Prevalence, virulence genes, and antimicrobial resistance of Vibrio species isolated from diseased marine fish in South China

Here, 70 potential Vibrio pathogens belonging to nine species, dominated by Vibrio harveyi, were isolated and identified from diseased aquacultured marine fish in South China. Subsequently, the prevalence of 11 virulence genes and the resistance to 15 antibiotics in these strains were determined. Most strains possessed atypical virulence genes in addition to typical virulence genes. Notably, hflk and chiA originating from V. harveyi, and flaC associated with V. anguillarum were detected in more than 40% of atypical host strains. Multidrug resistance was widespread: 64.29% strains were resistant to more than three antibiotics, and the multi-antibiotic resistance index ranged from 0.00 to 0.60. The proportions of strains resistant to the antibiotics vancomycin, amoxicillin, midecamycin, and furazolidone all exceeded 50%; nevertheless, all strains were sensitive to florfenicol, norfloxacin, and ciprofloxacin. Furthermore, both virulence genes and antibiotic resistance were more prevalent in Hainan than in Guangdong, owing to the warmer climate and longer annual farming time in Hainan. These results therefore suggest that warming temperatures and overuse of antibiotics are probably enhancing antibiotic resistance and bacterial infection. This study reveals that pathogenic Vibrio spp. with multi-antibiotic resistance are highly prevalent among marine fish in South China and thus warrant further attention. The results will provide helpful guidance for ecological regulation and local antibiotic use in the control of marine fish farming’ Vibrio diseases in South China, facilitating the implementation of national green and healthful aquaculture.

To meet the increasing demand for animal protein, the aquaculture industry has developed rapidly, and the proportion of fish farming (marine fish farming and freshwater fish farming) increased from 5% to more than 40% of global fish production since 1970 1 . Notably, in China, fish farming production accounted for as much as 75% of the total fish production in 2018 2 . Because of the advantage of the proximity to the South China Sea, farming of marine fish (including Lateolabrax japonicus, Panalichthys lethostigma, Pseudosciaena crocea, Rachycentron canadum, and Epinephelussp spp.) has rapidly developed in South China 2,3 , accounting for 78.26% of the total marine fish farming production in China in 2018 2 . In recent years, intensive and industrialized mariculture has gradually developed in South China to meet the needs of the national economy and provide food support 3 . Therefore, sustainable development of marine fish farming in South China is crucial to securing the food supply and strengthening the national economy of China.
Scientific RepoRtS | (2020) 10:14329 | https://doi.org/10.1038/s41598-020-71288-0 www.nature.com/scientificreports/ However, over-intensive aquaculture and harmful anthropogenic activities contribute to outbreaks of serious bacterial infections, thereby affecting both economic and social development 4,5 . Vibriosis is one of the most prevalent bacterial diseases affecting diverse marine fish and shellfish 6 . Chong et al. 7 have noted that approximately two-thirds of the diseases reported in Epinephelus spp. are vibrioses, which affect all stages of fish growth and lead to as much as 50% mortality among fish 8,9 . Several species of Vibrionaceae, including Vibrio harveyi, V. vulnificus, V. parahaemolyticus, V. alginolyticus, and V. anguillarum, are the most common species and are associated with health problems in marine animals 10 . For example, infection with V. alginolyticus and V. harveyi in marine cage-cultured P. crocea led to a mortality rate between 30 and 40%, and as high as 80% in Zhejiang province, China, between May 2000 and November 2003 11 .
The pathogenicity of Vibrio strains is facilitated by a broad range of virulence factors encoded by virulence genes 12 . In general, virulence factors allow pathogens to infect and damage the host, by enabling pathogenic adherence and entrance, establishment and multiplication, avoidance of host defenses, damage to the host, and finally exit from the infected host 13 . Five major virulence factors are found in vibrios: capsular polysaccharides, adhesive factors, cytotoxins, lipopolysaccharides, and flagella 14,15 . Bacteria acquire new (atypical) virulence genes in addition to their innate virulence genes (typical virulence genes), thus improving their virulence. These atypical virulence genes are acquired via horizontal gene transfer (HGT) 12 from the environment and/or other bacteria 16 . HGT, particularly the HGT of atypical virulence genes, is considered an important process influencing bacterial evolution and promoting bacterial virulence 17 . Global climate change, antibiotics, heavy metals, and nutrient pollutants have been reported to increase the pathogenicity and drug resistance of pathogens by affecting HGT 18,19 . Therefore, the investigation of virulence genes, particularly atypical virulence genes, and the analysis of factors influencing the presence of virulence genes should provide meaningful insights for the study of Vibrio pathogenesis and facilitate the establishment of ecological control systems.
Antibiotics have been extensively used against bacterial infections in the aquaculture industry 20 . For instance, florfenicol and oxolinic acid are mainly used to control vibriosis in cod fry in Norway 21 . Quinolones and flumequine are widely used to treat classical and cold water vibriosis 22 . However, the extensive use of antibiotics results in drug residues, thus promoting the development of antibiotic resistance and consequently contaminating food, water, and sediments 23 . Defoirdt et al. 24 have reported that with the increased use of antibiotics such as chloramphenicol, cotrimoxazole, erythromycin, and streptomycin to combat V. harveyi infection in giant tiger prawns, the bacteria have become resistant, and the antibiotics are no longer effective. In 2013, 54,000 tons of antibiotics were excreted into the environment by humans and animals in China, and this amount has gradually increased in subsequent years 25 , resulting in the selection and accumulation of severely resistant and multi-antibiotic resistant bacteria 26 . Moreover, strong drug resistance is likely to enhance bacterial virulence, making it difficult to treat infections 27,28 . Therefore, analyzing the drug resistance of pathogens in a specific area is crucial to formulating local antibiotic reduction policies and effective antibiotic use programs.
High-density farming, along with intense human activities and global climate change, has led to frequent vibriosis, antibiotic overuse, and antibiotic resistance in recent years 6,29 . To thoroughly deploy the "Green and Healthy Aquaculture" initiative launched in 2020, the Ministry of Agriculture and Rural Affairs decided to implement "Drug Reduction in Aquaculture" and "Ecological Health Breeding" initiatives for the development of aquaculture animal disease detection, pathogen resistance monitoring, determination of suitable local antimicrobial profiles for aquaculture, provision of guidance for scientific medication, and promotion of vaccine use for disease prevention. Previous studies have reported the virulence genes and antibiotic resistance of V. harveyi in South China 30,31 ; however, little information on other Vibrio spp. is available regarding the virulence genes and antimicrobial resistance patterns in South China. In this study, we assessed the variations in nine Vibrio species' virulence genes and antibiotic resistance, and identified the factors likely to influence the transmission of virulence and drug resistance. Our results may provide guidance for ecological regulation and local antibiotic use for sustainable and healthful breeding, facilitating the implementation of national green and healthful aquaculture.
In total, 38 resistance types (A to AL) were present among all strains, and the antibiotic resistance pattern abundance (ARPA = the number of resistance types/the number of strains) was 0.54. The ang_X14RP15 showed sensitivity to all 15 tested antibiotics. Six strains had resistance to one tested antibiotic, with resistance types of AD, AH, and AI, of which four were resistant to vancomycin, one was resistant to midecamycin, and one was resistant to furazolidone (Fig. 2B, Figure S2). Seven strains showed resistance to two tested antibiotics (AE, AG, and AK), all of which were resistant to vancomycin (Fig. 2B, Figure S2). In addition, three strains showed resistance to amoxicillin, one showed resistance to midecamycin, and three showed resistance to furazolidone (Fig. 2B, Figure S2). Eleven strains had resistance to three tested antibiotics (C, D, AB, AF, AJ, and AL), all of which were resistant to vancomycin and two of which were resistant amoxicillin, midecamycin, furazolidone, tobramycin, and gentamicin (Fig. 2B, Figure S2). The other 45 (64.29%) strains had multidrug resistance to more than three tested antibiotics (Fig. 2B, Figure S2). The alg_F14DM01 (AA) exhibited the highest MARI value of 0.60 and showed resistance to nine antibiotics ( Figure S2).

Discussion
The presence of pathogenic Vibrio spp. in marine fishes is gaining attention, because these organisms frequently cause of systemic infections resulting in fish death and even diseases in humans 32 . Here, we collected the dominant clones from diseased marine fishes. Nine Vibrio species that are potential pathogens in fish diseases were isolated. V. harveyi was most frequently isolated, followed by V. vulnificus and V. alginolyticus. Among them, V. harveyi, V. alginolyticus, V. rotiferianus, V. campbellii, V. parahaemolyticus, and V. communis (a total of 51 isolates, accounting for 72.86%) belonged to Harveyi clade strains, the most important pathogen clade in aquatic organisms 13 . Harveyi clade species prefer warm temperatures and are expected to be transmitted to higher latitudes with global climate change 33,34 . Vibriosis has been frequently recorded in tropical Malaysia, which has a year-round tropical climate of 28 °C, and V. harveyi is most frequently isolated in outbreaks, followed by V. parahaemolyticus, V. alginolyticus, and V. anguillarum, which affect perches and groupers 32,35 . Therefore, the warming temperatures in semitropical/tropical South China should contribute to the extensive isolation of the Harveyi clade, particularly V. harveyi, from diseased marine fishes. V. vulnificus and V. parahaemolyticus are also pathogenic to humans, causing serious diseases including seafood-borne gastroenteritis, wound infections, and septicemia 6 . The Harveyi clade, particularly V. harveyi, is prevalent in marine fish farming in South China and has led to dramatic losses in the aquaculture industry. www.nature.com/scientificreports/ Identification of virulence factors is essential for evaluating bacterial pathogenicity, because these factors allow bacteria to infect and damage hosts 32 . In this study, the high prevalence (> 70%) of hemolysin vhh, the quorum-sensing regulator luxR, chitinase chiA, and the transmembrane transcription regulator toxR Vh in the typical host V. harveyi was consistent with findings from previous reports, whereas the moderate presence of serine protease hflk (44.44%) was lower than that previously reported 30,31 . Except for the V. anguillarum flagella gene flaC identified in all V. anguillarum strains, V. parahaemolyticus hemolysin tdh and V. vulnificus hemolysin vhh were observed in less than one-third strains of V. parahaemolyticus and V. vulnificus, respectively. Previous studies have indicated that virulence genes can be inherent (typical virulence genes) and/or obtained by HGT (atypical virulence genes) 12 . The nine above-mentioned genes and two virulence genes specific to V. cholera (toxR Vc and hlyA) were detected in their atypical hosts; notably, hflK, chiA, and flaC were found in more than 40% of atypical host strains, findings similar to those from studies on strains isolated from regions including Mexico, USA, Thailand, Japan, and Spain 13 . We speculate that HGT plays an important role in the evolution and virulence development in Vibrio spp. Moreover, these virulence factors may be essential for virulence toward different hosts, because vibrios infect a wide range of aquatic hosts, including fish, shrimp, and mollusks 6,32,36 . The acquisition of atypical virulence genes may increase Vibrio virulence against a specific host, although establishing an obvious correlation between the pathogenicity and the number/kind of virulence genes in vibrios is difficult.
Antibiotics are widely used to prevent or treat bacterial diseases in aquaculture, resulting in an increase in antibiotic resistance and multidrug resistance in bacteria and making treatment of infections difficult 28 . In the present study, the ARPA of 0.54, the highest MARI of 0.60, and the multidrug resistance rate of 64.29% were all higher than findings from previous studies in the same area 37,38 , indicating that antimicrobial resistance is temporally different and is likely to increase with time. In this study area, compared with other areas, we found that resistance to vancomycin, amoxicillin, and furazolidone was most prevalent (> 50%), resistance to tetracycline was less prevalent (10%-50%), and sensitivity (< 10%) to chloramphenicol and norfloxacin was observed, results consistent with findings from studies in South India and the Persian Gulf [39][40][41] . The low resistance to gentamicin and erythromycin were similar to findings from studies in South India and Persian Gulf, respectively [39][40][41] . The moderate resistance to tobramycin was higher than that in South India 40 , whereas the low resistance to doxycycline was much lower than that in the Persian Gulf 41 . These results indicated that antimicrobial resistance shows spatial variation, probably because of the types of antibiotics used and the development of different antibiotic resistance mechanisms 42,43 . For example, extensive intrinsic resistance to vancomycin and amoxicillin has been reported 44,45 . Use of antibiotics for which high or moderate resistance have been observed (including vancomycin, amoxicillin, midecamycin, furazolidone, tobramycin, rifampicin, gentamicin, and tetracycline) is recommend to be reduced, whereas antibiotics for which low resistance and sensitivity has been reported (including erythromycin, trimethoprim-sulfamethoxazole, doxycycline, chloramphenicol, florfenicol, norfloxacin, and ciprofloxacin) are suggested to be used in marine fish farming to inhibit bacterial diseases. However, chloramphenicol and norfloxacin have been banned in the aquaculture industry by the Ministry of Agriculture and Rural Affairs, China.
In Hainan, compared with Guangdong, both virulence genes and antibiotic resistance were more prevalent, probably because of the warmer temperature and more useage of antibiotics in Hainan. Warming temperatures can directly induce the expression of antibiotic resistance genes and virulence genes 45 . For instance, the virulence factors involved in motility, host degradation, secretion, antimicrobial resistance, and transcriptional regulation are upregulated as much as 16 times in V. corallilyticus when the temperature is increased from 24 to 27 °C, concurrently with phenotypic changes in motility, antibiotic resistance, hemolysis, cytotoxicity, and bioluminescenc 46 . Temperature has also been reported to affect HGT, in an important mechanism of virulence and drug resistance transmission, through influencing functions including biofilm formation, membrane permeability, immune system activity, and HGT-related enzymes 47 . The conjugation efficiency of Pseudomonas spp. has been found to increase by 10,000 times after a temperature increase from 15 °C to 28 °C by promoting the biofilm formation of Pseudomonas spp 48 . Moreover, extensive vibriosis outbreaks in summer have been widely reported 32,49 . As the southernmost province in China, Hainan is located in a lower latitude (18°10′-20°10′ N, 108°37′-111°03′ E) than that of Guangdong (20°13′-25°31′ N, 109°39′-117°19′ E), and it consequently is usually approximately 3 °C warmer than Guangdong ( Figure S4A). Because of warming temperatures, Hainan became a major marine fish hatchery and usually has a longer annual breeding time than Guangdong, resulting in more usage of antibiotics ( Figure S4B, Table S3) and enhanced antibiotic resistance in Hainan, which has also been supported by the temporally increased antimicrobial resistance in South China 37 . Additionally, acid-base and organic pollution have also been reported to promote HGT and enhance bacterial virulence and antibiotic resistance 50 .
In conclusion, vibrios, particularly the Harveyi clade, cause serious diseases in marine fish, threatening the sustainable development of the aquaculture industry in South China. Virulence genes and antimicrobial resistance are prevalent in those isolated Vibrio strains. Notably, atypical virulence genes are presented in Vibrio strains, promoting bacterial virulence and broadening the host range. High multi-drug resistance makes it difficult to treat infections. Warm temperatures and continued use of antibiotics are likely to enhance the bacterial virulence and antimicrobial resistance. Our results may be helpful for evaluating Vibrio pathogenicity and the abuse of antibiotics. We expect that they can provide guidance for future disease treatment in aquaculture, and serve as a theoretical basis for the development of sustainable disease control methods. To prevent and control the diseases caused by Vibrio species in South China, we suggest the following. (1) In aquaculture, particularly in indoor culture, ecological control systems, including temperature control, should be established, and the use of antibiotics, particularly vancomycin, amoxicillin, midecamycin, furazolidone, tobramycin, rifampicin, gentamicin, and tetracycline, should be reduced to minimize the expression of virulence and resistance genes and decrease the transmission of virulence and resistance. Our results may be useful to support the actions of "Drug Reduction in Aquaculture" and "Ecological Health Breeding. " (2) In the absence of other eco-friendly control methods, erythromycin, trimethoprim-sulfamethoxazole, doxycycline, florfenicol, and ciprofloxacin should be used to inhibit Vibrio diseases. Our findings highlight the need for more detailed investigations to support the Scientific RepoRtS | (2020) 10:14329 | https://doi.org/10.1038/s41598-020-71288-0 www.nature.com/scientificreports/ control of antibiotic use, formulation of relevant laws, and improvements in consumer protection and public health safety. Future research should focus on the following: (1) the detailed virulence mechanism, with the aim of developing environmentally friendly treatments, such as vaccines, probiotics, and immunostimulants; and (2) the joint influence of environmental and anthropogenic factors on the virulence and antibiotic resistance of Vibrio populations in large areas over long time scales, to aid in establishing ecological regulations in the aquaculture industry.

Methods
Bacterial isolates. The diseased marine fishes (including Protonibea spp., Epinephelussp spp., Siganus spp., Trachinotus spp., and Nibea spp., etc.) were determined by clinical diagnosis, with showing a frail situation of sluggish action and reducing ingestion and having the typical symptoms of nodule, rotten body, ascites, ulcer, or enteritis etc. Then, the Vibrio species were isolated and identified from the diseased marine fishes collecting in Guangdong province (including the cities of Zhuhai, Zhanjiang, Huizhou, Chaozhou, and Shenzhen) and Hainan province (including Xincun bay and the cities of Sanya and Wenchang) with following procedures. The tissues (including the liver, spleen, kidney, intestines, and brain) of diseased marine fishes were homogenized. Then the homogenates were screened on 2216E, nutrient, and Brain-Heart Infusion (BHI) agar plates simultaneously and incubated at 28