








key: cord-284377-hsju2shr
authors: Li, Meng; Zhao, Lin; Ma, Jiajun; Zhao, Na; Luo, Jing; Wang, Chengmin; Chen, Lin; Ma, Guoyao; Wang, Yutian; He, Hongxuan
title: Vibrio vulnificus in aquariums is a novel threat to marine mammals and public health
date: 2018-07-26
journal: Transbound Emerg Dis
DOI: 10.1111/tbed.12967
sha: 
doc_id: 284377
cord_uid: hsju2shr

Vibrio vulnificus is a Gram‐negative, curved, obligate halophilic marine bacterium that exclusively exists in coastal seawaters. Previous studies revealed that V. vulnificus is one of the most dangerous foodborne zoonotic pathogens for human beings. However, it remains unknown whether marine mammals can be infected by V. vulnificus. In May 2016, a captive spotted seal (Phoca largha) died due to septicemia induced by V. vulnificus. Upon post‐mortem examination, V. vulnificus was isolated, identified, and named as BJ‐PH01. Further analysis showed that BJ‐PH01 belongs to biotype 1 and the Clinical genotype. Furthermore, we performed an epidemiological investigation of V. vulnificus in six aquariums in northern China. As a result, V. vulnificus was successfully isolated from all investigated aquariums. The positive rates ranged from 20% to 100% in each investigated aquarium. During the investigation, 12 strains of V. vulnificus were isolated, and all 12 isolates were classified into biotype 1. Eleven of the 12 isolates belonged to the Clinical genotype, and one isolate belonged to the Environmental genotype. All 12 isolated V. vulnificus strains showed limited antibiotic resistance. Overall, our work demonstrated that V. vulnificus is frequently distributed in aquariums, thus constituting a threat to captive marine mammals and to public health.

salinity, (b) season, (c) gender and age, (d) preexisting chronic diseases, and (e) bacterium virulence. As reported, the majority of human infections occur in the subtropical regions from April to November. People over the age of 40 are predominantly infected.

Moreover, males are more susceptible than females (Ito et al., 2012) , perhaps due to the role of estrogen in protecting against the bacterium's endotoxins (Miyamoto et al., 1999; Soucy, Boivin, Labrie, & Rivest, 2005) . As mentioned previously, patients with underlying chronic diseases, including alcoholism, diabetes, cancer, or renal diseases, are more susceptible to V. vulnificus (Bross, Soch, Morales, & Mitchell, 2007a) . Several other risk factors contribute to the high pathogenicity of V. vulnificus in humans, such as capsule, iron, and the vcg gene (Jones & Oliver, 2009 ).

Marine mammals are important inhabitants of tropical and temperate regions, especially offshore areas of the sea (Schipper et al., 2008) . There is an overlap in the distributions of V. vulnificus and marine mammals. No evidence has shown that marine mammals can be infected by V. vulnificus; however, the concerns cannot be excluded. Here, we provide evidence that marine mammals can also be infected by V. vulnificus. Further investigation showed that V. vulnificus is ubiquitous in aquariums, thus revealing a novel threat to captive marine animals and human beings in aquariums.

Animal studies were performed in strict accordance with the Guidelines for the Care and Use of Animals in Research, which is issued by the Institute of Zoology, Chinese Academy of Sciences. This study was evaluated and approved by the Animal Ethics Committee of Institute of Zoology, Chinese Academy of Sciences. All experiments were conducted in a Biosafety Level 2 (BSL-2) facility.

An autopsy was performed after the spotted seal died. Pathological lesions were observed and recorded. Tissue samples (i.e., lung, liver, stomach, spleen, intestine, kidney, and heart) were sterilely collected.

A festered sample was collected from the trauma injury. All samples were transported on ice and analyzed immediately.

Tissue samples were cultured on blood agar (Oxoid, UK), MacConkey agar (Oxoid, UK), chocolate agar (Oxoid, UK), and thiosulphatecitrate-bile-salt-sucrose (TCBS) agar (Oxoid, UK). All plates were placed in aerobic or anaerobic conditions at 37°C for 18-24 hr.

From each plate, at least 24 single colonies were selected for identification, and all the selected colonies were preliminarily identified by 16S rDNA sequencing. The results were further confirmed by the BD Phoenix automated Microbiology System (BD Diagnostic Systems, Sparks, MD.) (Stefaniuk, Baraniak, Gniadkowski, & Hryniewicz, 2003) and species-specific PCR amplification. Several known viral pathogens that have been reported to infect marine mammals were also detected by virus-specific PCR, including type A influenza virus (IAV), phocine distemper virus (PDV), coronavirus, and rotavirus. The primers used in this study are listed in Table 1 (Chatzidaki-Livanis, Jones, & Wright, 2006; Gómara, Wong, Blome, Desselberger, & Gray, 2002; Hill et al., 1991; Lau et al., 2005; Miller et al., 2011; Reynaud, Pitchford, De Decker, Wikfors, & Brown, 2013; Sea, 2015; Warner & Oliver, 2008 CCATCATCAGATAGAATCATCATA animals (i.e., spotted seal, turtle, dolphin, shark, whale, and saltwater fish) were kept (Table 2) . Animal (i.e., turtle, spotted seal) body surface swabs were collected using medical degreasing cotton. Freshwater samples and freshwater fish body surface swabs were also collected as negative controls. In total, 54 samples were collected.

All the samples were stored on ice, transported to the Institute of Zoology, Chinese Academy of Sciences, and analyzed immediately.

All samples were examined using procedures in the Bacteriological Analytical Manual of The Food and Drug Administration (FDA) (Kaysner & DePaola, 2004) . In detail, water samples were serially diluted and cultured by 1% NaCl alkaline peptone water (APW) at 37°C for 18-24 hr. Body surface swabs were diluted in a 5-ml volume of sterile phosphate-buffered saline (PBS) followed by vortexing for 45 s.

The supernatant was cultured in 1% NaCl alkaline peptone water (APW) at 37°C for 18-24 hr. The resulting products were diluted and cultured on blood agar and then detected by V. vulnificus vvhA gene-specific PCR (Warner & Oliver, 2008) , 16S rDNA sequencing, and the BD Phoenix Automated Microbiology System (BD).

Biotyping of isolated V. vulnificus was performed as previously reported (Bisharat, Agmon, Finkelstein, Raz, Ben-Dror, Lerner, Soboh, Colodner, Cameron, & Wykstra, 1999) . Briefly, ONPG testing, indole production, 1% NaCl ornithine decarboxylase testing, D-sorbitol fermentation, D-mannitol fermentation, and lactose fermentation analysis were performed to identify the biotypes of the isolated V. vulnificus.

Genotyping was conducted to identify the virulence genes of the isolated V. vulnificus. In our study, vcg (virulence-correlated gene), serE (serovar E) gene, cps (capsular polysaccharide) gene, bt2 (biotype 2) gene, ary (arylsulfatase) gene, mtlABC (mannitol/fructose-specific phosphotransferase system IIA protein) gene, and nanA (N-acetylneuraminate lyase) gene were detected using virulence gene-specific PCR as listed in Table 1 .

The V. vulnificus isolated from the seal (BJ-PH01) was cultured in 1% APW and quantified by a colony forming unit (CFU) assay on blood agar. The inoculum was serially diluted to 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , and 10 3 CFU/ml 6-week-old female BALB/c mice were intraperitoneally (for each group, n = 4) (Strom & Paranjpye, 2000) or intramuscularly (for each group, n = 4) ( 

Several virulence factors of V. vulnificus have been reported previously, such as toxin, LPS, and capsule (Jones & Oliver, 2009 ).

Possession of an antiphagocytic capsule is one of the absolute requirements for virulence. Encapsulated cells produce opaque colonies, and only opaque cells are able to utilize transferrin-bound iron.

As shown in Figure 2b , the V. vulnificus isolate BJ-PH01 grew opaque colonies on blood agar. This result prompted us to consider that BJ-PH01 is virulent in animal models (Simpson, White, Zane, & Oliver, 1987) . Several animal models have been constructed to explore the increased susceptibility to V. vulnificus infections after the injection of iron-containing compounds. Here, we conducted an 

Having demonstrated that V. vulnificus can be detected in aquariums, we next asked whether this lethal bacterium is widespread in aquariums. To explore the prevalence of V. vulnificus in aquariums, we performed an epidemiological investigation of V. vulnificus.

In this analysis, we successfully collected 54 samples from six aquariums in northern China ( genotypes based on its virulence-correlated gene (vcg) (Warner & Oliver, 2008) ; our analysis showed that 11 of the 12 isolated V. vulnificus strains were classified into the C genotype, and only one of the 12 belonged to the E genotype (Table 4) .

A previous study demonstrated that genotypic markers cannot unequivocally predict virulence, although several genes were putatively reported to be linked to virulence. Here, we investigated the genes ary (arylsulfatase), mtlABC (mannitol/fructose-specific phosphotransferase system IIA protein), and nanA (N-acetylneuraminate lyase)

by PCR in our study (Table 4 ). The ary gene has been demonstrated to be associated with virulence of clinical strains by providing a pathogen with sulfur within the host, thus providing an immune evasion approach (Morrison et al., 2012) . Positive Ary PCRs were obtained in all of the isolated strains. Although mtlABC appears to be linked to pathogen virulence, the precise role of mtlABC is not 

BJ-SAR01

| 1867 yet understood (Reynaud et al., 2013) . In our experiments, we obtained positive mtlABC results for all of the isolated strains. The nanA gene has been demonstrated to be involved in sialic acid metabolism and is essential for V. vulnificus virulence (Kim, Hwang, Kim, & Choi, 2011) . Positive nanA gene PCR results were obtained for all of the isolated strains. These results indicate that the 12 isolated V. vulnificus strains display a potential threat to mammals, including marine animals and humans.

The disease progression of V. vulnificus infection is often acute, and it is therefore important to provide timely treatment with proper antibiotics. We conducted a further analysis to understand its sensitivity to major antibiotics. The result showed that all isolated V. vulnificus strains were sensitive to antibiotics. In detail, V. vulnificus was sensitive to levofloxacin, tetracycline, piperacillin, and gentamicin (Table 5) .

Vibrio vulnificus is a foodborne pathogen of humans. Here, we provide evidence that marine mammals can also be infected by V. vulnificus. Epidemiological investigations of V. vulnificus showed that this lethal bacterium can frequently be detected in aquariums, thus constituting a novel threat to marine animals, workers, and tourists in relevant aquariums. Antibiotic drug resistance is an increasing concern due to the overuse of antibiotics. Fortunately, all of the isolated V. vulnificus isolates in our study were sensitive to levofloxacin, tetracycline, piperacillin, and gentamicin. No drug-resistant V. vulnificus isolates were isolated.

The minimum dose capable of causing human infection is currently unknown (Strom & Paranjpye, 2000) . Previous studies have suggested that the dose may be fewer than 1,000 organisms. Animal experiments have been helpful in researching disease syndromes produced by V. vulnificus. However, they have no instructive value for determining the infectious dose 50 (ID 50 ) for human infections (Strom & Paranjpye, 2000) . In our research, we found that the med- (Gao et al., 2017) . Vibrio fluvialis has been considered an emerging pathogen that induces foodborne diarrhea (Ramamurthy, Chowdhury, Pazhani, & Shinoda, 2014) . These results suggest that V. vulnificus is not the only threat to marine animals and public health in aquariums.

The major limitation of our research is that we failed to perform

Koch's postulate test because it was impossible for us to confirm the infection using a healthy seal. Due to limited background information, the typical clinical manifestations of V. vulnificus infected seals are poorly understood. We provided a diagnosis mainly based on the detection and isolation of V. vulnificus from the visceral tissues and blood, which indicated that V. vulnificus induced septicemia (Bross, Soch, Morales, & Mitchell, 2007b We demonstrated that V. vulnificus can infect and kill marine mammals. However, it was difficult to evaluate the risk of infection of marine mammals by V. vulnificus: (a) V. vulnificus is an opportunistic pathogen, and one or more predisposing factors are required to initiate disease; (b) the minimum dose for V. vulnificus to cause an infection is poorly understood, even in humans; (c) the interface between V. vulnificus and marine mammals in the wild has never been studied. Nevertheless, the prevalence of V. vulnificus is a persistent threat to marine mammals.

This work was funded by grants from the State Forestry Administration of China, Chinese Academy of Sciences (CZBZX-1).

No conflict of interest exists in the submission, and all authors approved the publication.

Li http://orcid.org/0000-0002-2453-1821

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