MIMET-04671; No of Pages 7 Journal of Microbiological Methods xxx (2015) xxx–xxx

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Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth

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Junko Sakata a,⁎, Kentaro Kawatsu a, Tadashi Iwasaki b, Yuko Kumeda a a

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a r t i c l e

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Article history: Received 19 March 2015 Received in revised form 16 June 2015 Accepted 16 June 2015 Available online xxxx

12 13 14 15 16 27

Keywords: Epitope mapping Identification Immunochromatographic assay Vibrio parahaemolyticus

i n f o

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Division of Bacteriology, Osaka Prefectural Institute of Public Health, Osaka, Japan Division of Veterinary Science, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka, Japan

a b s t r a c t

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To rapidly and simply determine whether or not bacterial colonies growing on agar were Vibrio parahaemolyticus, we developed an immunochromatographic assay (VP-ICA) using two different monoclonal antibodies (designated mAb-VP34 and mAb-VP109) against the delta subunit of V. parahaemolyticus-F0F1 ATP synthase. The epitopes recognized by mAb-VP34 and mAb-VP109 were mapped to sequences of eight (47LLTSSFSA54) and six amino acid residues (16FDFAVD21), respectively. An amino acid sequence similarity search of the NCBI database using BLASTP showed that both epitopic amino acid sequences were present together only in V. parahaemolyticus. When 124 V. parahaemolyticus strains and 94 strains of 27 other Vibrio species or 35 non-Vibrio species were tested using the VP-ICA, the VP-ICA identified V. parahaemolyticus with 100% accuracy. The VP-ICA rapidly and simply identified the pathogen directly from a single agar colony within 30 min, indicating that VP-ICA will greatly reduce labor and time required to identify V. parahaemolyticus compared with conventional biochemical tests. © 2015 Published by Elsevier B.V.

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1. Introduction

33 34

Vibrio parahaemolyticus is a halophilic bacterium and a major food-borne pathogen that causes diarrheal illness in humans through consumption of seafood (Blake et al., 1980; Su and Liu, 2007; Yeung and Boor, 2004). Reducing the risk of V. parahaemolyticus-associated food-borne illness therefore requires closely monitoring its presence in foods. The common method for detecting V. parahaemolyticus in foods involves enrichment cultures that are first plated on selective agar such as TCBS agar or CHROMagar Vibrio agar (Gomez-Gil and Roque, 2006; Hara-Kudo et al., 2001; Kaysner and DePaola, 2004). However, when grown on selective agar, certain Vibrio species such as Vibrio vulnificus, Vibrio mimicus, Vibrio harveyi, or Vibrio campbellii produce colonies similar to those of V. parahaemolyticus (Shima et al., 2011; Su et al., 2005), requiring the utilization of time-consuming (N 2 days) and laborintensive biochemical tests for reliable identification (Kaysner and DePaola, 2004; Ministry of Health, Labor and Welfare, 2004). A rapid and simple method that can be used to discriminately detect V. parahaemolyticus colonies growing on selective agar is therefore required. Assays using monoclonal antibodies (mAbs) are powerful tools for the rapid and simple identification of bacterial species, in comparison with culture methods (Marot-Leblond et al., 2006; Pongsunk et al.,

41 42 43 44 45 46 47 48 49 50 51 52 53

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Development of a rapid and simple immunochromatographic assay to identify Vibrio parahaemolyticus

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⁎ Corresponding author at: Division of Bacteriology, Osaka Prefectural Institute of Public Health, 1-3-69 Nakamichi, Higashinari-ku, Osaka 537-0025, Japan. E-mail address: [email protected] (J. Sakata).

1999; Qian et al., 2008). However, a mAb that reacts specifically with V. parahaemolyticus is not available. We generated a specific mAb (designated mAb-VP34) against the delta subunit of the V. parahaemolyticus F0F1 ATP synthase (designated δ-subunit in the text that follows) that we used to develop a dot-blot method (VP-Dot assay) for identifying V. parahaemolyticus (Sakata et al., 2012). The δ-subunit consists of 177 amino acids, couples ATP synthesis to proton transport, and maintains homeostasis (Sakai et al., 1990; Thompson et al., 2007). Because mAb-VP34 reacted in ELISAs with all 140 V. parahaemolyticus strains tested and cross-reacted only with Vibrio natriegens among 97 strains belonging to 57 unrelated species (Sakata et al., 2012), we judged this mAb suitable for use in the VP-Dot assay. However, aside from its cross-reactivity with V. natriegens, the VP-Dot assay is too complex for routine use (Sakata et al., 2012). The purpose of this study was to develop a more rapid, simple, and specific method for identifying V. parahaemolyticus compared with the VP-Dot assay. Because immunochromatography assays are rapid, simple, and do not require specialized equipment and technical skills (Bautista et al., 2002; Kawatsu et al., 2006; Park et al., 2003), we chose to develop such an assay. To this end, we generated a mAb (designated mAb-VP109) that recognizes an epitope different from that recognized by mAb-VP34 and successfully developed a sandwich-type immunochromatographic assay (VP-ICA) for rapid and specific identification of V. parahaemolyticus colonies growing on selective agar. Further, to support the specificity of the VP-ICA, we also characterized each epitope recognized by mAb-VP34 or mAb-VP109 and compared its sequence with that of the δ-subunits among V. parahaemolyticus strains and other bacterial species.

http://dx.doi.org/10.1016/j.mimet.2015.06.009 0167-7012/© 2015 Published by Elsevier B.V.

Please cite this article as: Sakata, J., et al., Development of a rapid and simple immunochromatographic assay to identify Vibrio parahaemolyticus, J. Microbiol. Methods (2015), http://dx.doi.org/10.1016/j.mimet.2015.06.009

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J. Sakata et al. / Journal of Microbiological Methods xxx (2015) xxx–xxx

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2. Materials and methods

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2.1. Bacterial strains

Species

Number of strains

Source or strain no.a

t1:3

84 85

Vibrio parahaemolyticusb

93 45

Clinical Seafood

93

The 138 V. parahaemolyticus strains, 54 strains of 27 other Vibrio species, and 40 strains of 35 unrelated species analyzed here are listed in Table 1. The V. parahaemolyticus strains, including 65 different O- and K-antigen serotypes, were isolated between 1983 and 2012 and were confirmed using the most accurate species-specific PCR assay available that detects toxR (toxR-PCR) (Croci et al., 2007; Kim et al., 1999). Ninety-three strains isolated from clinical samples were provided by Kansai Airport Quarantine Station (Osaka, Japan). The identity of a V. natriegens strain isolated from seafood was verified using nucleotide sequence analysis of atpA (Thompson et al., 2007).

94

2.2. Nucleotide sequence analysis of the δ-subunit

95 96

101 102

The genes encoding the δ-subunit from V. parahaemolyticus and V. natriegens were PCR-amplified using primers described by Sakata et al. (2012). The amplicons were sequenced using the primers described in Table 2 using a BigDye Terminator, version 3.1, Cycle Sequencing Kit (Life Technologies, Carlsbad, CA, USA) in accordance with the manufacturer's instructions. Sequences were determined using an Applied Biosystems 3130 Genetic Analyzer and analyzed with GENETYX software (GENETYX, Tokyo, Japan).

103

2.3. Epitope mapping

104 105

NBRC15629T NBRC15630T Unknown NBRC104587T NBRC15631T Seafood Clinical NBRC102076T NBRC103148T NBRC102218T NBRC101058 Clinical Seafood NBRC103151T NBRC102217T RIMD2224001T Seafood NBRC15847T NBRC102082T NBRC15635T Clinical Seafood Clinical Seafood NBRC15636T Seafood NBRC15637T NBRC15638T NBRC15640T NBRC13287T NBRC102084T NBRC101061 NBRC15644T NBRC15645T Clinical Seafood

125

To map the epitopes recognized by mAb-VP34 and mAb-VP109, we constructed a library of fragments representing segments of the δ-subunit as well as the full-length sequence (Fig. 1A, B) using PCR-amplification of V. parahaemolyticus (V2409, serotype O3:K6) genomic DNA and then ligated the products to a pET-SUMO vector using a TA cloning kit (Champion pET-SUMO Expression System; Life Technologies). The primer pairs used are listed in Table 2. Escherichia coli BL21(DE3) was transformed with each construct, and the transformants were treated with B-PER II Bacterial Protein Extraction Reagent (Life Technologies). The reactivity of each extract with its respective mAb was tested using a dot-blot assay (Sakata et al., 2012) with each of the peroxidase-labeled mAbs. Uninduced BL21(DE3) cells transformed by each of the pET-SUMO vectors served as negative controls. To identify the amino acid sequence recognized by each mAb, overlapping peptides (Fig. 1A, B) were synthesized, spotted on a nitrocellulose membrane (Pep-SPOT; JPT Peptide Technologies GmbH, Berlin, Germany), and tested for reactivity using a chemiluminescent dot-blot assay with each mAb according to the manufacturer's instructions. Epitope sequences were used as queries for BLASTP analyses of the National Center for Biotechnology Information (NCBI) nonredundant protein database (nr).

1 1 1 1 1 1 3 1 1 1 1 1 1 1 1 1 6 1 1 1 1 1 2 2 1 1 1 1 1 1 1 1 1 1 1 10

126 127

2.4. Production of mAbs against different regions of the epitope recognized by mAb-VP34

128

A recombinant δ-subunit was expressed using the Champion pETSUMO Expression System, as described previously (Sakata et al., 2012). The His-tagged SUMO-recombinant δ-subunit that was purified from whole cell lysates under nondenaturing conditions using a HisTrap HP Kit (GE Healthcare UK, Ltd., Little Chalfont, Buckinghamshire, UK) was used as an immunogen for the production of mAbs. Three female BALB/c mice (8 weeks old) were immunized intraperitoneally with 70 μg of the immunogen emulsified in Freund's complete adjuvant (Wako Pure Chemical Industries, Ltd., Osaka, Japan). After 2, 4, 6, and 8 weeks, the mice were boosted intraperitoneally with 70 μg of the immunogen emulsified in Freund's incomplete adjuvant (Wako Pure Chemical Industries Ltd.). At 14 weeks, antibody titers were determined using an

1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

IAM12087T Clinical IAM1646 IAM12333 IAM12369T Food ATCC6633 ATCC43478 ATCC33560T NBRC12681T Clinical NBRC102416T Clinical JCM1235T IAM12348T IAM12349T IAM14238T Clinical Food Clinical JCM1665T JCM1663T NBRC14940T Food NBRC13266T NBRC15639T JCM1672T NBRC15633T Clinical IAM12542T Clinical IAM1514T ATCC31898T ATCC43176

t1:4 t1:5 t1:6 t1:7 t1:8 t1:9 t1:10 t1:11 t1:12 t1:13 t1:14 t1:15 t1:16 t1:17 t1:18 t1:19 t1:20 t1:21 t1:22 t1:23 t1:24 t1:25 t1:26 t1:27 t1:28 t1:29 t1:30 t1:31 t1:32 t1:33 t1:34 t1:35 t1:36 t1:37 t1:38 t1:39 t1:40 t1:41 t1:42 t1:43 t1:44 t1:45 t1:46 t1:47 t1:48 t1:49 t1:50 t1:51 t1:52 t1:53 t1:54 t1:55 t1:56 t1:57 t1:58 t1:59 t1:60 t1:61 t1:62 t1:63 t1:64 t1:65 t1:66 t1:67 t1:68 t1:69 t1:70 t1:71 t1:72 t1:73 t1:74 t1:75 t1:76 t1:77

110 111 112 113 114 115 116 117 118 119 120 121 122 123 124

129 130 131 132 133 134 135 136 137 138 139

F O

Vibrio gazogenes Vibrio halioticoli Vibrio harveyi

R O

Vibrio cholerae Vibrio comitans Vibrio diazotrophicus Vibrio ezurae Vibrio fischeri Vibrio fluvialis

P

Vibrio ichthyoenteri Vibrio inusitatus Vibrio mediterranei Vibrio metschinikovii

D

Vibrio nereis Vibrio orientalis Vibrio penaeicida Vibrio proteolyticus Vibrio rarus Vibrio splendidus Vibrio tubiashii Vibrio vulnificus

E

T

C

108 109

Vibrio azureus Vibrio campbellii

Vibrio natriegens

E

106 107

Other Vibrio species Vibrio aestuarianus Vibrio alginolyticus

Vibrio mimicus

R

99 100

R

97 98

O

91 92

C

89 90

N

87 88

U

86

t1:1 t1:2

Table 1 Bacterial strains used in this study.

Other species Acinetobacter calcoaceticus Aeromonas caviae Aeromonas hydrophila Aeromonas sobria Alcaligenes faecalis Bacillus cereus Bacillus subtilis Campylobacter coli Campylobacter jejuni Citrobacter freundii Citrobacter koseri Cronobacter sakazakii Edwardsiella tarda Enterobacter aerogenes Enterobacter cloacae Enterobacter intermedius Escherichia coli Grimontia hollisae Klebsiella oxytoca Klebsiella pneumoniae subsp. ozaenae Klebsiella pneumoniae subsp. pneumoniae Listeria monocytogenes Listonella anguillarum Listonella pelagia Morganella morganii Photobacterium damselae subsp. damselae Plesiomonas shigelloides Proteus vulgaris Providencia alcalifaciens Pseudomonas aeruginosa Raoultella ornithinolytica Raoultella planticola

Please cite this article as: Sakata, J., et al., Development of a rapid and simple immunochromatographic assay to identify Vibrio parahaemolyticus, J. Microbiol. Methods (2015), http://dx.doi.org/10.1016/j.mimet.2015.06.009

J. Sakata et al. / Journal of Microbiological Methods xxx (2015) xxx–xxx

consisting of the membrane (5 × 25 mm) with the absorbent pad 165 (5 × 17 mm). A 1.0-μl aliquot of the mAb solution (2 mg/ml) was 166 deposited on the membrane as a 1-mm-wide line at about the midpoint 167

140

157 158

ELISA to detect the immunogen. At 15 weeks, the 2 mice with the highest titers were injected intraperitoneally with 210 μg of the immunogen in phosphate-buffered saline. After 4 days, splenocytes of the mice were fused with P3-X63-Ag8.U1 myeloma cells by a modification of a protocol described previously by Galfre and Milstein (1981), and hybridomas were cloned as described by Kawatsu et al. (2008). Hybridoma culture supernatants were screened using a direct ELISA with cell extracts of V. parahaemolyticus strain (V2409) prepared with the B-PER II Bacterial Extraction Reagent as the coating antigen. To identify hybridomas producing the desired mAbs, hybridoma culture supernatants were screened using a sandwich-ELISA with the mAb-VP34 F(ab′)2 fragment as a capture antibody and the cell extracts of V. parahaemolyticus strain (V2409) and V. natriegens NBRC15636T as antigens. The mAb-VP34 F(ab′)2 fragment was prepared by a modification of a protocol described previously by Kawatsu et al. (1997). Production, purification, and isotyping of the mAbs were performed as described by Kawatsu et al. (2008). To determine whether or not the selected mAb recognized δ-subunit, the recombinant protein was tested using Western blotting as described previously (Sakata et al., 2012).

159

2.5. VP-ICA

160

163 164

The strips and mAb-colloidal-gold conjugates were prepared in accordance with the protocol described by Kawatsu et al. (2008). Briefly, an absorbent pad was attached to a laminated membrane (HiFlow Plus HF135; Merck Millipore, Darmstadt, Germany) so that the pad slightly overlapped the membrane, and the assembly was then cut into strips

t2:1 t2:2

Table 2 Nucleotide sequences of primers used in this study.

151 152 153 154 155 156

161 162

C

149 150

E

147 148

R

145 146

R

143 144

N C O

141 142

O

a Sources of the strains were as follows: American Type Culture Collection (ATCC), Manassas, VA, USA; Institute of Applied Microbiology Culture Collection (IAM), Tokyo, Japan; Japan Collection of Microorganisms (JCM), Saitama, Japan; NITE Biological Resource Center (NBRC), Chiba, Japan; Research Institute for Microbial Diseases (RIMD), Osaka, Japan. Superscript T denotes a type strain. b All strains were detected using PCR analysis of toxR.

T

t1:82 t1:83 t1:84 t1:85 t1:86 t1:87

R O

ATCC33628T Clinical ATCC51192T Food

P

1 1 1 1

D

Raoultella terrigena Salmonella enterica serotype Enteritidis Shewanella algae Staphylococcus aureus

Source or strain no.a

E

t1:78 t1:79 t1:80 t1:81

Number of strains

F

Table 1 (continued) Species

3

t2:3 t2:4

Primers used to synthesize the DNAs of F0F1 ATP synthase δ-subunits of V. parahaemolyticus and V. natriegens

t2:5

Primer name

t2:6 t2:7 t2:8 t2:9

VP-delta-seq-F VP-delta-seq-R

t2:10

Primer name

t2:11 t2:12 t2:13 t2:14 t2:15 t2:16 t2:17 t2:18 t2:19 t2:20 t2:21 t2:22 t2:23 t2:24

VP-ATP synthase-delta-F VP-ATP delta half-R VP-ATP delta half-F VP-ATP synthase-delta-R VP-ATP synthase-delta-F VP-ATP delta (1–46)-R VP-ATP delta (46–87)-F VP-ATP delta half-R VP-ATP delta (16–73)-F VP-ATP delta (16–73)-R VP-ATP delta (12–73)-F VP-ATP delta (16–73)-R VP-ATP delta (26–73)-F VP-ATP delta (16–73)-R

t2:25

a

Sequence (5′-3′) cttttgctgc tgaagttgcc ctacactgca attcagcttc

U

Primers used for epitope mapping of mAb-VP34 and mAb-VP109 Sequence (5′-3′)

Fragment namea

atgtctgatt tgactacaat cgc ttacgctaaa cgaccattct cag atggctgaga atggtcgttt ttaagactgc aatgcatcgc atgtctgatt tgactacaat cgc ttactcattc atttgttcgt ttttgg atggagcttc taaccagttc attctctg ttacgctaaa cgaccattct cag atgttcgact ttgcggtaga taa ttaaccgtgc gcatcaactt gt atggctaaag cagccttcga ctt ttaaccgtgc gcatcaactt gt atggaccaat ggggtcaaat gc ttaaccgtgc gcatcaactt gt

Alpha

Details are described in Fig. 1A and B.

Beta A B C D E

Fig. 1. Diagram of the strategy used to identify epitopes recognized by mAbs on the V. parahaemolyticus F 0F 1 ATP synthase delta subunit. Lines represent fragments of the δ-subunit, and the flanking numbers indicate the positions of the amino acid residues. Epitope mapping was carried out in two stages. In stage I, a library of fragments was created covering the entire δ-subunit, and in stage II, overlapping synthetic peptides covering the potential region containing each epitope were tested for reactivity with each mAb. (A) Diagram of the strategy used to identify the epitope recognized by mAb-VP34. (B) Diagram of the strategy used to identify the epitope recognized by mAb-VP109.

Please cite this article as: Sakata, J., et al., Development of a rapid and simple immunochromatographic assay to identify Vibrio parahaemolyticus, J. Microbiol. Methods (2015), http://dx.doi.org/10.1016/j.mimet.2015.06.009

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Of the 218 bacterial strains tested, 124 V. parahaemolyticus strains and 94 strains of 27 and 35 other Vibrio or unrelated species were

196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212

To determine the sensitivity of the VP-ICA, 10 V. parahaemolyticus strains (6 clinical strains and 4 seafood strains) were cultured on TCBS agar (Eiken Kizai Co., Ltd., Tokyo Japan) at 37 °C overnight. Briefly, using a 1-μl disposable inoculating loop (Nunc), up to 1 loopful of cells picked from a single colony on TCBS agar was suspended in 1 ml of sterile saline. The suspensions were diluted with sterile saline, and 50 μl of 4.0% n-octyl-β-D-glucopyranoside was added to 50 μl of each suspension. After mixing for 1 min, the mixtures were centrifuged for 5 min at 21,900 ×g at 4 °C, and the supernatants (cell extracts) were tested using the VP-ICA. The cell concentrations of the suspensions were estimated by counting colonies on agar plates.

213 214

E 1

194 195

2.7. VP-ICA detection limits

T C

187 188

E

185 186

R

183 184

R

181 182

O

179 180

C

177 178

215 216 217 218 219 220 221 222 223

2.8. Accuracy of VP-ICA in identifying colonies of V. parahaemolyticus 224 grown on TCBS 225 The 124 V. parahaemolyticus strains along with 2 V. natriegens strains, which cross-react with mAb-VP34, as well as 3 V. vulnificus and 2 V. harveyi strains, which both produce colonies similar to those produced by V. parahaemolyticus on TCBS agar, were cultured on TCBS agar at 37 °C overnight. Using a 1-μl disposable inoculating loop (Nunc), up to 1 loopful of cells was picked from a single colony on TCBS agar and suspended in 100 μl of 2.0% n-octyl-β-D-glucopyranoside. After mixing for 1 min, the suspensions were centrifuged at 21,900 ×g at 4 °C for 5 min. The resulting supernatants (cell extracts) were then tested using the VP-ICA.

226 227

3. Results

236

228 229 230 231 232 233 234 235

3.1. Comparison of amino acid sequences of δ-subunit of V. parahaemolyticus 237 strains with those of unrelated bacterial species 238

N

175 176

U

174

F

2.6. VP-ICA specificity

172 173

O

191

170 171

cultured on nonselective agar medium containing Soybean-Casein Digest Agar (SCD Agar; Wako Pure Chemical Industries Ltd.), SCD agar containing 2% NaCl, or 325 agar (15.0 g Bacto Agar, Becton, Dickinson and Company, Franklin Lakes, NJ, USA; 10.0 g of BactoPeptone, Becton, Dickinson and Company; 2.0 g of yeast extract, Becton, Dickinson and Company; 0.5 g of MgSO4·7H2O, Nacalai Tesque, LTD., Kyoto, Japan; 750 ml of seawater; 250 ml of water; pH 7.2–7.4), respectively and 37 °C or 25 °C for 18 h. Two Campylobacter species were cultured on SCD agar at 42 °C for 24 h in a microaerobic atmosphere using a humidified CO2 AnaeroPack-Microaero gas system (Mitsubishi Gas Chemicals, Tokyo, Japan). Listeria monocytogenes was cultured on SCD agar at 25 °C for 48 h. One loopful of cells picked from bacterial colonies on each agar plate using a 1-μl disposable inoculating loop (Nunc; Life Technologies) was suspended in 100 μl of 2.0% n-octyl-β-D-glucopyranoside (Dojindo, Kumamoto, Japan). After mixing for 1 min, the suspensions were centrifuged at 21,900 ×g at 4 °C for 5 min. The resulting supernatants (cell extracts) were tested using the VP-ICA.

P

189 190

of the length of the membrane to serve as the detection zone of the V. parahaemolyticus antigen. As the control zone for confirmation of test performance, 1.0 μl of a goat anti-mouse immunoglobulin (Ig) G solution (1 mg/ml; Sigma-Aldrich, St. Louis, MO, USA) was deposited on the membrane as a 1-mm-wide line 5 mm below the detection zone. After drying, the test strips were stored at 4 °C. To prepare the mAb-colloidal-gold conjugates, 1.0 ml of a mAb solution (0.2 mg/ml) was added to 10 ml of a colloidal-gold suspension (40 nm; BBI Solutions, Cardiff, UK). After incubation and blocking, the mAb-gold conjugates were suspended in 1.0 ml of 50 mM Tris–HCl–50 mM NaCl buffer, pH 8.2, containing 1% bovine serum albumin and 20% glycerol and then stored at −30 °C. The VP-ICA procedure was as follows: A 25-μl sample and 25 μl of the mAb-gold conjugate (diluted 1:10 in 100 mM Tris–HCl buffer, pH 10.0, containing 2% bovine serum albumin and 2% Triton X-100, 0.2% Lipidure®-BL405; NOF Corporation, Tokyo, Japan) were added to a well of a 96-well general assay plate (flat-bottom type; Corning Inc., Corning, NY, USA). After immediate mixing by repeated aspiration and ejection with a micropipette, the test strip was inserted into the mixture in the well. After migration of the mixture through the membrane for 15 min at room temperature, the appearance of red lines at the detection and control zones was interpreted as detection of the δ-subunit (Fig. 2).

D

168 169

J. Sakata et al. / Journal of Microbiological Methods xxx (2015) xxx–xxx

R O

4

2

Fig. 2. VP-ICA. Positive (1: V. parahaemolyticus AQ4037) and negative (2: V. natriegens NBRC15636T) VP-ICA test results.

To observe strain-to-strain variation in the amino acid sequence of the δ-subunit, the genes encoding the δ-subunit of 25 V. parahaemolyticus strains (14 clinical and 11 seafood strains) were sequenced, and their predicted amino acid sequences were compared (Fig. 3). The sequences of 23/25 strains were 100% identical, and the δ-subunit of the other two strains differed from the others by a single amino acid residue as follows: S193, serotype O2:KUT, E102D; and V146, serotype O8:K21, G23D. To determine whether the amino acid sequence was suitable for developing an immunochromatogaraphic assay specific for V. parahaemolyticus, the amino acid sequences of 18 δ-subunits of representative species were compared with that of V. parahaemolyticus (Fig. 3). The δ-subunits of two V. natriegens strains that cross-reacted with mAbVP34 were sequenced along with 16 others retrieved from the GenBank database. The amino acid sequence of V. parahaemolyticus was 96.0%

Please cite this article as: Sakata, J., et al., Development of a rapid and simple immunochromatographic assay to identify Vibrio parahaemolyticus, J. Microbiol. Methods (2015), http://dx.doi.org/10.1016/j.mimet.2015.06.009

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285

To map the epitope recognized by mAb-VP34, fragments of recombinant δ-subunit were expressed in BL21(DE3) and tested using the dot-blot assay with peroxidase-labeled mAb-VP34 (Fig. 1A). In stage I of this process, two fragments designated as alpha and beta were generated, and only the alpha fragment was detected. Three fragments (designated as A, B, and C) covering the alpha fragment were constructed, and fragments B and C reacted with mAb-VP34, indicating that the epitope resided within their region of overlap (46ELLTSSFSAEKMAEIFVAVCGEQVDAHG73). In stage II, to identify amino acid sequence of mAb-VP34 epitope, 25 overlapping peptides covering the sequence of the region (peptides 46 to 59) were prepared and tested using the dot-blot assay. Two peptides (peptides 46 and 47) reacted with mAb-VP34, indicating that the epitope is present in the N-terminal region of the sequence 47 LLTSSFSAEKMAEI 60 . We next generated sequential C-terminal truncations of the peptide (designated peptides 47-1 to 47-11). Seven peptides (47-4, 47-5, and 47-7 to 47-11) reacted with mAb-VP34. These findings identify 47LLTSSFSA54 as the epitope recognized by mAbVP34, which is present in all 25 V. parahaemolyticus and 2 V. natriegens strains sequenced here (Fig. 3). When we performed BLASTP searches of the NCBI database using the sequence of mAb-VP34 epitope LLTSSFSA, the results showed that 9 isolates of V. parahaemolyticus, 1 isolate of V. natriegens, 1 isolate of Vibrio sp., and 8 isolates of non-Vibrio isolates (Bifidobacterium spp., Brukholderia glathei, Lactobacillus curvatus, Pseudomonas monteilii, Sediminibacterium sp., and Gemmatimonadetes sp.) had the identical sequence.

286 287

3.3. Production and characterization of mAbs that recognize other δ-subunit epitopes

288

We screened 3800 hybridoma clones and observed that 124 culture supernatants reacted with the cell extracts of V. parahaemolyticus strain

268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284

289

C

E

R

R

266 267

N C O

264 265 Q9

U

262 263 Q8

290 291

3.4. Characterization of the epitope recognized by MAb-VP109

300

Fragments of recombinant δ-subunit were expressed in BL21(DE3) and tested using the dot-blot assay with peroxidase-labeled mAb-VP109 (Fig. 1B). This was performed using fragments A, B, and C described above. Only fragments A and C reacted with mAb-VP109. Subsequently, fragments D and E were generated, and only the D fragment reacted with mAb-VP109. To identify the amino acid sequence of the MAb-VP109 epitope, 15 overlapping peptides covering the sequence 4 LTTIARPYAKAAFDFAVDKGQLDQWGQML32 were generated (peptides 4 to 18), and ten peptides (peptide 7 to 16) reacted with mAb-VP109 in the dot-blot assay. These findings identify the sequence 16FDFAVD21 within the δ-subunit as the epitope recognized by mAb-VP109. The peptide sequence is present in all 25 V. parahaemolyticus but not in the 2 V. natriegens strains sequenced here (Fig. 3). The mAb-VP109 epitope sequence FDFAVD was not present in any available V. natriegens δ-subunit sequence or in the nine isolates of different species containing the sequence of mAb-VP34 epitope (LLTSSFSA).

301

3.5. Development of the VP-ICA and evaluation of its specificity

317

Using mAb-VP109 as the antibody immobilized on test strips and mAb-VP34 conjugated with colloidal gold particles as the detection antibody, we established a sandwich-type immunochromatogaraphic assay to identify V. parahaemolyticus (VP-ICA). To assess the ability of the VP-ICA to distinguish V. parahaemolyticus from other species, 124 V. parahemolyticus strains and 94 strains including 27 other Vibrio species and 35 unrelated species were analyzed using the VP-ICA. Table 3 presents data demonstrating that the VP-ICA detected all

318

D

3.2. Characterization of the epitope recognized by mAb-VP34

(V2409) in direct ELISAs, and one mAb (designated mAb-VP109) reacted with the cell extracts of V. parahaemolyticus but not with those of V. natriegens NBRC15636T in a sandwich-ELISA using the mAb-VP34 F(ab)2 fragment as the capture antibody. Western blot analysis revealed that mAb-VP109 (IgG1κ) recognized the SUMOrecombinant δ-subunit that migrated as a band of about 33 kDa (data not shown). In contrast, specific bands were not detected in extracts prepared from bacterial cells transformed with the pET-SUMO/ CAT control vector, which expresses an N-terminally tagged chloramphenicol acetyl transferase (CAT) fusion protein.

E

259

255 Q5 256

T

257 Q6 258 Q7

(NBRC15636 T ) and 96.6% (S22-88) identical to those of the two V. natriegens sequences. In contrast, the sequence of the δ-subunit was 94.4% identical to that of V. harveyi (accession number ZP_01986973) and 61.0% identical to those of Aeromonas hydrophila (accession number YP_858682) and Klebsiella pneumoniae (accession number; YP_002241284).

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Fig. 3. Alignment of partial amino acid sequences of bacterial δ-subunits. The genes encoding the δ-subunit of 25 V. parahaemolyticus and two V. natriegens strains were sequenced, and amino acid sequences of other species were retrieved from the GenBank database. aThese strains included 13 clinical and 10 seafood strains. bThe strain was isolated from seafood (serotype O2:KUT, 2009). cThe strain was isolated from patients with diarrhea (serotype O8:K21, 1995). dThe strain was isolated from seafood, and its identity was determined using sequence analysis of atpA.

Please cite this article as: Sakata, J., et al., Development of a rapid and simple immunochromatographic assay to identify Vibrio parahaemolyticus, J. Microbiol. Methods (2015), http://dx.doi.org/10.1016/j.mimet.2015.06.009

292 293 294 295 296 297 298 299

302 303 304 305 306 307 308 309 310 311 312 313 314 315 316

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Number VP-ICA-positive

t3:4 t3:5 t3:6 t3:7 t3:8

V. parahaemolyticus (clinical)a V. parahaemolyticus (seafood)a V. natriegens Other Vibrio species (n = 26)b Non-Vibrio species (n = 35)b

79 45 2 52 40

79 c 45 0 0 0

t3:9 t3:10 t3:11

a b c

All strains were detected using toxR-PCR. Details are described in Table 1. mAb-VP34 reacted weakly with strain V146, clinical, 1995, serotype O8:K21.

V. parahaemolyticus strains regardless of serotype or origin but none of the other strains.

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3.6. Limit of detection of the VP-ICA for V. parahaemolyticus TCBS colonies

329

341 342

The minimum detectable limits of the VP-ICA ranged from about 1.3 × 106 colony-forming units (CFU) to 1.9 × 106 CFU per 100 μl of cell suspensions containing 2.0% n-octyl-β-D-glucopyranoside. In contrast, the number of up to 1 loopful of V. parahaemolyticus cells picked from a single colony on TCBS agar using a 1-μl disposable inoculating loop ranged from about 2.6 × 108 to 3.8 × 108 CFU. Thus, the VP-ICA is sufficiently sensitive to identify V. parahaemolyticus directly from a single colony growing on selective agar. To determine whether the VP-ICA identified V. parahaemolyticus directly from a single colony regardless of colony morphology, 124 V. parahemolyticus strains and 7 other representative strains of Vibrio were analyzed using the VP-ICA. All V. parahaemolyticus strains were detected, regardless of colony morphology or size. In contrast, no other strains reacted.

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4. Discussion

344 345

The VP-ICA developed here represents a rapid and simple approach for identification of the pathogen. Sequence comparisons of the F0F1 ATP synthase delta subunit revealed that its amino acid sequence is highly conserved among V. parahaemolyticus strains. Because the δ-subunit maintains homeostasis, its gene is expected to be distributed and expressed among all strains of V. parahaemolyticus and will therefore be retained in the host's genome (Sakai et al., 1990; Thompson et al., 2007). For these reasons, the immunological method described here using monoclonal antibodies targeting this protein appears to have low risk of generating false-negatives caused by a mutation in the region of the epitopic polypeptide or loss of the gene. We showed that mAb-VP34 cross-reacted only with V. natriegens (NBRC15636T) among other Vibrio or unrelated strains (Sakata et al., 2012). V. natriegens is a close relative of V. parahaemolyticus (Thompson and Jean, 2006). Indeed, epitope mapping of mAb-VP34 and DNA sequence analysis confirmed that the V. natriegens gene encodes the identical sequence, LLTSSFSA, which is recognized by mAb-VP34 (Fig. 3). Therefore, the VP-Dot assay using mAb-VP34 did not distinguish between V. parahaemolyticus and V. natriegens. Because V. natriegens can be easily distinguished from V. parahaemolyticus strains by its characteristic yellow colony color caused by fermentation of sucrose on TCBS agar (Farmer et al., 2005), this disadvantage was not an obstacle to the development of the VP-Dot assay in our previous study (Sakata et al., 2012). However, we found here that V. natriegens strain S22-88, isolated from seafood, produced colonies similar to those of V. parahaemolyticus on CHROMagar Vibrio agar and X-VP agar, although the colony color of the V. natriegens strain on TCBS agar was different from that of V. parahaemolyticus (data not shown). We are unable to explain why the strain produced colonies similar to those produced by V. parahaemolyticus on this chromogenic agar, because the identities of the component of these agars are not available. Therefore, development

348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374

C

E

346 347

R

339 340

R

337 338

O

335 336

C

333 334

N

332

U

330 331

Acknowledgments

423

T

326 327

F

Number of strains tested

O

Species (origin)

375 376

R O

t3:3

of a new method for identifying V. parahaemolyticus that does not cross-react with V. natriegens was required. Further, because the VP-Dot assay is complicated, we developed a simple sandwich-type immunochromatographic assay by generating mAb-VP109 that reacted with V. parahaemolyticus but not V. natriegens using a sandwich ELISA with the mAb-VP34 F(ab)2 fragment as a capture antibody. The mAb-VP109 recognizes the sequence 16FDFAVD21, which is perfectly conserved among V. parahaemolyticus and certain bacterial spp. such as V. harveyi but not in V. natriegens (FDFAVE) (Fig. 3). Qian et al. reported that changing a single amino acid residue abolished the reactivity of a mAb (Qian et al., 2008). Therefore, the change of one amino acid residue (D21E) likely accounted for the lack of reactivity of mAb-VP109 with V. natriegens. BLASTP analyses of the NCBI database using the epitope sequences recognized by each mAb revealed that only V. parahaemolyticus harbors both epitopes. Moreover, the available sequences of the V. parahaemolyticus δ-subunit are exact matches to those of the epitopes recognized by each mAb. These results indicate the high specificity, sensitivity, and consistency of the immunochromatographic assay using a combination of mAb-VP34 and mAb-VP109 to identify of V. parahaemolyticus. The VP-ICA detected all 124 V. parahaemolyticus strains tested but reacted weakly with 1 strain (V146, clinical, 1995, serotype O8:K21) (Table 3). This may be explained by the glycine residue two positions downstream of the mAb-VP109 epitope (FDFAVD, underscored) ‘16FDFAVDKG23’ being substituted with D (Fig. 3). Further, when 14 other clinical strains representing seroptype O8:K21 (clinical, isolated from 1983 to 2006) were tested using the VP-ICA, only 1 strain (AQ3785, 1983) was detected with certainty, while the others were weakly positive (data not shown). The G23D mutation is present in all 13 strains identified from 1985 onward. Therefore, the decrease in their reactivities can be attributed to steric hindrance caused by the amino acid substitution (Nicolaisen-Strouss et al., 1987). The VP-ICA can be completed within 30 min, including the time required to prepare the cell extracts, which is much shorter than the minimum of 3 days required for the conventional biochemical tests. Further, the VP-ICA is performed more easily and quickly than its predecessor, the VP-Dot assay. Specifically, VP-ICA testing of a cell extract can be completed using a one-step incubation after the test strip is inserted into the mixture of the cell extract and the mAb-VP34-colloidal-gold conjugate, while the VP-Dot assay requires multiple incubations and washing steps after a cell extract is spotted onto the nitrocellulose membrane. Moreover, the VP-ICA provides advantages over PCR assays (Croci et al., 2007; Kim et al., 1999), as this assay does not require any specialized skills or equipment such as a thermal cycler nor any timeconsuming agarose gel electrophoresis. Thus, the VP-ICA shows potential for rapid and unambiguous identification of V. parahaemolyticus colonies on selective agar and monitoring food for contamination with this pathogen.

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Table 3 VP-ICA specificity.

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t3:1 t3:2

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377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 Q10 409 410 411 412 413 414 415 416 417 418 419 420 421 422

We are grateful to the staff of the Kansai Airport Quarantine Station 424 for providing the Vibrio strains. This work was supported by JSPS 425 KAKENHI Grant Number 24780206. 426 References

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