of V. cholerae.^53,54 In contrast, seasonal variations in tropical and subtropical regions, like Bangladesh, are mainly influenced by alternating rainy and dry seasons, with increased detection rates during the rainy season possibly linked to elevated nutrient levels and altered hydrological conditions due to rainfall.^55,56 Besides temperature and precipitation, factors such as salinity, pH, and plankton abundance may also influence the seasonal distribution of V. cholerae.⁵⁷ This seasonal pattern not only reflects the adaptability of non-O1/non-O139 V. cholerae to environmental conditions but may also be related to its ability to enter a viable but non-culturable (VBNC) state and subsequent reactivation.^1,2 Understanding these seasonal patterns is crucial for developing targeted monitoring and prevention strategies, with intensified surveillance and preventive measures during high-risk seasons potentially helping to mitigate public health risks. Overall, the seasonal pattern of non-O1/non-O139 V. cholerae is an essential component of its ecological characteristics, reflecting the complex interactions between these bacteria and their environment. This provides important clues for understanding and predicting the environmental dynamics of V. cholerae, while also offering valuable guidance for public health management.

3.4. Influence of Environmental Factors

Multiple studies have shown that environmental factors significantly influence the distribution and abundance of non-O1/non-O139 V. cholerae. Water temperature is one of the most critical factors, with optimal growth occurring between 30°C and 37°C, although V. cholerae can grow at temperatures ranging from 10°C to 43°C.^53,56 A significant increase in V. cholerae abundance has been observed when water temperatures exceed 17°C.⁵⁸ Salinity is also an important parameter, with V. cholerae tolerating a wide range from 0 to 45 parts per thousand (ppt), but optimal growth occurs at salinities between 2 and 14 ppt.^59,60 Additionally, factors such as pH, dissolved oxygen, and organic matter content affect the survival and reproduction of these strains. V. cholerae can survive in pH ranges from 5.0 to 9.6, with optimal growth at pH 7.6 to 8.6.^61,62 As a facultative anaerobe, V. cholerae grows best in aerobic conditions, with oxygen concentrations above 0.5 mg/L supporting growth.^60,63 High levels of organic matter, particularly chitin, promote V. cholerae growth, with concentrations of dissolved organic carbon above 3 mg/L associated with increased abundance.^53,64

It’s noteworthy that global climate change may alter these environmental factors, thereby impacting the prevalence dynamics of non-O1/non-O139 V. cholerae.⁶⁵ The complex interactions between these factors create specific ecological niches favorable for V. cholerae growth. For instance, Huq et al.⁵⁶ found that the combination of water temperatures above 25°C, pH between 7 and 8.5, and salinity between 0.2 and 2.0 ppt was particularly conducive to V. cholerae growth in aquatic environments.

In recent years, the application of new technologies such as metagenomics and ecological network analysis has enabled a comprehensive understanding of the position and role of non-O1/non-O139 V. cholerae in aquaculture ecosystems.⁶⁶ These studies not only reveal the complex relationships between strains and environmental factors but also provide new perspectives for understanding their interactions with other microbial communities.⁶⁷ For instance, Nandi et al.⁶⁸ found that certain non-O1/non-O139 V. cholerae strains may play important ecological roles in aquatic environments.

Overall, the epidemiological characteristics of non-O1/non-O139 V. cholerae in aquatic animals present complex spatiotemporal dynamics.⁶⁹ Future research needs to further integrate multidisciplinary approaches from molecular epidemiology, ecology, and climate science to better predict and control the spread of these potential pathogens.⁵⁸ Simultaneously, establishing long-term, systematic monitoring networks is crucial for assessing the potential risks of non-O1/non-O139 V. cholerae to the aquaculture industry and public health.⁷⁰ Recent studies have also highlighted the potential role of non-O1/non-O139 V. cholerae in the spread of antibiotic resistance, further emphasizing the importance of continuous monitoring.⁷¹

4. Impact of Non-O1/non-O139 V. cholerae on Aquatic Animals

4.1. Pathogenesis Mechanism

The pathogenesis mechanism of non-O1/non-O139 V. cholerae in aquatic animals is a complex multi-stage process involving the synergistic action of multiple virulence factors. This process begins with the adhesion and colonization of bacteria to host surface structures through MSHA pili, TCP (toxin co-regulated pilus), and other adhesion factors such as OmpU, which is a crucial step in establishing infection.^72,73 Subsequently, bacteria secrete enzymes such as hemolysin (HlyA), extracellular protease (PrtV), and chitinase to break down host tissue barriers, promoting further invasion and spread.⁷⁴ HlyA has been shown to form pores in erythrocytes and other cell types, contributing to tissue damage and inflammation.⁷⁵ Various toxins directly act on host cells, leading to cellular dysfunction and tissue damage. These include the heat-stable enterotoxin (Stn/Sto), Cholix toxin, and the multifunctional autoprocessing repeats-in-toxin (MARTX) toxin.^76,77 The MARTX toxin, for example, has been found to disrupt the actin cytoskeleton of host cells, facilitating bacterial invasion.⁷⁸

Some strains may also evade immune clearance by altering surface antigens or interfering with host immune responses. The O-antigen capsule and the VPS exopolysaccharide have been implicated in immune evasion and biofilm formation, respectively, enhancing bacterial survival in the host.^79,80 Additionally, the quorum sensing system, regulated by the CAI-1 and AI-2 autoinducers, allows bacteria to coordinate group behavior, expressing virulence genes at appropriate times to enhance pathogenicity.⁸¹ This system has been shown to control the expression of virulence factors such as proteases and the cholera toxin gene in a density-dependent manner.⁸² Recent studies have also highlighted the role of type VI secretion systems (T6SS) in bacterial competition and virulence. T6SS has been found to deliver toxic effectors to both prokaryotic and eukaryotic cells, potentially contributing to the pathogenesis of non-O1/non-O139 V. cholerae in aquatic hosts.⁸³

Understanding these complex pathogenesis mechanisms is crucial for developing effective prevention and treatment strategies for non-O1/non-O139 V. cholerae infections in aquaculture and for assessing their potential impact on public health. The multifaceted nature of non-O1/non-O139 V. cholerae pathogenesis, involving adhesion, tissue invasion, toxin production, immune evasion, and coordinated virulence expression, underscores the adaptability and potential threat of these strains in aquatic environments. Future research should focus on elucidating the specific roles of these virulence factors in different aquatic hosts and environmental conditions, as well as exploring potential targets for intervention strategies to mitigate the impact of non-O1/non-O139 V. cholerae in aquaculture settings.

4.2. Common Symptoms and Pathological Changes

Non-O1/non-O139 V. cholerae infections in aquatic animals manifest with diverse symptoms and pathological changes, varying according to host species, infection site, and severity. Fish, crustaceans, and mollusks, as the main groups of aquatic animals, present different clinical manifestations and pathological features upon infection (Table 1). This diversity reflects the variation in host physiological structures and immune responses, and is closely related to environmental factors and pathogen strain variability.^58,69

In terms of symptom development speed and external manifestations, fish and shrimp typically present more acute and obvious symptoms, such as skin ulcers, fin rot, and abnormal movement, usually appearing within 2-7 days post-infection.^84,85 In contrast, mollusks and some crab species tend to exhibit a more chronic disease course, with less obvious external symptoms like weakened shell closure response and mantle retraction, which may become apparent only weeks after infection.^87,88 This difference partly stems from the varying complexity and efficiency of immune systems among species, with fish typically showing a more pronounced inflammatory response, while mollusks demonstrate a relatively weaker immune reaction.⁸⁹

Internal pathological changes and histological features also vary among species. Fish commonly exhibit enlargement and lesions in the liver, kidney, and spleen; crustaceans may show hepatopancreas enlargement and muscle liquefaction; while mollusks often present with digestive gland atrophy and soft tissue liquefaction.^37,86,90 These differences are closely related to the unique physiological structures of each species, for example, the hepatopancreas in crustaceans and the digestive gland in mollusks are often primary target organs. Histological examination typically reveals cell degeneration and necrosis in corresponding organs, as well as varying degrees of inflammatory response and immune cell infiltration. Understanding these species-specific responses is crucial for developing effective diagnostic and control strategies, and provides important insights for assessing the potential impact of non-O1/non-O139 V. cholerae on aquaculture and public health.⁹¹

It should be noted that the severity of symptoms and pathological changes may vary depending on environmental conditions, host health status, and the virulence of the pathogen strain. Moreover, some symptoms may overlap among different species, necessitating comprehensive consideration of multiple factors in actual diagnosis. Future research should further explore the molecular mechanisms underlying these differences to develop more precise and species-specific intervention measures.

5. Overview of Virulence Factors in non-O1/non-O139 V. cholerae

Although non-O1/non-O139 V. cholerae do not produce the classical cholera toxin, they possess various virulence factors that enable them to cause disease in aquatic animals and potentially pose a threat to human health. This section provides an overview of the virulence factors of non-O1/non-O139 V. cholerae from two aspects: virulence genes and their mechanisms of action and pathogenesis, and the diversity and adaptability of virulence genes.

5.1. Virulence genes and their mechanisms of action and pathogenesis

Non-O1/non-O139 V. cholerae strains possess a diverse array of virulence factors that contribute to their pathogenicity. These virulence genes encode various proteins and toxins that play crucial roles in bacterial colonization, invasion, and the induction of host cellular responses (Table 2). The virulence factors can be broadly categorized into toxins, adhesins, secretion systems, and regulatory proteins. Collectively, these factors enable non-O1/non-O139 V. cholerae to attach to host cells, evade host immune responses, disrupt cellular functions, and cause tissue damage.^75,84

The expression of these virulence factors is controlled by a complex regulatory network, including quorum sensing (QS) systems, ToxR regulatory systems, and various global regulators.¹⁰² Environmental factors such as temperature, salinity, and pH also significantly affect the expression of virulence genes, allowing non-O1/non-O139 V. cholerae to adapt to different ecological niches.⁵⁴

In addition to the well-established virulence factors, recent studies have identified new mechanisms contributing to the pathogenicity of non-O1/non-O139 V. cholerae. Outer Membrane Vesicles (OMVs) have been found to play a significant role in bacteria-host interactions, carrying various virulence factors including proteases, hemolysins, and DNA.^1,2 Some strains also possess CRISPR-Cas systems, which not only provide defense against phage infection but may also be involved in virulence regulation.¹⁰⁴ Furthermore, the discovery of small RNAs such as VqmR and TarA has revealed an additional layer of virulence regulation in these bacteria.¹⁰⁵

The complexity of virulence regulation in non-O1/non-O139 V. cholerae extends beyond these newly identified factors. In addition to the well-known ToxR system, other two-component systems like VarS/VarA have been implicated in virulence regulation.¹⁰⁶ This diversity and complexity of virulence factors underscore the pathogenic potential of non-O1/non-O139 V. cholerae. Understanding the functions and regulatory mechanisms of these factors is crucial for developing targeted prevention and treatment strategies. Future research should focus on exploring the expression patterns of these virulence factors under various host and environmental conditions, as well as their interactions. Such studies will provide new insights for controlling non-O1/non-O139 V. cholerae infections and may lead to novel therapeutic approaches.

5.2. Diversity and Adaptability of Virulence Genes

Non-O1/non-O139 V. cholerae strains exhibit remarkable diversity and adaptability in their virulence genes, which is key to their successful colonization and pathogenicity. These strains possess a highly plastic genome, capable of rapidly acquiring or losing virulence factors through mechanisms such as horizontal gene transfer, genetic recombination, and mutation.¹⁰⁷ This genetic plasticity allows non-O1/non-O139 V. cholerae to adapt to various ecological niches, including aquatic environments and human hosts.⁶⁹ The expression of virulence genes is controlled by complex regulatory networks, including quorum sensing systems, ToxR regulatory pathways, and other global regulators, enabling bacteria to flexibly adjust their virulence phenotypes in response to environmental conditions such as temperature, pH, and osmotic pressure.^102,108 For instance, the ToxR system not only regulates classical virulence genes like cholera toxin (CT) and toxin co-regulated pilus (TCP) but also influences the expression of other virulence factors such as the outer membrane protein OmpU.¹⁰⁹ Moreover, these strains often carry multiple virulence factors with overlapping functions, providing functional redundancy and enhancing their survival capabilities in different environments.

In recent years, the application of high-throughput sequencing technologies has enabled a more comprehensive understanding of the diversity of virulence genes in non-O1/non-O139 V. cholerae.¹¹⁰ Studies have found that even within the same geographical region, there can be significant differences in virulence gene composition between different strains. Kirchberger et al.¹¹¹ used whole-genome sequencing to analyze non-O1/non-O139 V. cholerae strains from coastal environments, revealing high diversity in virulence gene composition and identifying new virulence factors. Furthermore, the adaptability of non-O1/non-O139 V. cholerae is also reflected in their antibiotic resistance. Research has shown that these strains can develop multiple drug resistance by acquiring resistance genes or mutations, further increasing their survival ability and public health threat.¹¹² Another noteworthy aspect is the interaction between non-O1/non-O139 V. cholerae and environmental microbial communities. These interactions may influence the expression and spread of virulence genes. Purdy et al.¹¹³ found that certain environmental factors can induce non-O1/non-O139 V. cholerae to enter a viable but non-culturable (VBNC) state, which may affect the expression and detection of their virulence genes.

In conclusion, non-O1/non-O139 V. cholerae possess diverse virulence factors and complex pathogenic mechanisms. The diversity and adaptability of their virulence genes enable them to be widely distributed in aquatic animals and potentially pose a threat to human health. Future research should focus on elucidating the mechanisms of action of these virulence factors in different hosts, exploring the influence of environmental factors on virulence gene expression, and studying the horizontal transfer and evolution of virulence genes. Additionally, developing new rapid detection methods and effective prevention strategies are important directions for future research. These studies will provide important bases for developing targeted prevention and control strategies and assessing public health risks. The diversity of virulence genes also leads to a variety of clinical manifestations in non-O1/non-O139 V. cholerae infections, ranging from mild gastrointestinal symptoms to severe systemic infections.¹¹⁴ This genetic and phenotypic diversity not only increases the environmental adaptability and pathogenic potential of non-O1/non-O139 V. cholerae but also poses challenges for disease diagnosis, treatment, and prevention, highlighting the importance of continuous monitoring and research on these emerging pathogens.

6. Antibiotic Resistance in non-O1/non-O139 V. cholerae

6.1. Common Resistance Patterns

Antibiotic resistance in non-O1/non-O139 V. cholerae (non-O1/non-O139 strains) has become a major challenge for global aquaculture and public health sectors. In recent years, these strains have exhibited complex and diverse resistance patterns, with a significant increase in the proportion of multidrug-resistant (MDR) strains. Liu et al.¹¹⁵ reported that the percentage of MDR strains isolated from coastal regions in China rose from 15% in 2010 to 35% in 2020. Resistance is not only observed in commonly used antibiotics such as β-lactams, tetracyclines, and fluoroquinolones but also shows distinct geographical distribution differences and host specificity.^110,116

6.2. Resistance Mechanisms

These bacteria acquire resistance through various mechanisms, primarily including enzyme-mediated resistance (e.g., production of β-lactamases and aminoglycoside-modifying enzymes), target site alterations (e.g., mutations in DNA gyrase and topoisomerase IV), enhanced efflux pump systems (e.g., RND family efflux pumps), and reduced membrane permeability (Table 3). Wang et al.¹¹⁷ also discovered a novel tetracycline resistance mechanism involving tet(X) gene mutations, further complicating resistance control efforts.

The diversity and complexity of these resistance mechanisms highlight the severity of antibiotic resistance in non-O1/non-O139 V. cholerae strains. Understanding these mechanisms is crucial for developing new antibiotics and formulating effective resistance control strategies. Future research should continue to monitor resistance trends, explore new resistance mechanisms, and develop innovative treatment approaches to address this increasingly serious problem.⁹¹

6.3. Transmission and Evolution of Resistance

The transmission and evolution of antibiotic resistance in non-O1/non-O139 V. cholerae is a complex process, primarily achieved through various mechanisms of horizontal gene transfer. Ceccarelli et al.¹¹⁰ found that up to 70% of non-O1/non-O139 V. cholerae strains isolated from the Chesapeake Bay carried multiple resistance genes, typically located on mobile genetic elements such as integrons and transposons. Rapa et al.¹²⁴ further confirmed the importance of integrons, particularly class 1 integrons, in facilitating the horizontal transfer of resistance genes in the environment. Beyond integrons, Spagnoletti et al.¹²⁵ studied the role of SXT/R391 family integrative conjugative elements (ICEs) in V. cholerae, discovering that these ICEs could efficiently transfer multiple antibiotic resistance genes between different V. cholerae strains. Additionally, Wang et al.¹²⁶ identified multiple transferable resistance plasmids in non-O1/non-O139 V. cholerae isolated from coastal regions of China, further illustrating the diversity of horizontal gene transfer. Environmental factors, such as excessive antibiotic use and high-density aquaculture, significantly influence the spread of resistance. Ramamurthy et al.¹²⁷ pointed out that the improper use of antibiotics in aquaculture may accelerate the selection and spread of resistant strains, posing a potential threat to public health.

Globalization and climate change also profoundly impact the spread of resistance. Baker-Austin et al.⁹¹ reviewed how climate change might influence the ecology and epidemiology of V. cholerae through various mechanisms, including altering its geographical distribution and seasonal patterns. Vezzulli et al.⁵⁸ demonstrated a significant correlation between rising water temperatures in the coastal North Atlantic over the past 50 years and an increase in V. cholerae abundance, which may accelerate the spread of resistance genes. Furthermore, Verma et al.¹¹² revealed how the genomic plasticity of V. cholerae facilitates its acquisition of new antibiotic resistance, enabling these bacteria to survive in different environments and acquire new resistance characteristics through adaptive evolution.

Addressing the challenge of antibiotic resistance in non-O1/non-O139 V. cholerae requires a multi-pronged, comprehensive strategy. The World Health Organization¹²⁸ emphasized the importance of establishing comprehensive resistance monitoring systems in its Global Antimicrobial Resistance and Use Surveillance System (GLASS) report. Meanwhile, new detection technologies are continually developing. For example, Li et al.¹²⁹ developed a microfluidics-based method for real-time study of the rapid spread of antibiotic resistance plasmids in biofilms. This innovative technology provides new tools for understanding the dynamics of resistance spread in microenvironments. Only through multidisciplinary collaboration, integrating microbiology, ecology, epidemiology, and policy research, can we more comprehensively understand and control the antibiotic resistance of non-O1/non-O139 V. cholerae, providing strong support for the sustainable development of aquaculture and public health safety.

7. Prevention and Control Measures

The prevention and control of non-O1/non-O139 V. cholerae strains pose significant challenges for both the aquaculture industry and public health sectors. Effective prevention and control strategies require comprehensive measures, including aquaculture environment management, vaccine development, biological control, and rational use of antibiotics.

7.1. Aquaculture Environment Management

Aquaculture environment management is fundamental to controlling the spread of non-O1/non-O139 V. cholerae. Hossain et al.¹³⁰ demonstrated that implementing good aquaculture management practices can significantly reduce the number of V. cholerae strains in the environment. Specific measures include optimizing water quality parameters (such as pH, salinity, and dissolved oxygen), regular disinfection and substrate improvement, and implementing biosecurity measures. For example, Krummenauer et al.¹³¹ found that using biofloc technology in shrimp farming can effectively improve water quality, reduce the number of pathogens, and enhance the health status of cultured organisms. These environmental management strategies not only directly reduce the number of pathogens but also indirectly lower antibiotic use by improving the health of cultured organisms, thereby reducing the development and spread of resistance. Furthermore, Rameshkumar et al.¹³² showed that implementing comprehensive aquaculture management measures, including proper water quality management and probiotic supplementation, can significantly decrease the prevalence of Vibrio species in shrimp aquaculture systems. These measures can also help maintain the balance of aquaculture ecosystems, reducing the occurrence of environmental conditions favorable for the growth and gene transfer of V. cholerae.

7.2. Progress in Vaccine Development Research

Vaccine development is a crucial direction for preventing non-O1/non-O139 V. cholerae infections. Although no commercial vaccines are currently available for non-O1/non-O139 V. cholerae, research progress is encouraging. Yan et al.¹³³ studied the adhesion characteristics of pathogenic V. alginolyticus to the intestinal mucus of large yellow croaker, providing an important foundation for developing adhesion inhibitors and vaccines against Vibrio species. Furthermore, Luan et al.¹³⁴ expressed and characterized a metalloprotease from Vibrio parahaemolyticus, which could potentially serve as a vaccine target. However, developing a broad-spectrum effective vaccine remains challenging due to the high genetic diversity of non-O1/non-O139 V. cholerae.

7.3. Biological Control Strategies

Biological control strategies, as environmentally friendly alternatives, have gained widespread attention in recent years. These strategies mainly include probiotics, plant extracts, and phage therapy. Sha et al.¹³⁵ found that lactic acid bacteria isolated from healthy Litopenaeus vannamei could effectively inhibit the growth of Vibrio species and enhance host immune responses. Regarding plant extracts, Packiavathy et al.¹³⁶ demonstrated that curcumin has significant inhibitory effects on biofilm formation of V. cholerae. As an emerging strategy, phage therapy has shown promise, with Yen et al.¹³⁷ successfully isolating phages that specifically lyse V. cholerae, providing potential for developing new biological control agents.

7.4. Rational Use of Antibiotics

The rational use of antibiotics is key to controlling the spread of resistance. Many countries have begun implementing stricter antibiotic use management policies.Watts et al.¹³⁸ reviewed the status and trends of antibiotic use in aquaculture, emphasizing the importance of implementing antibiotic stewardship programs. Santos and Ramos¹³⁹ proposed a precision medication model based on rapid pathogen diagnosis and antibiotic sensitivity testing, which can significantly reduce antibiotic use while maintaining therapeutic efficacy. Furthermore, Deng et al.¹⁴⁰ demonstrated that the stock density in shrimp aquaculture affects the microbial community in biofloc water and shrimp gut microbiota, which has implications for disease management and antibiotic use strategies.

8. Zoonotic Potential of non-O1/non-O139 V. cholerae

As potential zoonotic pathogens, non-O1/non-O139 V. cholerae warrant in-depth research into their transmission routes from aquatic animals to humans, public health risks, and food safety control measures.

8.1. Transmission Routes from Aquatic Animals to Humans

Non-O1/non-O139 V. cholerae primarily spread to humans through consumption of contaminated seafood or contact with polluted water bodies. Halpern et al.¹⁴¹ found that fish can act as reservoirs for V. cholerae, including non-O1/non-O139 strains, potentially facilitating their transmission to humans. Additionally, occupational exposure (such as fishermen and aquatic product processing workers) is also an important transmission route. Senderovich et al.⁸⁴ demonstrated that non-O1/non-O139 V. cholerae could be isolated from fish in freshwater habitats, suggesting a potential risk for individuals in close contact with these environments.

8.2. Public Health Risk Assessment

Assessing the public health risks of non-O1/non-O139 V. cholerae is fundamental to developing prevention and control strategies. Baker-Austin et al.⁹¹ reviewed the increasing incidence of Vibrio infections, including those caused by non-O1/non-O139 V. cholerae, in the context of climate change. They highlighted that warming coastal waters could lead to increased exposure risks, particularly in temperate regions. However, actual infection numbers may be underestimated due to often mild symptoms and potential misdiagnosis.

8.3. Food Safety Control Measures

Given that food is the main route of transmission for non-O1/non-O139 V. cholerae, strengthening food safety control is crucial. Wei et al.¹⁴² developed multiplex PCR assays for the simultaneous detection of several Vibrio species, including V. cholerae, V. parahaemolyticus, V. vulnificus, and V. alginolyticus, with an internal amplification control. This method provides a rapid, sensitive, and specific tool for detecting potentially pathogenic Vibrio species, including non-O1/non-O139 V. cholerae, in various samples. The inclusion of an internal amplification control enhances the reliability of the assay by reducing false-negative results. This rapid detection technology offers a powerful tool for food safety regulation, allowing for the efficient identification of potentially harmful non-O1/non-O139 V. cholerae strains in food products. The authors reported that the detection limit of their multiplex PCR assay was 10³ CFU/mL for pure cultures and 10⁴ CFU/mL for spiked oyster samples, demonstrating its potential for practical application in food safety monitoring. Additionally, Pruzzo et al.¹⁴³ reviewed the persistence mechanisms of Vibrio species in marine environments, which has implications for developing effective control strategies in aquaculture and seafood processing. Their work highlights the importance of understanding the ecological factors that contribute to the survival and spread of non-O1/non-O139 V. cholerae in aquatic environments and food chains.

9. Future Perspectives

While significant progress has been made in the study of non-O1/non-O139 V. cholerae, numerous challenges remain. Future research should focus on three main areas: genomics, host-pathogen interaction mechanisms, and the development of novel prevention and control strategies. In the field of genomics, researchers should concentrate on comparative genomics analysis to understand evolutionary relationships between different serotypes and ecotypes, pan-genome analysis to identify core and accessory genes contributing to virulence and environmental adaptation, and functional genomics studies to elucidate gene expression regulation under various environmental conditions. Additionally, metagenomics approaches will be crucial in understanding the role of non-O1/non-O139 V. cholerae in microbial communities.

Exploring host-pathogen interaction mechanisms is vital for developing effective prevention and control strategies. Future studies should utilize advanced in vitro models to simulate the infection process in different host species, investigate how non-O1/non-O139 V. cholerae evade host immune systems, and explore their interactions with the host microbiome. Identifying new virulence factors and their roles in pathogenesis will also be crucial. In terms of prevention and control strategies, research should focus on developing next-generation broad-spectrum vaccines, exploring the application of immunomodulators to enhance host resistance, developing smart drug delivery systems for targeted therapy, and utilizing synthetic biology techniques to design functional probiotics. Novel biocontrol methods, such as phage therapy or CRISPR-based approaches, also warrant investigation.

Future research should also address improving rapid detection methods for non-O1/non-O139 V. cholerae in environmental and clinical samples, developing predictive models for outbreaks based on environmental and climatic factors, studying the impact of climate change on the distribution and virulence of these strains, and investigating their role in horizontal gene transfer of virulence and antibiotic resistance genes in aquatic environments. This field is full of opportunities and challenges, requiring multidisciplinary collaboration and international cooperation to integrate research efforts from microbiology, ecology, immunology, genomics, and other fields. This comprehensive approach will lead to a better understanding of this complex pathogen and the development of more effective prevention and control strategies, ultimately contributing to the sustainable development of aquaculture and global public health security.

Acknowledgments

This work was supported by the Natural Science Foundation of Hunan Province (Grant no. 2023JJ30435), the Research Project of Education Department of Hunan Province (Grant no. 24C0679 and no. HNJG-2020-0719), and Changde Vocational and Technical College 2024 School-Enterprise Cooperation Project (XQ2401).

Authors’ Contribution

Conceptualization: Qing Tan (Equal), Rong-hua Wang (Equal). Funding acquisition: Qing Tan (Equal), Rong-hua Wang (Equal). Writing – original draft: Qing Tan (Equal), Man Xu (Equal), Xue-Xian Li (Equal). Writing – review & editing: Qing Tan (Equal), Rong-hua Wang (Equal). Data curation: Ya-jun Chen (Equal), Lin Tang (Equal), Jian Liu (Equal).

Competing Interest

The authors declare no conflict of interest.

Informed Consent Statement

All authors and institutions have confirmed this manuscript for publication.

Data Availability Statement

All are available upon reasonable request.