Characteristics and Phylogeny of Shewanella haliotis Isolated from Cultivated Shellfish in Taiwan

Shewanella haliotis is an emerging human pathogen. Many infectious cases were linked to shellfish ingestion or aquatic exposure. Therefore, it is important to study the phylogeny and distribution of S. haliotis in shellfish aquaculture. We investigated the distribution of S. haliotis in cultivated shellfish farming in Taiwan in which S. haliotis was found in the shellfish from all sampling sites. S. haliotis was identified in cultivated shellfish by 16S rRNA gene sequencing, such as abalone (Haliotis diversicolor), clam (Meretrix lusoria), and oyster (Crassostrea gigas). This study highlighted the contamination of S. haliotis in cultivated shellfish and importance of further study regarding the biodiversity and pathogenesis of S. haliotis.


Introduction
Genus Shewanella is a member of the class Gammaproteobacteria and comprises a group of Gram-negative, nonfermentative, and facultative anaerobic motile bacilli [1]. More than 50 species of Shewanella have been reported [2,3]. ey can grow anaerobically by reduction of various sulfur compounds to H 2 S, including thiosulfate and sulfite [4].
Most of the organisms detected were found in marine environments, and four of these species were commonly found in clinical specimens (S. algae, S. putrefaciens, S. haliotis, and S. xiamenensis). Among these four, S. putrefaciens and S. algae have been found increasingly in human infections [5]. Shewanella infection is associated with direct contact with the organism through seawater or ingestion of raw seafood [3,6]. In addition, a subject infected by Shewanella spp. is usually associated with hepatobiliary disease [3,6].
S. haliotis was first isolated from gut microflora of abalone in 2007 [7] and was first reported to be associated with human infections in Japan [8,9]. As a marine microorganism, it has been rarely reported in humans until recently [10,11]. During 2012 and 2013, Liu et al. reported that S. haliotis is the causative organism for 5 (56%) out of 9 Shewanella bacteremia cases in Taiwan [12]. e increasing case reports included bacteremia, abdominal infection, and soft tissue infections [9,13]. e risks of Shewanella infection caused by seafood consumption are rarely discussed and limited in the literature. In addition, the importance of ecological distribution of S. haliotis in the marine environment has not yet been recognized. In this study, we report the distribution of S. haliotis in cultivated shellfish farming in aquaculture and diverse water sources in Taiwan. e findings from this study will serve as the basis of further understanding of the relationship between different sources of S. haliotis for future studies.

Sampling Sites.
According to the Fisheries Statistical Yearbook published by the Fisheries Agency of Taiwan [14], eight largest cultivated shellfish sites around Taiwan's coastal areas from twenty cultivated fishing districts were included in this study (Table 1). A total of 78 cultivated shellfish samples, including abalones (Haliotis diversicolor), clams (Meretrix lusoria), and oysters (Crassostrea gigas) were collected and analyzed from 2012 to 2014. e sample numbers of abalones, clams, and oysters were 12, 30, and 36, respectively. All of the abalone samples were obtained from Toucheng and Chenggong, two major townships for abalone cultivation, both located on the east coast of Taiwan. Clams and oysters farms are widely distributed on the west coast. Wuqi, Kouhu, Dongshih, Budai, Zihguan, and Fangliao were sampling sites for oysters. e clam samples were collected from Wuqi, Kouhu, Budai, Zihguan, and Fangliao.

2.2.
Isolation of Shewanella haliotis. We followed the guidelines described in U.S. FDA Bacteriological Analytical Manual [15] for shellfish sampling and preparation of sample homogenate. Six samples of shellfish were selected randomly from each sampling site by hand fishing. Samples were packed into sterile plastic bags and then transported to laboratory under refrigerated condition. All the samples were stored at 0-4°C until analyzed and were examined within 6 h of collection. Shells were removed by using the sterilized appliance. One gram of sample was mixed with 100 ml of saline and then homogenized in a sterile blender. One ml of the resultant mixture was transferred and enriched in 10 ml of marine broth (BD, Sparks, USA) and incubated at 30°C for 2-5 days. After incubation, isolation was carried out by the aerobic plate count according to methods described in the Association of Official Analytical Chemists's (AOAC's) guidelines [16]. Serially, decimal dilutions of enriched bacterial culture (from 10 −1 to 10 −6 ) were prepared by transferring 1 ml of previous dilution to 9 ml of marine broth. One ml of each dilutions was spread across onto separate marine agar (BD, Sparks, USA) and then incubated at 30°C for 72 h. According to the description by Kim et al., the typical colony morphology of S. haliotis is circular, convex, entire margin, smooth, opaque, and pinkorange color [7]. e cells are Gram-negative bacillus (rodshaped) and 0.5-0.7 × 2.0-4.3 μm in size [7]. All eligible colonies were picked for further species identification using 16S rDNA sequence analysis. Biochemical characteristics were analyzed by API 20NE (bioMérieux, Marcy-l'Étoile, France) and API ZYM (bioMérieux, Marcy-l'Étoile, France) following the manufacturer's protocol.

DNA Extraction and 16S rRNA Gene Sequencing.
Bacterial DNA was purified with DNeasy Blood and Tissue Kit (QIAGEN, Hilden, Germany). Two universal primers, 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGGCTACCTTGTTACGACTT-3′), were performed for 16S rRNA gene amplification. Each PCR reaction panel contained 10 mM of Tris-HCl (pH 9.0), 1.5 mM of MgCl 2 , 0.2 mM of dNTP, 50 ng of chromosomal DNA template, 20 mM each of primers, and 1 U of Taq DNA polymerase in a final volume of 25 μl. A full PCR cycle following a 30 s denaturation at 94°C included 25 cycles at 94°C for 1 min, 55°C for 1 min, 72°C for 2 min, and a final extension at 72°C for 5 min. All the processes mentioned above were performed on PerkinElmer GeneAmp 9600 PCR system (PerkinElmer, Norwalk, USA). Sequencing of these amplicons was completed by a sequencing company (MB MISSION BIOTECH, Taipei, Taiwan) using ABI 3730 × l DNA Analyzer (Applied Biosystems, Foster City, USA). Species identification was performed by NCBI BLAST (http://blast.ncbi.nlm.nih. gov/Blast.cgi).

Phylogenetic
Analysis. S. haliotis shellfish isolates, four human clinical isolates, and one type strain, S. haliotis DW01 (JCM 14758), were included in the phylogenetic analysis. 16S rRNA gene sequence analysis of clinical isolates was carried out as described above. e sequence of S. haliotis DW01 was obtained from the NCBI database (accession number NR044134.1). e 16S rRNA gene sequences were first aligned by the Clustal W method by using the MegAlign program (DNASTAR Lasergene v. 7.1.0). e phylogenetic analysis was performed by using the neighbor-joining method [17] and constructed by MEGA 6.0 software [18] (http://www.megasoftware.net). e resultant neighborjoining tree topology was evaluated by bootstrap analyses  Figure 1 shows the 8 study sampling sites, and the 17 isolates are shown in Table 1. e S. haliotis isolation percentages of abalones, clams, and oysters samples were 16.7% (2/12), 20% (6/30), and 25% (9/36), respectively ( Table 2). All isolates grew at 4°C and 37°C and expressed typical morphological characteristics of colonies and cells.
Based on the phylogenetic tree (Figure 2), shellfish 3 of clam origin and shellfish 3, 12, 14, and 17 of oyster origin are most related with the S. haliotis type strain DW01 (grouped as cluster 1), followed by the isolates included in cluster 2 and cluster 3. Among all shellfish isolates, shellfish 5 (cluster 2) of clam origin possessed the closest similarity with these two clinical isolates: patient 1 and patient 2. As also shown in the phylogenetic tree, the other isolates were more diverse from these two clinical isolates. e only two abalone origin isolates, shellfish 1 and shellfish 2, were grouped together with three other clam (shellfish 6, 7, and 8) origin isolates in cluster 3. Other isolates could not represent closely related evolutionary relationships to group into any cluster. Overall, the data revealed that S. haliotis is a phylogenetically complex and diverse species.

Discussion
is study is the first evidence of the linkage between human and environmental isolates. e phylogenetic analysis also Wuqi (3) Kouhu (5) Dongshi (1) Budai (3) Zihguan (2) Fangliao (1) Chenggong (  revealed the highly diverse nature of S. haliotis, which warrants further study of optimal typing scheme. e wide distribution of positive sampling sites further highlights the possibility of extensive contamination of the zoonotic pathogens. Shellfish, such as abalones, clams and oysters, is commonly served in cuisine in Taiwan. However, the risk of Shewanella infection caused by seafood consumption as well as the ecological distribution of S. haliotis in marine environment are still unclear [21,22]. To date, S. haliotis has caused several cases of soft tissue infections and bacteremia, of which most were seen in Asian countries during warm seasons [23]. Most patients lived in coastal areas and had a history of seawater contact or seafood consumption [10,12]. e causal link between direct seawater contact and soft tissue infection has been established [3,6]. e entry route and primary infectious loci for S. haliotis bacteremia, however, remains unknown. In this study, we provided results to assess the prevalence of S. haliotis in the distribution of aquaculture farm in Taiwan. We collected 78 samples; 21.8% (17/78) of isolates from shellfish was identified as S. haliotis (Tables 1 and 2). In 2007, S. haliotis was first isolated from the gut microflora of abalone in Korea [7] which might be misidentified as Shewanella algae due to biochemical test results in some Shewanella infection cases [10].
is implied S. haliotis infection might have been underestimated [10].
100% 100% In our study, 17 isolates from shellfish were collected and compared with four isolates from clinics in their morphological, physiological, and biochemical characteristics. ey shared a high percentage of identical biochemical characteristics in API 20NE and API ZYM tests (Table 3). In addition, 16S rDNA and sequencing analyses all revealed a rather close relationship with S. haliotis. As previous study pointed out that 16S sequence similarities of ≥97% should be considered as the same species [24], the 17 isolates in our study were identified as S. haliotis base on the 16S sequence. is close relationship was further confirmed by the phylogenetic clustering of isolates with the reference strain of S. haliotis DW01 (Figure. 2).
Based on the phylogenic tree, the phylogenetic relationship between different sources of S. haliotis is extremely multivariate. In the other words, there are many different types of S. haliotis present in the environment, and there might be more isolates of S. haliotis that can cause human diseases. e causal link between ingesting undercooked or even raw seafood, including shellfish, and the development of Shewanella bacteremia has been identified [25]. Byun et al. raised the concerns that this dietary habit may increase the risk to develop Shewanella bacteremia among people with underlying hepatobiliary diseases [10]. Of note, hepatobiliary diseases are common in Taiwan with around 8000 new hepatocellular carcinoma cases annually [26]. In addition, according to the results, we found that the profiles between clinical isolates (patient 1 and patient 2) and environmental isolates (shellfish 5) were similar, suggesting that patients may have acquired infection from cultivated shellfish. It will be helpful to study the environmental habitat and virulence factor of this group of isolates to understand why they are emerging in temperate climates, which may be an indication of the geographical distribution of S. haliotis in the world. Based on the analysis of 16S rRNA gene sequence for S. haliotis in this study, we speculate a high diversity of S. haliotis with various molecular types present in aquaculture environment of Taiwan.
ere are some limitations of our present study, which can be readily resolved in the future. For example, the number of S. haliotis in shellfish could be determined by a quantitative culture or by employing quantitative real-time PCR technique. In addition, whole genomic sequencing may fill in some phylogenetic gap that could be missing based on our current strategy of 16S ribosomal RNA sequencing. Furthermore, extensive and continuous surveillance would be helpful in the future to define the evolution and spread of the disease.

Conclusion
In this study, we aimed to establish the existence of S. haliotis in cultivated shellfish commonly consumed, such as abalones  (Haliotis diversicolor), clams (Meretrix lusoria), and oysters (Crassostrea gigas). Furthermore, we investigated the phylogenetic relationship between clinical and environmental isolates in Taiwan. Analysis of the data suggests the linkage between human and environmental isolates. Shewanella spp. have been recently associated with a number of new disease syndromes, including bacteremia and gastrointestinal infection. Patients with underlying diseases in the hepatobiliary tract seem to be susceptible to these marine pathogens. Our data support the hypothesis of S. haliotis as a foodborne pathogen. Further study is needed to refine our knowledge of the pathogenesis of S. haliotis infection.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
is research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.