Marine Pollution Bulletin 76 (2013) 85–88

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Marine Pollution Bulletin

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Antibiotic resistance of Shewanella putrefaciens isolated from shellfishcollected from the West Sea in Korea

0025-326X/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.marpolbul.2013.09.025

⇑ Corresponding author. Tel.: +82 32 860 8666, mobile: +82 10 2395 7516; fax:+82 32 872 4046.

E-mail address: sjaeseon@inha.ac.kr (J.-S. So).

Chang-Ho Kang, YuJin Shin, HanEul Jeon, Jae-Ho Choi, SuYeon Jeong, Jae-Seong So ⇑Department of Biological Engineering, Inha University, 100 Inha-ro, Nam-gu, Incheon 402-751, Republic of Korea

a r t i c l e i n f o a b s t r a c t

Keywords:Shewanella putrefaciensShellfishgyrBAntibiotic resistance

In this study, we isolated and characterized Shewanella putrefaciens from shellfish harvested from theWest Sea in Korea. For the initial isolation of S. putrefaciens, LB agar plates supplemented with ferroussulfate and sodium thiosulfate were inoculated with shellfish homogenates, incubated for 24 h, and thenblack colonies were selected. Gram-negative and catalase-positive colonies were subsequently confirmedby PCR assays and API 20E kit test system. The Shewanella-specific 16S rRNA and gyrB genes were used todesign S. putrefaciens-specific PCR primers. From 6 species of shellfish tested, 24 S. putrefaciens strainswere isolated. These 24 isolates had the following profiles of resistance against 16 antibiotics: all the iso-lates were resistant to cephalothin and vancomycin and 95.8% were resistant to ampicillin. Here, wereport the isolation of S. putrefaciens from shellfish and our results point to a new possible route forexposing healthy individuals to S. putrefaciens.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

The genus Shewanella has been studied for decades and has his-torically been classified taxonomically as Achromobacter, Pseudo-monas, Alteromonas, or Shewanella (Venkateswaran et al., 1999).The potential of Shewanella spp. to mediate the co-metabolic biore-mediation of halogenated organic pollutants (Petrovskis et al.,1994) and the destructive souring of crude petroleum has alsobeen reported. Moreover, the human pathogen Shewanella putre-faciens has been associated with septicemia (Brink et al., 1995;Chen et al., 1997), and recently, it has been isolated repeatedlyfrom food and environmental samples (e.g. Gram and Huss,1996; Semple et al., 1989; Stenstrom and Molin, 1990; Vogelet al., 2005). S. putrefaciens is a gram-negative, oxidative, H2S-pro-ducing rod bacterium. It is well known for being involved in fishspoilage under various conditions (Dalgaard, 1995; Feldhusen,2000). S. putrefaciens was originally isolated in 1985 from diseasedcultured rabbit fish (Siganus rivulatus) (Salunkhe et al., 2011).Moreover, studies have reported on S. putrefaciens infections inshellfish.

Recently, bacterial populations in the marine environment havegrown because of global warming. In addition, the emergence ofpathogens with antibiotic resistance (ABR) has raised concernsover public health and the health of marine animals (Al-Bahryet al., 2009; Lima-Bittercourt et al., 2007). The results of studies

on fish and marine mammals support the hypothesis that marinespecies harbor antibiotic-resistant microbes and that these speciesmay serve as reservoirs for ABR (Miranda and Zemelman, 2001;Johson et al., 1998).

In this paper, we report the first isolation of S. putrefaciens inshellfish collected from the West Sea in Korea. Initially, we testedfor the presence of S. putrefaciens in shellfish and analyzed the anti-biotic resistance profiles of 24 isolated strains as part of a 1-yearsurvey of shellfish harvested from the West Sea in Korea. Firstputative Shewanella species, including some species that are hu-man pathogens, were isolated and the efficacy of conventional bio-chemical methods for detection and differentiation of these specieswere evaluated. In addition, we used API 20E kit test system andsequence analyses to confirm the species identity of some of theseisolates. This study was aimed to gather information about the po-tential bacterial pathogens in shellfish, and to determine the anti-microbial susceptibility patterns of S. putrfaciens isolates.

2. Materials and methods

2.1. Preparation of shellfish

Shellfish samples were collected during monthly samplingcruises conducted from May 2012 to December 2012 on the WestSea in South Korea. The accurate position in which the shellfishsamples were collected is as follows: 37�11033.1600N (latitude)and 126�37012.2400E (longitude), 37�34044.0900N (latitude) and126�26022.5800E (longitude). The samples were collected by handfishing and immediately placed in autoclaved stainless-steel

A9%

B33%

C17%

D33%

E4%

F4%

Fig. 1. Percentage frequency of isolates from shellfishes harvested from the westSea. A = Mytilus edulis, B = Cyclina sinensis, C = Crassostrea gigas, D = Ruditapesphilippinarum, E = Neptunea (Barbitonia) arthritica cumingii, and F = Dosinorbisjaponicus.

86 C.-H. Kang et al. / Marine Pollution Bulletin 76 (2013) 85–88

bottles. These were then transported to the laboratory in an icechamber to prevent spoilage. Before homogenizing the shellfishsamples, their shells were removed using autoclaved knives. Shell-fish meat (200 g) was transferred to an autoclaved beaker andmixed with 200 mL of phosphate-buffered saline (PBS, 2.5 mMKH2PO4; pH 7.2). The mixture was ground in an autoclaved stain-less-steel blender (7011S, Waring, Torrington, CT) for 90 s (30 sat low speed and 60 s at high speed), and the shellfish juice wasserially diluted 10�1 to 10�2 into PBS. Next, 100 lL of shellfishhomogenates and the serial 10-fold dilutions (in 0.1% peptonewater) of the homogenates were spread on tryptic soy agar (Difco,Detroit, MI, USA) containing 0.5% NaCl (1% total) (TSA-N) and incu-bated at 37 �C for 24 h. Colonies producing H2S were identified dueto their black color and enumerated. Three replicates of at least 3dilutions were enumerated. All plates were visually examined fortypical colony morphology and morphological characteristics asso-ciated with the growth medium.

2.2. Isolation of Shewanella spp.

Biochemical testing was performed using the API 20E system(BioMerieux, France) and the Kovac’s Reagent (Sigma–aldrich,St.Louis, MO, USA). Because TSA-N was non-selective, many marinebacteria and potential pathogens, including Shewanella spp. grewrapidly on this medium. Therefore, Shewanella spp. were only iden-tified if they were among the predominant colonies on these platesbecause biochemical testing was performed only on coloniespicked from countable plates.

2.3. Sequence analysis of the 16S rRNA gene and gyrB gene

To further identify strains, DNA from 5 mL bacterial cultureswas isolated and purified on hydroxyapatite as described by Cash-ion et al. (1997). 16S rRNA and the gyrB gene were amplified fromthe purified DNA by using the polymerase chain reaction (PCR)method. For amplification of Shewanella-specific 16S rRNA, 2 uni-versal primers, She211f (50-CGC GAT TGG ATG AAC CTA G-30) andShe1259r (50-GGC TTT GCA ACC CTC TGT A-30), were used (Todor-ova and Costello, 2006). For amplification of the amplicons of422 bp from the gyrB gene, 2 universal primers, SP-1 (50-TTC GTCGAT TAT TTG AAC AGT-30) and SP-2r (50-TTT TCC AGC AGA TAATCG TTC-30), were used (Venkateswaran et al., 1999). PCR amplifi-cation was performed under the following conditions: 30 cycles at94 �C for 1 min, 51.2 �C for 50 s, and 72 �C for 1.5 min followed by afinal extension step at 72 �C for 7 min. PCR products were analyzedby agarose gel electrophoresis, and amplification of a predicted422 bp amplicon indicated the presence of S. putrefaciens DNA ina given sample.

2.4. Antimicrobial susceptibility testing of S. putrefaciens isolates

Susceptibility to several antimicrobial agents was determinedusing a disk-diffusion technique (NCCLS, 2003). Bacterial suspen-sions were spread onto Müller-Hinton agar (Difco, Detroit, MI,USA) plates onto which antibiotic disks (Oxoid, UK) were thenplaced. The plates were incubated at 28 �C for 18–24 h under aer-obic conditions. The diameter of the zone of inhibition around eachdisk was measured and recorded. Each bacterial species was clas-sified as resistant (R), intermediately resistant (I), or susceptible(S) according to the guidelines of the Clinical and Laboratory Stan-dards Institute (NCCLS, 2003). The following antibiotics, with theirconcentrations given in parentheses, were tested ampicillin (AM;10 lg), cefotaxime (CTX; 30 lg), cefotetan (CTT; 30 lg), cephalo-thin (CF; 30 lg), chloramphenicol (C; 30 lg), ciprofloxacin (CIP;5 lg), cefepime (CEP; 30 lg), erythromycin (E; 15 lg), gentamicin(GM; 10 lg), kanamycin (K; 30 lg), nalidixic acid (NA; 30 lg),

rifampicin (RA; 5 lg), streptomycin (S; 10 lg), tetracycline (TE;30 lg), trimethoprim/sulfamethoxazole (SXT; 1.25 lg and23.75 lg, respectively), and vancomycin (VA; 30 lg).

3. Results and discussion

3.1. Isolation of S. putrefaciens

A total of 80 bacterial colonies isolated on the TSA-N plateswere subjected to biochemical identification using the API 20E sys-tem; 24 of these colonies (30% of all picked colonies) were identi-fied as presumptive S. putrefaciens isolates. These 24 isolates wererecovered from the following shellfish species: Mytilus edulis(N = 2), Cyclina sinensis (N = 8), Crassostrea gigas (N = 4), Ruditapesphilippinarum (N = 8), Neptunea (Barbitonia) arthritica cumingii(N = 1), Dosinorbis japonicas (N = 1) (Fig. 1).

3.2. Molecular phylogenetic analysis

One of the major conclusions drawn from this work is as thatthe species S. putrefaciens needed to be better defined. This conclu-sion was reached after an unexpected result in an experimentusing a molecular biological approach. A primer pair (SP-1 andSP-2r) was designed to specifically recognize the gyrB sequencein S. putrefaciens KCTC 12458. When this PCR primer pair wastested against the type strains of all 24 known shewanellae(Fig. 2), a 422 bp amplicon was detected only in S. putrefaciens.The primer set consisted of the primers She211f and She1259r thattogether amplify an approximately 1048 bp fragment of theShewanella-specific 16S rRNA gene.

3.3. Characterization of S. putrefaciens

The 24 newly isolated S. putrefaciens strains, along with the typestrains of S. putrefaciens, were characterized as follows: all isolatesconsisted of gram-negative, rod-shaped, non-spore-forming. Cellsgrown on the TSA-N medium produced circular, smooth, and con-vex colonies. S. putrefaciens was isolated by selecting black colonieson spread plated LB agar supplemented with ferrous sulfate andsodium thiosulfate after incubation for 24 h. All strains were posi-tive for oxidase and catalase activity. All isolates grew optimally at25 �C, pH 7.0 and at 0.085 M NaCl concentration.

3.4. Antibiotic susceptibility

S. putrefaciens is commonly susceptible to aminoglycosides, car-bapenems, erythromycin, and quinolones, but resistant to

Fig. 2. Identification of S. putrefaciens isolates by using polymerase chain reaction(PCR). The PCR products were analyzed by 1% agarose gel electrophoresis. Lanes: M,1 kb ladder; C, PCR product of S. putrefaciens KCTC 12458; 1–2, PCR product ofisolates from Mytilus edulis; 3–10, isolated from Cyclina sinensis; 11–14, isolatedfrom Crassostrea gigas; 15–22, isolated from Ruditapes philippinarum; 23, isolatedfrom Neptunea (Barbitonia) arthritica cumingii; 24, isolated from Dosinorbisjaponicas.

Table 1Antibiotic resistance of S. putrefaciens isolates from shellfish collected from the WestSea.

S. putrefaciens Source Resistant toa

Reference strainsKCTC 12458 KF, VA, AMP

Shellfish isolatesHS-29 Mytilus edulis KF, VA, AMPHS-30 Crassostrea gigas KF, VAHS-31 Ruditapes philippinarum KF, VA, AMPHS-34 Ruditapes philippinarum KF, VA, AMPHS-36 Crassostrea gigas KF, VA, AMPHS-37 Ruditapes philippinarum KF, VA, AMPHS-38 Mytilus edulis KF, VA, AMPHS-39 Ruditapes philippinarum KF, VA, AMPHS-42 Cyclina sinensis KF, VA, AMPHS-45 Cyclina sinensis KF, VA, AMPHS-47 Cyclina sinensis KF, VA, AMPHS-51 Dosinorbis japonicas KF, K, VA, AMPHS-54 Ruditapes philippinarum KF, VA, AMPHS-56 Cyclina sinensis KF, VA, AMPHS-57 Cyclina sinensis KF, VA, AMPHS-59 Ruditapes philippinarum KF, VA, AMPHS-60 Cyclina sinensis KF, VA, AMPHS-62 Cyclina sinensis KF, VA, AMPHS-63 Ruditapes philippinarum KF, VA, AMPHS-74 Crassostrea gigas KF, VA, AMPHS-75 Cyclina sinensis KF, VA, AMPHS-76 Neptunea (Barbitonia) arthritica cumingii KF, VA, AMPHS-78 Crassostrea gigas KF, VA, AMPHS-80 Ruditapes philippinarum KF, VA, AMP

a Abbreviated as shown in materials and methods.

C.-H. Kang et al. / Marine Pollution Bulletin 76 (2013) 85–88 87

penicillin (e.g. Chen et al., 1997; Holt et al., 1997, 2004; Kim et al.,1989; Richard et al., 1985; Vogel et al., 1997). Susceptibilities toampicillin and cephalosporins are variable, with more S. putrefac-iens isolates being susceptible to 3rd- and 4th- than to 1st- and2nd-generation cephalosporins (e.g. Holt et al., 1997, 2004; Kimet al., 1989; Richard et al., 1985). All Danish Shewanella algae iso-lates were susceptible to piperacillin, aminoglycosides, ciprofloxa-cin, erythromycin and tetracycline, whereas they displayedvariable susceptibilities to ampicillin and cephalosporins (Holtet al., 2004). Table 1 shows that the S. putrefaciens isolates were

sensitive to AM, VA, and CF and resistant to CTX, CTT, C, CIP, CEP,E, GM, K, NA, RA, S, TE, and SXT. According to the literature, mostShewanella infections are treated easily by a combination of surgi-cal therapy and drainage and antibiotics (e.g. Bulut et al., 2004;Dominguez et al., 1996; Holt et al., 1997, 2004; Paccalin et al.,2001; Yohe et al., 1997). Poor outcome is associated most oftenwith underlying disease (e.g. Brink et al., 1995; Chen et al., 1997;Kim et al., 1989). Treatment options include b-lactams (providedthat the strain is susceptible), aminoglycosides, and quinolones.

4. Conclusions

In this study, we isolated S. putrefaciens from shellfish collectedfrom the West Sea in Korea. The isolates were identified bysequencing their Shewanella-specific 16S rRNA and gyrB genes;currently, this is the most suitable method for identification ofShewanella spp., because conventional biochemical methods didnot provide satisfactory results. Although S. putrefaciens has al-ready been isolated from various sources in water environments,to the best of our knowledge, isolation of S. putrefaciens from shell-fish has not yet been reported.

In conclusion, this paper identified potentially pathogenic spe-cies of Shewanella for the first time in the West Sea, Korea.Gram-negative, oxidase and catalase positive and H2S producingrods were isolated from shellfish and identified as S. putrefaciensby using both API 20 E test system and PCR analysis. We furtheridentified hemolytic activity in some S. putrefaciens isolates, indi-cating the pathogenic potential of these bacteria. High numbersof S. putrefaciens isolates in shellfish during monthly samplingssuggest that monitoring the levels of pathogenic bacterial speciesand strains should be continued in the West Sea and should be ex-panded to other geographic locations.

Acknowledgement

This study was supported by an Inha University Research Grant.

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