Comparison of molecular and biochemical heterogeneity of Yersinia ruckeri strains isolated from Turkey and the USA

Aquaculture 450 (2016) 80–88

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Comparison of molecular and biochemical heterogeneity of Yersinia ruckeri strains isolated from Turkey and the USA Ilhan Altinok ⁎, Erol Capkin, Halis Boran Karadeniz Technical University, Faculty of Marine Science, Department of Fisheries Technology Engineering, 61530 Surmene, Trabzon, Turkey

a r t i c l e

i n f o

Article history: Received 7 July 2015 Received in revised form 15 July 2015 Accepted 16 July 2015 Available online 22 July 2015 Keywords: Pulsed-field gel electrophoresis Lipopolysaccharide Outer membrane protein Biotype 2

a b s t r a c t Forty isolates of Yersinia ruckeri from Italy (4), Turkey (24), the USA (10), and two type strains were compared by using biochemical test, pulsed-field gel electrophoresis (PFGE), and lipopolysaccharide (LPS) and outer membrane protein (OMP) profiles. Two biotype 1 and biotype 2 Y. ruckeri were determined based on motility and phospholipase activity. Twenty-two different pulse types were observed by cutting the DNA with SpeI restriction enzyme and running on PFGE. Four major clusters were generated using the UPGMA technique: cluster A contained Italian, Turkish and USA strains; cluster B included all the USA strains; and cluster C and cluster D contained all the Turkish strains. Based on the OMP profile, Y. ruckeri strains were divided into 4 clusters with 30 OMP types. A total of 30 different LPS types were observed. All the Italian, the USA, and most of the Turkish isolates were grouped together to form cluster A that consisted of 26 LPS types. It seems that all the four typing methods are highly discriminatory to distinguish even closely related strains. The overall similarities among the strains were 32.4 ± 6.1%, 58.7 ± 11.1%, and 79.5 ± 3.9% in LPS, PFGE, and OMP profiles, respectively. The PFGE, biochemical, OMP, and LPS profiles of none of the strains were found to be similar. Hence, each typing method showed its own discriminatory characteristics to distinguish between the individual strains. It is apparent that there is sufficient genetic diversity to justify using PFGE for analysis of horizontal transmission of Y. ruckeri in trout rearing facilities. It is possible that diverse environmental conditions resulted in a relatively high degree of genetic diversity in Y. ruckeri. Statement of relevance We believe that this manuscript entitled “Comparison of molecular and biochemical heterogeneity of Yersinia ruckeri strains isolated from Turkey and the USA” is relevant to aquaculture because Y. ruckeri is one of the most common bacterial fish diseases in salmonid culture. In the present study, Y. ruckeri strains were compared by using biochemical test, pulsed-field gel electrophoresis (PFGE), and lipopolysaccharide (LPS) and outer membrane protein (OMP) profiles to see which typing method has the most discriminatory power to distinguish strains in order to study epizootiology of yersiniosis. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Yersinia ruckeri causes enteric redmouth disease (ERM) or yersiniosis, which is sometimes a problem in salmonid aquaculture. This bacterial species most commonly infects salmonids but has also been isolated from several non-salmonid fishes, earthworms, birds, and mammals (Willumsen, 1989; Horne & Barnes 1999), including one isolation from human bile (Farmer et al., 1985). It was first isolated in Idaho, USA, from rainbow trout Oncorhynchus mykiss (Ross et al., 1966) and has subsequently been found in most areas where salmonids are cultured including Turkey (Kayis et al., 2009; Ozturk and Altinok, 2014). The phenotypic characteristics of Y. ruckeri have been widely studied to characterize the isolates. Y. ruckeri strains can be divided into two ⁎ Corresponding author. E-mail address: [email protected] (I. Altinok).

http://dx.doi.org/10.1016/j.aquaculture.2015.07.011 0044-8486/© 2015 Elsevier B.V. All rights reserved.

biotypes: biotype 1, generally motile and has phospholipase activity and isolates belonging to biotype 2 are nonmotile and do not have phospholipase activity (Davies and Frerichs, 1989). Traditionally, the diagnosis of this disease is carried out by agar cultivation and determining the phenotypic and serological properties of the pathogen (Pazos et al., 1996). Furthermore, some of the phenotypically similar bacteria such as Cytophaga and Flavobacterium could not be differentiated from each other by conventional diagnostic methods (Shewan and Mcmeekin, 1983). With the help of DNA-based approaches in recent decades, many genotypic methods have been used effectively in taxonomic and identification studies of a range of bacterial genera (Falcao et al., 2006; Kingston et al., 2009). Pulsed-field gel electrophoresis (PFGE) has only been used in epidemiological studies. Pulsed-field gel electrophoresis can be used to compare genotypic characteristics within the same species (Johnson et al., 1995). This method has high sensitivity, reproducibility, and discrimination ability compared with other molecular methods (Meays et al., 2004). In previous studies, biotype 2 Y. ruckeri

I. Altinok et al. / Aquaculture 450 (2016) 80–88

isolated from Finland and UK was characterized by using PFGE (Strom-Bestor et al., 2010; Wheeler et al., 2009). As a result, little is known about the ability of different molecular typing methods to discriminate between different Y. ruckeri strains isolated from different countries. The objectives of this study were to describe the phenotypic and genetic characterization of Y. ruckeri isolates from rainbow trout with yersiniosis in Turkey between 1994 and 2010, and to compare Turkish strains with the strains of Y. ruckeri isolated from different cases of yersiniosis in the USA and Italy. The cell envelope of Gram-negative bacteria is composed of the inner membrane and the outer membrane (OM). The OM consists of lipopolysaccharide (LPS), phospholipids, outer membrane proteins, and lipoproteins. Outer membrane proteins comprise almost 50% of the bacterial membranes of Gram-negative bacteria (Koebnik et al., 2000). Lipopolysaccharide is the major component of the outer membrane of Gram-negative bacteria, contributing greatly to the structural integrity of the bacteria, and protecting the membrane from certain kinds of chemical attack (Koebnik et al., 2000). The cell membrane is of crucial importance to Gram-negative bacteria, which upon mutation or removal leads to fatal consequences. The components of the bacterial capsules, LPS, and outer-membrane proteins are responsible for the complement resistance of bacteria (Rautemaa and Meri, 1999). The outer membrane proteins (OMPs) of Gram negative bacteria play an important role as virulence factors (Zierler and Galan, 1995) and are associated with pathogenicity and protective antigenicity (Lutwyche et al., 1995). These proteins have been used as epidemiological markers for animal pathogens (Barenkamp et al., 1981). Since LPS and outer

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membrane proteins (OMPs) have immunogenic properties (Hirst and Ellis, 1994; Kozinska and Guz, 2004), it is important to determine the virulent and avirulent strains based on OMP and LPS profiles. In the present study, we aimed to determine the LPS and outer membrane protein profiles of different strains and to compare them with PFGE and other biochemical tests. 2. Materials and methods 2.1. Bacterial isolates Forty isolates of Y. ruckeri were studied (Table 1). Strains of Y. ruckeri were isolated from rainbow trout during epizootic outbreaks of yersiniosis during the period between 1994 and 2010, from different fish farms located in 7 different regions of Turkey (Turkey is divided into 7 geographic regions). Y. ruckeri isolated from Italy and the US was kindly provided by A. Manfrin (Istituto Zooprofilattico Sperimentale delle Venezie, Adria, Italy) and K. Hayden (Auburn University, Department of Fisheries and Allied Aquaculture, Auburn, AL), respectively. All the bacteria were subcultured on trypticase soy agar (TSA) to ensure purity of the culture. 2.2. Biochemical test After purification, the following biochemical characteristics were used to identify Y. ruckeri: Gram staining, cytochrome oxidase, oxidation/fermentation, catalase, hemolysis on sheep blood agar,

Table 1 Yersinia ruckeri strains, sources, country, isolation year and pulse field type (PT), outer membrane type (OMT), lipopolysaccharide type (LPST) and biotype (BT). All Turkish strains were isolated from rainbow trout and from 7 different regions. All Turkish strains of Y. ruckeri were isolated from yersiniosis outbreaks. Isolates (strains)

Country, city and regions

Year of isolation

Provided from

PT

OMT

LPST

BT

GA97126 GA99045 GA9804 MSA3016 ATCC29473 MO688 TMP089 TSE075 RARDE071 TA0712 L14 TCO0710 691/97 NCIMB1315 M22 M45 GA97016 KM27 GA9707 SAYr14 IYr2 Yrnif ANT10 M72 M84 KA012 3019 ME128 3020 3018 ALG9488 LV01 ALG94883 ANT117 TT072 DEN12 ME17 Yr118 EL14 ISYr1

USA USA USA USA USA USA Trabzon, Black Sea Region, Tr Trabzon, Black Sea Region, Tr Rize, Black Sea Region, Tr Trabzon, Black Sea Region, Tr Zonguldak, Black Sea Region, Tr Trabzon, Black Sea Region, Tr Italy USA Mugla, Aegean Region, Tr Mugla, Aegean Region, Tr USA K. Maras, SE Anatolia Region, Tr USA Sakarya, Marmara Region, Tr Istanbul, Marmara Region, Tr Izmir, Aegean Region, Tr Antalya, Mediterranean Region, Tr Mugla, Aegean Region, Tr Mugla, Aegean Region, Tr Kayseri, Central Anatolia Region, Tr Italy Mersin, Mediterranean Region, Tr Italy Italy USA USA USA Antalya, Mediterranean Region, Tr Trabzon, Black Sea Region, Tr Denizli, Aegean Region, Tr Mersin, Mediterranean, Tr Antalya, Mediterranean, Tr Elazig, East Anatolia Region, Tr Isparta, Mediterranean Region, Tr

1997 1999 1998 2006 1975 1997 2007 2007 2007 2007 2006 2007 1998 1965 1999 2000 1997 2002 1997 2004 2008 2000 2005 2002 2005 2008 2003 2007 2003 2003 1994 2008 1994 2003 2007 2005 2007 2007 2001 2006

K. Hayden K. Hayden K. Hayden K. Hayden ATCC K. Hayden Laboratory collection Laboratory collection Laboratory collection Laboratory collection Laboratory collection Laboratory collection A. Manfrin NCIMB H. Cagirgan H. Cagirgan K. Hayden Laboratory collection K. Hayden Laboratory collection N. Turk N. Turk Laboratory collection H. Cagirgan H. Cagirgan Laboratory collection A. Manfrin S. Ozer A. Manfrin A. Manfrin K. Hayden K. Hayden K. Hayden Laboratory collection Laboratory collection Laboratory collection S. Ozer Laboratory collection E. Seker Laboratory collection

A1 A1 A1 A2 B C D E1 E1 E2 E3 E3 E3 E4 F1 F2 G H1 H2 H3 H3 H3 H3 H4 J K L1 L2 M M O1 O1 O2 P R S T U V Y

O22 O21A O24 O23A O7 O24 O8A O8A O26 O9 O8A 011 O27 O5 O25 O2 O12 O10 O21A O16A O4 O17 O16A O16A O19 O8A O14 O3 O13 O8A O23A O16A O6 O18 O30 O1 O28 O20 O29 O8A

L4 L4 L1 L8 L9 L1 L18 L5 L29 L19 L5 L6 L15 L15 L15 L24 L15 L30 L3 L20 L28 L12 L13 L20 L27 L26 L6 L17 L23 L11 L10 L2 L10 L22 L17 L16 L25 L21A L14 L7

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

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arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, citrate utilization, H2S production, esculin hydrolysis, urease, tryptophane deaminase, indole production, Voges–Proskauer, gelatinase, fermentation of glucose, mannitol, inositol, sorbitol, sucrose, xylose, rhamnose, amylose, melibiose, lactose, methyl red, and arabinose tests. The isolates were identified by standard bacterial taxonomy procedures (Ross et al., 1966; Horne & Barnes 1999). All biochemical tests were carried out at 22 °C for 48 h. The identification was confirmed by PCR as described by Altinok et al. (2001). Biotyping was done according to Davies and Frerichs (1989) with isolates designated biotype 1 (BT1) or biotype 2 (BT2), according to motility and phospholipase activity, which characterize BT1 (isolates being motile and having phospholipase activity) and BT2 (isolates being non-motile with no detectable phospholipase). Bacteria were stabinoculated with a needle into the bottom of API M medium (BioMérieux) to confirm motility. Lipase activity was examined using TSA plates supplemented with 0.1% (v/v) Tween 20 or Tween 80. After incubation at 22 °C for 48 h, the presence of opaque zones surrounding the growth was indicative of positivity (Tinsley et al., 2011). Motility was determined in wet mounts with phase-contrast microscopy and in semisolid glucose motility deep cultures (Walters and Plumb, 1978). 2.3. DNA extraction from bacterial cultures Y. ruckeri was grown in tryptic soy broth at room temperature for 24 h, transferred to 1.5 mL centrifuge tubes, and centrifuged at 9000 ×g for 5 min. The supernatant was discarded and the pellet was dissolved in 100 μL distilled water and heated at 100 °C for 10 min, followed by immediate cooling on ice for 2 min. The suspension was centrifuged at 17,000 ×g for 3 min, and the supernatant was stored at −20 °C for PCR assays. 2.4. PCR assay All of the Y. ruckeri isolates were confirmed by PCR assay as described by Altinok et al. (2001). Briefly, each 25 μL of PCR reaction mixture (prepared on ice) contained 100 ng of the sample DNA, 12.5 μL of 2× Master Mix PCR mixture (Qiagen Master PCR Kit, Qiagen Molecular Biochemicals), 100 pmol of each primer, and distilled water. Thermal cycling was performed with a Thermo Hybaid thermal cycler (Thermo Electron Inc., Waltham, MA, USA). The PCR amplification conditions consisted of initial denaturation at 95 °C for 5 min, with 30 cycles of denaturation at 95 °C for 30 s, annealing at 60 °C for 30 s, extension at 72 °C for 45 s, and final cycle of amplification at 72 °C for 10 min. 5 μL of PCR reaction mixture was subjected to electrophoresis in 1% agarose gel prepared in 0.5× Tris–Acetate–EDTA buffer; and gels were run at 90 V for 1 h. Then the gels were stained with ethidium bromide, and viewed by UV transillumination. 2.5. Analysis of chromosomal DNA restriction patterns by PFGE PCR confirmed that Y. ruckeri isolates were retrieved from stock cultures stored at −70 °C and grown on TSA agar at 22 °C for 24 h. Single colonies were spread on TSA agar with sterile cotton swabs and incubated at 22 °C for 24 h. Cells from plates were then suspended with sterile cotton swabs in 5 mL of cell suspension buffer (100 mM Tris, 100 mM EDTA, pH 8.0) and adjusted to an optical density of 0.8 at 610 nm measured using a 2500 model Shimadzu spectrophotometer (Shimadzu, Chiyoda-Ku, Tokyo, Japan). In each tube, 240 μL of cell suspension was gently mixed with 240 μL of 1.2% (w/v) Bio-Rad low melting agarose (Bio-Rad Laboratories, Hercules, CA, USA) and 40 μL of proteinase K (20 mg/mL). The cell–agarose mixture (240 μL) was immediately loaded onto a disposable plug mould (Bio-Rad Laboratories) and allowed to solidify for 10 min at 4 °C. The plugs were then transferred to 50 mL sterile tubes each containing 5 mL of lysis buffer (50 mM

Tris, pH 8.0; 1% SDS; 50 mM EDTA, pH 8.0) and 60 μL of proteinase K (20 mg/mL) and incubated for 2 h at 55 °C in a shaking incubator set at 85 rpm. The plugs were then washed three times for 30 min each, with preheated (55 °C) DNase, RNase free dH2O followed by washing three times with TE buffer (100 mM Tris, pH 8.0; 100 mM EDTA, pH 8.0). After the final wash, the plugs were stored in TE buffer at 4 °C. SpeI (Takara) was used for restriction endonuclease digestion in accordance with the manufacturer's instructions. The fragments were resolved by PFGE with PFGE-grade agarose (1%; Bio-Rad) by using a Rotaphor 6.0 PFGE system (Biometra GmbH, Goettingen, Germany). The following parameters were used: running time: 21 h; temperature: 14 °C; buffer: 0.5 × TBE buffer (Tris–borate–EDTA); voltage gradient: 180 V; initial pulse time: 0.4 s; final pulse time: 40 s; and included angle: 120°. The gels were stained with ethidium bromide (0.5 mg/mL) for 15 min, destained in distilled water, and photographed under UV light. A lambda ladder PFGE marker (Bio-Rad) was used for molecular size determination.

2.6. Lipopolysaccharide (LPS) isolation LPS was isolated from bacteria based on the method developed by Romalde et al. (1993) with slight modifications. Briefly, bacteria were incubated on TSA at 22 °C for 24 h. Cultures were suspended in phosphate-buffered saline (PBS) to an optical density (OD) of 1.0 at 520 nm measured with a Shimadzu spectrophotometer. 1.5 mL of the suspension was pipetted into 1.5 mL centrifuge tubes and centrifuged at 4000 ×g for 3 min. The pellets were suspended in 50 μL of sample buffer (125 mM Tris–HCl, 10% glycerol, 10% SDS, 130 mM dithiothreitol, 1% mercaptoethanol) and boiled for 10 min, and then the samples were centrifuged at 18,000 ×g for 10 min and the supernatants were retained. After adding 10 μL (20 mg/mL) of proteinase K, the supernatant was incubated at 60 °C for 60 min. The samples were centrifuged at 3000 ×g for 4 min to remove any debris and loaded onto 4–12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. Following electrophoresis, the gels were fixed and stained with silver stain (Tinsley et al., 2011). 2.7. Outer membrane proteins (OMPs) Bacteria were incubated on TSA at 22 °C for 24 h. The cultures were suspended in 5 mL of 50 mM Tris–HCl buffer (pH 7.4) to an OD of 1.5 at 590 nm measured with a Shimadzu spectrophotometer. The suspended bacteria were lysed by sonication over ice. After centrifugation at 5000 ×g for 10 min to remove cell debris, the OMPs were separated from the supernatant by adding 0.5 mL of 20% w/v sarkosyl and incubating at 22 °C for 30 min. Outer membrane proteins were collected by centrifugation at 50,000 ×g for 1 h at 4 °C and then resuspended in 50 μL of 50 mM Tris–HCl buffer. Single strength sample buffer (150 μL) was added and the samples were heated to 80 °C for 10 min. Outer membrane proteins were separated on 4–12% SDS-PAGE gels by loading 15 μg of protein per lane. Molecular size standards (Bio-Rad) were run concurrently. Following electrophoresis, the gels were fixed and stained with Coomassie Brilliant Blue G stain.

2.8. Analysis of PFGE, LPS and OMP patterns Band patterns were analyzed with Bionumerics GelCompar II (Applied Maths, Sint-Martens-Latem, Belgium). The similarities between band patterns of different isolates were calculated using the Dice similarity coefficient with a 2.5% tolerance and 1% optimization. The dendrogram was constructed using cluster analysis [unweighted pair-group method with arithmetic mean (UPGMA)]. Discriminatory indices (DIs) of the typing methods were calculated based on the formula developed by Hunter and Gaston (1988).

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3. Results All the isolates were confirmed to be Y. ruckeri by PCR amplification. Based on biochemical characteristics of Y. ruckeri, different strains showed variable results on arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, citrate utilization, esculin hydrolysis, urease, Voges–Proskauer, gelatinase, glucose, mannitol, sorbitol, xylose, rhamnose, sucrose methyl red, Tween 80, and arabinose tests. All of the Y. ruckeri were negative for cytochrome oxidase, melibiose, arabinose, amylose, hydrogen sulfur, inositol, indole, tryptophane deaminase, and lactose, and positive for catalase, alpha hemolysis, and ornithine positive except for GA9804 which was ornithine negative. Although most of the strains were oxidative and fermentative, only M84 strain was non-oxidative (Table 2). All the isolates produced alpha hemolysis on sheep blood agar. They were also catalase positive and cytochrome oxidase, H2S, inositol, rhamnose, saccharose, amygdalin, melibiose, and lactose negative. All isolates were motile except for Iyr1 and ISYr1 strains. All of the Y. ruckeri strains were oxidative/fermentative except for IYr2, ISYr1, ME17, and EL14. Based on motility and phospholipase activity, 22 strains including 9 Turkish strains belong to BT1 and the rest of them to BT2 (Tables 1 & 2). The samples were screened against Y. ruckeri type strain (ATCC29473) and TCO0710 strain to select an appropriate restriction enzyme for

83

PFGE assay. Ten restriction enzymes (NotI, ApaI, XbaI, SpeI, BamHI, SacI, SmaI, KpnI, HindIII, and EcoRI; New England Biolabs) were tested (data not shown). Some of the restriction enzymes yielded smear, while others yielded too many bands of insufficient size to be useful in a PFGE assay. Therefore, those enzymes were not used for PFGE analysis. SpeI was selected because of its ability to produce resolvable bands between 2 and 160 kb. SpeI enzyme generated 22 different pulse types with 11 to 29 bands, and the remaining five strains showed unique fingerprint profiles. By UPGMA technique, 4 major clusters were generated: cluster A contained Italian, Turkish and US strains; cluster B included all US strains; and cluster C & cluster D contained all Turkish strains (Fig. 1). PT10 was the most common fingerprint that included four strains. Seventeen of the Turkish strains located in cluster A were closely related to the Italian strains with 82.4 ± 4.3 similarity but they are significantly far from ALG9488, LV01, and ALG94883 US strains which formed cluster B with 92.5 ± 1.0% similarity. Six of the US isolates were grouped together in cluster A, and they were related to TMP089 Turkish strain with a similarity of 85.6 ± 2.5%. Italian strains 3018 and 3020 were similar and closely related to the other Italian strain 3019 and Me128 Turkish strain with 95.7 ± 0.1% similarity. Similarities between Turkish, Italian and US strains were found to be 58.7 ± 11.1%. Based on OMP profile, Y. ruckeri strains were divided into 4 clusters with 30 different OMP types. Cluster A consists of Italian, US, and

Table 2 Variable biochemical characteristics of Yersinia ruckeri. Biochemical tests such as ornithine decarboxylase (O), arginine dihydrolase (ARG), lysine decarboxylase (Lys), citrate (Cit), urease (U), tryptophane deaminase (TDA), Voges–Proskauer (VP), gelatinase (GEL), esculin hydrolysis (ESC), motility (MOT), fermentation of glucose (GLU), mannitol (MAN), sorbitol (SOR), sucrose (SUC), xylose (XYL), methyl red (MR), and arabinose (ARA) were used to identify Y. ruckeri. All isolates produced alpha hemolysis on sheep blood agar. They were also catalase positive and cytochrome oxidase, H2S, inositol, rhamnose, saccharose, amygdalin, melibiose and lactose negative. All of the Y. ruckeri were oxidative/fermentative except IYr2, ISYr1, ME17, and EL14. Strain

NCIMB1315 3020 3018 L14 GA97016 TA0712 GA97126 MSA3016 TT072 ATCC29473 TSE075 3019 ALG94-88 LV-01 ALG94-883 MO688 GA9707 GA99045 GA9804 RARDE071 TCO0710 TMP0809 691/97 M22 M45 M72 M84 KM27 Yr118 SAYr14 ANT117 IYr2 Yrnif ANT10 DEN12 KA012 ISYr1 ME17 EL14 ME128

Location

USA Italy Italy Zonguldak USA Trabzon USA USA Trabzon USA Trabzon Italy USA USA USA USA USA USA USA Rize Trabzon Trabzon Italy Mugla Mugla Mugla Mugla Maras Antalya Sakarya Antalya Istanbul Izmir Antalya Denizli Kayseri Isparta Mersin Elazig Mersin

Biochemical tests O

ARG

LYS

CIT

U

TDA

VP

GEL

GLU

MAN

SOR

SUC

ARA

GAS

MOT

MR

XY

ESC

TW20

TW80

+ + + + + + + + + + + + + + + − + + + + − + + + + + − + − + − − + + + − + + + −

− − − − − − − − − − + − − − − − − − − − − + − − − − − − + − + + − − − + − − + +

− − − + − + + + + + − − + + + + + + + − + + − + + − + + − + − − + + + − + + + +

+ + + − + + + − + − − + + + + + + + + − − − + + + + − + + − + + + + + + + + + +

− − − + + + − − + − − − + + + + + − + − − − − − − − − − + − + + − − + + − + + −

− − − − − − − − − − − − − − − − − − − − − − − − − − − − − − + + + + − − − − − −

+ + + + + − + + − − + + + + + + + + + + + + + + + + − + + + − + − − − − + − − +

+ − − + − − − + − − − − + + + + − + − − − − + − + + − + + + + + + + − + − − − −

+ + + + + + + + + + + + + + + + + + + + + + + + + + − + + + + + + + + − + + + +

+ + + + + + − + + + + + + + + + − − − + + + + + + + − + − + − − + + + − + + + +

+ + + + + + + + + − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − −

− − − − − + + − + − + − − − − − + + + − − − − − − − − − − − − − − − − − − + + −

− − − + − + − − + − − − − − − − − − − − + + − − − − − − + − + − − − − − − + + +

− − − + − − + − − − + − − − + + − − − − − − − − − − − − − − − − − − − − − − − −

+ + + + + − − + − + − + + + − − + + − + + − − + + + + − − − − − − − + + − + + −

+ + + + − − + + − + − + + + + + + + + − + − + + + + − + + + + + + + + + + + + +

− − − − − + + − + − + − − − − − + + + − − − − − − − − − − − − − − − − + − + + +

+ − − + − − − − − − − − − + − − − − − − − − − − − − − + − − − − − − − − − + + −

+ + + + + − − + − + − + + + − − + + − + + − − + + + + − − − − − − − + + − + + −

+ + + + + − − + − + − + + + − − + + − + + − − + + + + − − − − − − − + + − + + −

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Fig. 1. PFGE dendrogram of Yersinia ruckeri isolates based on UPGMA cluster analysis.

Turkish strains with 86.03 ± 3.75% similarity. Cluster B included M22 (Mugla, Tr), RARDE071 (Rize, Tr), and 691–97 (Italy) strains with 91.2 ± 2.1% similarity. ME17 (Mersin, Tr) and El14 (Elazıg, Tr) formed

cluster C with 91.4 ± 0.5% similarity, and TT071 (Trabzon, Tr) strain alone formed cluster D. All the 4 clusters together showed 79.5 ± 3.9% similarity with each other (Fig. 2).

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Fig. 2. Outer membrane protein dendrogram of Yersinia ruckeri isolates based on UPGMA cluster analysis.

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A total of 30 LPS types were observed in this study. All of the Italian, the US, and most of the Turkish isolates were grouped together to form cluster A that consists of 26 LPS types with 65.1 ± 10.2% similarity. The

rest of the Turkish strains such as M84, IYr2 (cluster B), RARDE071 (cluster C) and KM27 (cluster D) formed a different cluster (Fig. 3). Similarity between the Turkish, the Italian and the US strains was 32.4 ± 6.1%.

Fig. 3. Lipopolysaccharide dendrogram of Yersinia ruckeri isolates based on UPGMA cluster analysis.

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The discriminatory power of the different typing methods such as PFGE typing, OMP typing, and LPS typing was 0.997, 0.983 and 0.971, respectively. It seems that all the three typing methods have high discriminatory power to distinguish between strains. The overall similarities among strains were 32.4 ± 6.1%, 58.7 ± 11.1%, and 79.5 ± 3.9% in LPS, PFGE, and OMP profiles, respectively. Although GA97126 (USA), GA99045 (USA), and GA9804 (USA) strain have identical PFGE profiles, their biochemical tests were different. Also the PFGE profile of TSE075 and RARDE071 strain was similar; their one or more biochemical tests were different from each other. The PFGE, biochemical, OMP, and LPS profiles of none of the strains were similar. Hence, it is clear that each typing method had its own characteristics to discriminate between the strains. 4. Discussion In the same region, both biotype 1 and biotype 2, classified based on motility and phospholipase activity, can be found. This is the first report of isolation of biotype 2 Y. ruckeri from rainbow trout in Turkey. Y. ruckeri can be biotyped based on sorbitol fermentation or motility and Tween 80 (Davies and Frerichs, 1989); however, these two tests are not compatible to each other because BT2 Y. ruckeri based on motility and Tween 80 (phospholipase activity) test can be BT1 based on sorbitol fermentation or vice a versa. For example, sorbitol positive BT1 TA0712 strain is nonmotile and Tween 80 negative or sorbitol negative ALG94-88 USA strain is motile and Tween 80 positive. Furthermore, only 9 strains including 3 Turkish strains (L14, TA0712, TT072) were fermented sorbitol but 22 strains were motile and had phospholipase activity. Maybe for this reason motility and phospholipase activity are commonly used to biotype Y. ruckeri instead of sorbitol fermentation (Calvez et al., 2014; Wheeler et al., 2009). Although Altun et al. (2013) found that 15 Turkish and a strain each from Denmark and Belgium of Y. ruckeri were very similar in terms of phenotypes; we found that all Turkish strains were different from each other except for Yrnif and Ant10 strains. In a similar study, Davies and Frerichs (1989), Arias et al. (2007), Bastardo et al. (2011), Huang et al. (2013) and Calvez et al. (2014) found that Y. ruckeri strains had different biochemical characteristics. Yersiniosis is one of the most infectious diseases resulting in significant economic losses for fish farms in many countries during the summer including Turkey (Ozturk and Altinok, 2014). Hence, many studies were aimed at the epidemiological characterization of Y. ruckeri. However, only a few research methods have been employed to study the epidemiological relation between Y. ruckeri isolates from Turkey. In Turkey, a few researchers to date have used the phenotypic, serotypic, and genotypic methods such as random amplified polymorphic DNA, outer membrane protein, and ERIC-PCR to discriminate between Y. ruckeri strains (Altun et al., 2013; Onuk et al., 2011) but none of them used PFGE and LPS profiles to discriminate between different strains of Y. ruckeri. Also this research was the first attempt to compare the strains from Turkey and the USA. Although PFGE has also been applied for the molecular fingerprinting of Y. ruckeri (Lucangeli et al., 2000), in this study, we developed an entirely novel PFGE method for Rotaphor 6.0 PFGE system (Biometra) other than CHEF-DR systems by utilizing the SpeI digesting patterns of Y. ruckeri. This method is efficient enough to discriminate the wholegenome variations of the strains of Y. ruckeri. The discriminatory indices (DIs) of the biochemical test, PFGE, OMP, and LPS were 0.979, 0.997, 0.983 and 0.971, respectively. According to the discriminative powers of the typing methods, the most discriminative method for typing of Y. ruckeri isolates was PFGE (DI = 0.997) followed by OMP (DI = 0.983), biochemical test (DI = 0.97.9) and LPS (DI = 0.971) typing based on the present study. Huang et al. (2013) reported that the discriminatory power of API 20E, lipopolysaccharide and outer membrane protein ranged between 0.71 and 0.7. Furthermore, Onuk et al. (2011) and Bastardo et al. (2012) reported that the discriminatory power of

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OMP is 0.55 and 0.70, respectively, which is much lower than that of our findings. They concluded that the discriminative power of the typing methods that they used for Y. ruckeri strains was low; hence they suggested the use of a combination of the typing methods (DI = 0.94 & 90). Although they used combinations of the typing methods, the DI value was lower than that of any of the individual typing method explored in this study. The differences may arise due to the difference in strains that were studied and also due to the use of different methods for typing Y. ruckeri. Based on our results, any of the typing methods can be used to discriminate between the strains of Y. ruckeri. In our study, PFGE proved to be a useful technique to distinguish intraspecific genetic variation among Y. ruckeri isolates from Italy, Turkey and the USA fish farms. A widespread distribution of Y. ruckeri exists among trout farms (Ozturk and Altinok, 2014). Isolates from Turkey (Antalya, Elazig, Isparta, Istanbul, Kahramanmaras, Kayseri, Mersin, Mugla, Rize, Sakarya, Trabzon, and Zonguldak), Italy, and the US were analyzed, and no geographic correlation was observed based on biotype, OMP-type, and LPS-type among the isolates. However, based on PFGE, all Italian and USA isolates were clustered together to form subgroup A with type strains (ATCC29479 and NCIMB1315) with 92% similarity, suggesting a common ancestor for these isolates except for 3 USA strains (ALG9488, ALG94883, and Lv01) that formed the subgroup B. Six of the Turkish isolates (Ant117, TT072, Den12, Me17, Yr118, El14) were clubbed together in subgroup C, and IsYr1 alone formed the subgroup D suggesting no ancestry among these isolates. Italian strains 3018, 3019, and 3020 were grouped in the same biotype and PFGE cluster with 92.5 ± 1.0%; while each of them was included in different clusters based on OMP typing and LPS typing. Overall, the results of the present study indicate that biochemical test, OMP, LPS, and PFGE-based fingerprinting may be useful in studying the epidemiology of yersiniosis. However, some of them were not as discriminatory as the others. PFGE profiles of Y. ruckeri were clearly related to the geographic origin of the isolates; in that each of them was specific to the strains isolated from a certain geographic area of Turkey. OMP-typing and LPS-typing methods were not capable of discriminating between the Y. ruckeri isolated from different geographic regions of Turkey. Hence, this result suggests that PFGE might be a useful tool in tracing the epizootic of yersiniosis or for genetically fingerprinting Y. ruckeri. Epizootiology of Y. ruckeri in Turkey can be explained using the PFGE profile. In Turkey, Y. ruckeri was first isolated in 2001 in Mugla, Turkey (Cagirgan and Yurekliturk, 1991). Based on the PFGE profile, isolates from Mugla were closely related to the Aegean region, the Mediterranean region, and the Italian strains. These isolates were also related to the US isolates. It is apparent that there is sufficient genetic diversity to justify using PFGE to analyze the horizontal transmission of Y. ruckeri at trout rearing facilities. It is possible that diverse environmental sources resulted in a relatively high degree of genetic diversity in Y. ruckeri. Conflict of interest The authors declare that there is no conflict of interests regarding the publication of this paper. We alone are responsible for the content and writing of the paper. Acknowledgments This project was funded by Karadeniz Technical University, Scientific Research Fund (KTU-BAP-project no: 2007117012). References Altinok, I., Grizzle, J.M., Liu, Z.J., 2001. Detection of Yersinia ruckeri in rainbow trout blood by use of the polymerase chain reaction. Dis. Aquat. Org. 44, 29–34.

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