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Genotyping of outbreak-associated and sporadic Yersinia pseudotuberculosis strains by novel multilocus variable-number tandem repeat analysis (MLVA)
Genotyping of outbreak-associated and sporadic Yersinia pseudotuberculosis strains by novel multilocus variable-number tandem repeat analysis (MLVA) Jani Halkilahti, Kaisa Haukka, Anja Siitonen PII: DOI: Reference:
S0167-7012(13)00290-X doi: 10.1016/j.mimet.2013.09.007 MIMET 4225
To appear in:
Journal of Microbiological Methods
Received date: Revised date: Accepted date:
10 July 2013 9 September 2013 9 September 2013
Please cite this article as: Halkilahti, Jani, Haukka, Kaisa, Siitonen, Anja, Genotyping of outbreak-associated and sporadic Yersinia pseudotuberculosis strains by novel multilocus variable-number tandem repeat analysis (MLVA), Journal of Microbiological Methods (2013), doi: 10.1016/j.mimet.2013.09.007
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REVISED
Genotyping of outbreak-associated and sporadic Yersinia
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pseudotuberculosis strains by novel multilocus variable-number
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tandem repeat analysis (MLVA)
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Jani Halkilahti1#, Kaisa Haukka1,2 and Anja Siitonen1 1 Bacteriology Unit, Department of Infectious Disease Surveillance and Control, National Institute for Health and Welfare (THL), P.O. Box 30, FI-00271 Helsinki, Finland 2 Division of Microbiology, Department of Food and Environmental Sciences, P.O. Box 56, FI00014 University of Helsinki, Finland
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#National Institute for Health and Welfare Bacteriology Unit
P.O. Box 30, 00271 Helsinki, Finland Email: [email protected] Tel: +358 29 52 48172
ACCEPTED MANUSCRIPT Abstract Yersinia pseudotuberculosis human infections caused by serotype O:1 and O:3 isolates have been
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common in Finland and have also caused outbreaks. Epidemiological studies on the outbreaks have
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been limited by the lack of accurate typing methods. During the recent years, multilocus variable-
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number tandem repeat analysis (MLVA) has been successfully applied for molecular typing of several bacterial pathogens. We designed a MLVA scheme based on seven loci for Y. pseudotuberculosis. The method was able to discriminate clinical isolates of serotypes O:1 and O:3
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into several MLVA types. The MLVA profiles were based on the number of 6 to 9 bp long tandem
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repeats in each locus. The number of repeats varied from 1 to 23 depending on the locus. The loci were all located in the bacterial chromosome for stability of the markers. The MLVA method
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developed was serotype-specific and will be a new additional tool for the epidemiological
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investigations of isolates associated with disease outbreaks and for comparison of sporadic isolates. Keywords
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1. Introduction
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Genotyping, MLVA, VNTR, Yersinia pseudotuberculosis
The genus Yersinia contains three species pathogenic for humans: Yersinia pestis, Yersinia enterocolitica and Yersinia pseudotuberculosis (Brubaker, 1991). Y. pseudotuberculosis consists of 15 O-serotypes (O:1-O:15) and 10 subtypes (O:1a-O:1c, O:2a-O:2c, O:4a-O:4b, O:5a-O:5b) according to their varying lipopolysaccharide O-antigen structure (Skurnik et al., 2000). Different serotypes are common in different geographical areas. Serotypes O:1 and O:3 are most common in Europe whereas serotypes O:4 and O:5 are more prevailing in the Far East. Serotypes O:6-O:14 have not been isolated from human infections (Fukushima et al., 2001). Y. pseudotuberculosis infection in humans is associated with fever and abdominal pain due to mesenteric lymphadenitis, which are easily interpreted as symptoms of acute appendicitis 2
ACCEPTED MANUSCRIPT leading to unnecessary surgery. Post-infectious complications may include erythema nodosum and reactive arthritis (Tertti et al., 1989, 1984). In Finland, the annual incidence of yersiniosis caused by
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Y. pseudotuberculosis has varied from 28 to 252 according to the Statistical Database of the
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Infectious Diseases Register (www3.thl.fi/stat/). In addition, several outbreaks caused by strains of
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serotypes O:1 or O:3 have occurred (Jalava et al., 2004; Rimhanen-Finne and Sihvonen, 2010; Vasala et al., 2013). In the outbreaks, various vegetables have been vehicles of the pathogen but the sources of sporadic infections are largely unknown (Hallanvuo et al., 2003; Kangas et al., 2008;
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Nuorti et al., 2004; Rimhanen-Finne et al., 2009).
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Epidemiological studies have been limited by the lack of accurate molecular biology typing methods (Vincent et al., 2008; Wobeser et al., 2009) to compare Y. pseudotuberculosis
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isolates from humans and from the various environmental sources (Fukushima et al., 2001;
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Niskanen et al., 2003). The method that has commonly been applied to study the genotypes of Y. pseudotuberculosis, is pulsed-field gel electrophoresis (PFGE) (Tenover et al., 1995). PFGE has
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traditionally been the golden standard for epidemiological studies of Y. pseudotuberculosis and several other bacteria. However, PFGE is time-consuming, labour-intensive and difficult to equalize
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between laboratories. Furthermore, PFGE does not offer the desired resolution at subtyping level for epidemiological study purposes (Jalava et al., 2006; Niskanen et al., 2002; Nuorti et al., 2004). Therefore, a method that has high level of discrimination, low cost and is suited for epidemiological investigation is required. Bacterial genomes contain variable number tandem repeats (VNTR), which typically are highly polymorphic regions and consequently suited for subtyping of strains. Multilocus variable-number tandem repeat analysis (MLVA) is based on detecting size variations of several VNTR loci. These VNTR regions are utilized in MLVA, which is most commonly done using capillary electrophoresis and fluorescent dye labelled primers (Lindstedt, 2005). VNTR-based methods have already been published for Y. pestis, Y. enterocolitica, Escherichia coli, Francisella 3
ACCEPTED MANUSCRIPT tularensis, Mycobacterium tuberculosis, Haemophilus influenzae, Bacillus anthracis and Salmonella enterica (Gierczyński et al., 2007; Johansson et al., 2001; Keim et al., 2000; Klevytska
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et al., 2001; Lindstedt et al., 2003; Noller et al., 2003; Sihvonen et al., 2011; Skuce et al., 2002; van
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Belkum et al., 1997) among others. Although some studies have utilized the VNTR regions in
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characterization of Y. pseudotuberculosis strains (Platonov et al., 2013), no detailed MLVA method for epidemiological purposes has been described for the species this far. Especially the recent outbreaks in Finland have shown that with the current methods it is not possible to confirm whether
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certain strains belong to an outbreak or not. Therefore, the aim of this study was to develop a
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MLVA method suitable for more comprehensive subtyping of Y. pseudotuberculosis strains of
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2.1. Bacterial strains
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2. Materials and methods
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serotypes O:1 and O:3.
A set of 104 human isolates of Y. pseudotuberculosis serotypes O:1 (n=61) and O:3 (n=43) were selected from the 551 Y. pseudotuberculosis strains (394 O:1 and 127 O:3 strains) isolated in
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Finland in 1997 – 2012. All strains were stored at -70oC in the culture collection of the Bacteriology Unit of the National Institute for Health and Welfare (THL), Helsinki. Only one isolate per patient was permitted. The isolates studied included 17 serotype O:1 isolates associated with an outbreak of 38 laboratory-confirmed cases in Finland in 2008 (Ruska Rimhanen-Finne and Sihvonen, 2010). These 17 isolates were chosen due to a high frequency of reactive arthritis associated with this outbreak (Vasala et al., 2013). In addition, 32 randomly selected isolates (29 O:1, 3 O:3) from four small infection clusters were included (within these clusters the isolates had temporal and spatial linkage between each other). All other 55 isolates were from sporadic human infections and represented dispersed geographical occurrence and the year (between 1997 and 2012) of isolation. Three additional isolates were previously obtained from Prof. Mikael Skurnik (Department of 4
ACCEPTED MANUSCRIPT Bacteriology and Immunology, Haartman Institute, University of Helsinki) representing serotypes O:1 (Skurnik laboratory strain collection no. 2060 and 2061) and O:3 (Skurnik laboratory strain
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collection no. 2066) (Skurnik et al., 2000) and used as controls. The isolates were stored in the THL
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culture collection as FE100991, FE100992 and FE100994 bringing the total number of isolates in
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this study to 107. These three isolates and two randomly selected serotype O:1 isolates (FE73488 and FE82911) and one serotype O:3 isolate (FE81617) were used for the VNTR region sequencing.
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2.2. VNTR markers
The four complete genome sequences of Y. pseudotuberculosis available in the GenBank database
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in 2011 were used for primer design: Y. pseudotuberculosis IP32953, Y. pseudotuberculosis IP31758, Y. pseudotuberculosis PB1/+ and Y. pseudotuberculosis YPIII (GenBank accession
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numbers NC_006155.1, NC_009708.1, NC_010634.1 and NC_010465.1, respectively). The
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genomes were analysed to locate candidate VNTR markers using a tool provided by the
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Microsatellites/Tandem Repeats database (Bikandi et al., 2004). A total of 42 markers were found. The potential markers were compared with the Tandem Repeats Finder program (Benson, 1999). The tandem repeats that contained a minimum of four bases, were repeated at least four times in
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tandem and were located in the chromosome of Y. pseudotuberculosis were selected. The selected repeats were present in at least two of the four complete genome sequences examined. In all, 16 VNTR markers were obtained that did not contain any mismatches or gaps using the Tandem Repeats Finder program. The primers for the 16 VNTR markers were designed by using the tool provided by the Microsatellites/Tandem Repeats database. An in silico PCR (Bikandi et al., 2004) was performed to narrow the potential options using Y. pseudotuberculosis IP32953 and Y. pseudotuberculosis PB1/+ complete genome sequences as template. The in silico PCR amplification products were compared with each other (data not shown). Ten VNTR marker candidates that had identical flanking regions per definite locus and differed only in the number of tandem repeats were selected for further analysis. Primer3 software 5
ACCEPTED MANUSCRIPT (Rozen and Skaletsky, 2000) was used to locate suitable binding sites for 10 new primer pairs on the complete genome sequences of Y. pseudotuberculosis IP32953 and Y. pseudotuberculosis
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PB1/+. The number of loci was reduced to seven during the study. It was ensured that the tandem
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repeats would not be located in a plasmid. Theoretical annealing temperatures were calculated for
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the primers (SantaLucia, 1998). The PCR thermal cycling conditions were optimized by increasing the annealing temperature of each primer pair higher, than the calculated values to obtain PCR products with specific, single bands detectable in gel electrophoresis. For capillary electrophoresis,
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the forward primers were labelled with a fluorescent 6-FAM dye (Table 1).
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2.3. DNA purification
The 107 isolates were grown on Drigalski agar plates at 30 °C for 24 h. A loopful of biomass was
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inoculated into Penassay broth and incubated at 30 °C for another 24 h. The genomic DNA
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purification was carried out from 1 ml of the bacterial cell suspension using the GeneJET Genomic
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DNA Purification Kit (Fermentas, St. Leon-Rot, Germany) according to the manufacturer’s instructions. The precipitation phase of the DNA isolation was performed twice to ensure maximum yield of DNA. DNA quality and quantity were assessed with NanoDrop ND-1000
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Spectrophotometer (NanoDrop Technologies Inc., Wilmington, Delaware, USA) using ND-1000 software version 3.1.0. The genomic DNA had an A260/280 ratio of ≥1.8 and a concentration of >10 ng/μL.
2.4. PCR The PCR amplification was performed with a BioRad DNA Engine Dyad Peltier thermal cycler (Hercules, California, USA). The PCR reactions were carried out in the total volume of 25 μl containing 1 U of Taq DNA polymerase (Thermo Scientific, Massachusetts, USA), 1x Taq buffer supplied with the enzyme and 1.5 mM MgCl2, 5 pmol of each primer and approximately 15 ng of
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ACCEPTED MANUSCRIPT template DNA. The thermal cycling conditions were 95°C initial denaturation for 3 min, 35 cycles of 95°C denaturation for 30 s, 60°C annealing for 30 s and 72°C extension for 60 s ending with a 5
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min final extension step at 72°C. Primer YPb9 had an annealing temperature of 65°C.
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2.5. Capillary electrophoresis
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The PCR products were diluted in a ratio of 1:85 in sterile water and run in capillary electrophoresis at SeqLab of the Institute for Molecular Medicine Finland (FIMM, Helsinki) on an ABI3730xl
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DNA Analyzer (Applied Biosystems, Foster City, CA). The GeneScan™ 600 LIZ ® (Applied Biosystems) was used as an internal size standard. The electropherograms were analysed using the
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Local Southern size calling method in the Peak Scanner 1.0 software (Applied Biosystems).
2.6. DNA sequencing
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Sequencing of the seven VNTR markers was performed for six isolates (FE100991, FE100992, FE10094, FE73488, FE82911 and FE81617) on both strands using a Big Dye Terminator v1.1
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Cycle Sequencing Kit (Applied Biosystems) with an ABI 3730xl DNA Analyzer (Applied Biosystems). The sequencing was done with the MLVA primers without the fluorescent label
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(Table 1). The sequences were aligned by using BioEdit version 7.1.3.0 (Hall, 1999). The location of the fragments on the chromosome of Y. pseudotuberculosis IP32953 were determined by Basic Local Alignment Search Tool (BLAST) (Zhang et al., 2000) search (Table 1). The sizes of the flanking regions of each locus were calculated from the obtained sequencing data and from the genome sequences in the GenBank. In addition, locus YPb10 was sequenced from 20 randomly selected isolates to investigate the stability of the 3’ flanking region of the locus.
2.7. Data analysis Simpson’s diversity index (DI) (Hunter and Gaston, 1988) was calculated for each of the loci by using the PHYLOViZ software (Francisco et al., 2012) version 1.0. All the generated MLVA
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ACCEPTED MANUSCRIPT profiles were used for the calculations. The data on serotypes O:1 and O:3 were processed
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separately.
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3. Results
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Ten VNTR loci fulfilled our criteria for minimum of four bases that were repeated at least four times in a tandem repeat. Three of these VNTR markers were omitted due to their poorly repeatable PCR-results, leaving us with the seven loci suitable for genotyping of the Y. pseudotuberculosis
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isolates (Table 1). The different MLVA profiles were named as a string of seven numbers
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representing the actual number of tandem repeat units in each locus. The profiles were based on the number of 6 to 9 bp long tandem repeats in each locus, and the flanking regions of the loci ranged
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from 127 to 175 bp (Table 1).
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Comparison of the complete Y. pseudotuberculosis genomes available in the GenBank revealed that locus YPb5 had a flanking region of either 152 or 153 bp long (Table 1). Alignment of
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the 3’-flanking region of locus YPb5 of all six partially sequenced isolates revealed a one-base deletion in the same position of the region. This one base difference was observed in 50% of all
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available complete genome sequences of Y. pseudotuberculosis and also in the six partially sequenced isolates.
The fragment sizes obtained from sequencing (EMBL Nucleotide Sequence Database accession numbers HF584708 - HF584747) and from capillary electrophoresis differed from each other in all of the cases (data not shown). However, the differences between the sequencing and capillary electrophoresis results were always less than one tandem repeat in any of the loci with a mean value of 2,1 bp. Isolate FE100992 had a 7 bp deletion in the 3’-flanking sequence at locus YPb10 when compared to the 25 other YPb10 loci sequences (data not shown).
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ACCEPTED MANUSCRIPT The groups of 63 serotype O:1 isolates and 44 serotype O:3 isolates were both discriminated into 12 unique, serotype-specific MLVA profiles (Table 2). The number of repeats
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varied from 1 to 23 depending on the locus. The most frequent, MLVA profiles were obtained for
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52 serotype O:1 and 18 serotype O:3 isolates. There was no correlation between the MLVA profiles
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and the time of isolation (Table 3).
To study the stability of the VNTR markers, 17 isolates from an outbreak at NorthEastern part of Finland in 2008 were examined. These 17 outbreak isolates represented serotype
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O:1 and all of them had an identical MLVA profile 4-7-3-5-5-5-2 (Table 3). This profile occurred
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previously in 2000 and since then every year. In 2000, it was found among six of the randomly selected seven O:1 isolates. In 2001, one multilocus variant (4-7-7-3-2-2-3) was found among 11 O:1 isolates that were thought to belong to the same cluster. Similarly in 2004, one single locus
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variant (4-7-2-5-5-5-2) was found among seven O:1 isolates. Three of the O:3 isolates previously thought to represent a small outbreak in 2001 belonged to two single locus variants (5-3-9-2-1-3-2
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and 5-7-9-2-1-3-2) (Table 3).
Diversity indices were calculated for individual loci to investigate their discriminatory
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power. The DIs for serotype O:1 isolates for the loci YPb1, YPb3, YPb5, YPb7, YPb8, YPb9 and YPb10 were 0,7727; 0,8788; 0,8939; 0,7576; 0,8485; 0,8636 and 0,8788, respectively. The DIs for serotype O:3 isolates for the loci YPb1, YPb3 and YPb5 were 0,6212; 0,5758; 0,7576, respectively, and 0,0 for the loci YPb7 to YPb10.
4. Discussion We designed a MLVA method based on seven loci in the chromosome of Y. pseudotuberculosis for comparison of the clinical isolates. The method is based on capillary electrophoresis separation of PCR products labelled with a fluorescent dye. In assigning the tandem repeat numbers, we followed the style used for Salmonella Typhimurium (Larsson et al., 2009). A minimum of four bases, that 9
ACCEPTED MANUSCRIPT were repeated at least four times in a tandem repeat were selected. The prokaryotic microsatellites have been proposed to be connected to the slippage of the DNA polymerase, which is required for
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the polymorphism of the VNTR loci and is more frequent with short repeats (Noller et al., 2003).
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Therefore, short tandem repeats were chosen as VNTR markers in this study. In the previous five-
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loci MLVA method designed for Salmonella Typhimurium, one of the VNTR markers is located in a plasmid (STTR10pl) (Lindstedt et al., 2003). The locus in question had the highest percentage (48 %) of missing PCR products in comparison to all the other loci used in the method. The locus
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STTR10pl also had the highest polymorphism rate. However, plasmids are gained and lost quite
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easily by Yersinia (Eppinger et al., 2007), especially in laboratory cultures. Therefore, we did not include any VNTR markers possibly located in Y. pseudotuberculosis plasmids, although they
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might have added to the polymorphism.
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The VNTR loci showed variety of polymorphism within our set of Y. pseudotuberculosis isolates: the analysis of the 107 isolates yielded altogether 24 distinct MLVA
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patterns. The 12 MLVA patterns obtained for both serotypes O:1 and O:3 showed that the method presented here is capable of discriminating within as well as between the isolates of the two
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serotypes; namely, all of the MLVA profiles detected from 107 isolates were serotype-specific. Partial sequencing of six isolates were used to cross-check the theoretical and actual sizes of the flanking regions in each loci. The theoretical sizes of each locus, which were calculated from the complete genome sequences, matched the actual flanking region sizes obtained via sequencing. Thus, it is apparent that the flanking region sizes are conserved throughout the studied isolates. As an exception, the isolate FE100992 (serotype O:1) had a 7 bp deletion in the 3’ flanking sequence at locus YPb10 when compared to the other 25 YPb10 loci sequenced. Also, the occurrence of flanking regions of the two slightly different sizes (1 bp) at locus YPb5 was observed in our study with the partially sequenced isolates. These findings should be accounted for when
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ACCEPTED MANUSCRIPT analysing the results to avoid incorrect size calling and binning of the capillary electrophoresis results.
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In epidemiological investigation, the attention should always be focused on the
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combination of both pheno- and/or genotypic features of the causative agent as well as on the
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analytical epidemiological data. The possibility that one strain evolves into several genotypes during an outbreak can pose a problem in the use of the MLVA method (Noller et al., 2003) as in
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the use of other genotypic subtyping methods. In theory, the cellular mechanisms that increase or decrease the number of repeats by one in a given loci can also alter the tandem repeat number by
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several steps (Bichara et al., 2006). Therefore, an isolate with a change in the copy number in one locus might still be part of an outbreak. Common guidelines, similar to those described by Tenover
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et al. (1995) for PFGE, aiding the interpretation of the different MLVA profiles in the
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epidemiological investigations should be created as more isolates are analysed.
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Several investigations for epidemiology of Y. pseudotuberculosis have been conducted in Finland. They have shown that the non-outbreak isolates were genetically very closely related to the observed outbreak isolates (Hallanvuo et al., 2002). In the 2000’s, multiple Y.
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pseudotuberculosis outbreaks, mainly caused by O:1 isolates, occurred in Finland (Jalava et al., 2006, 2004; Kangas et al., 2008; Rimhanen-Finne and Sihvonen, 2010). Grated carrot was recognized as a vehicle in some of the outbreaks (Kangas et al., 2008; Rimhanen-Finne et al., 2009; Rimhanen-Finne and Sihvonen, 2010) but in some the vehicle of the pathogen remained unknown (Hallanvuo et al., 2002; Jalava et al., 2006; Rimhanen-Finne and Sihvonen, 2010). For example in 2001, there were altogether 55 cases caused by serotype O:1 isolates and 34 cases caused by serotype O:3 isolates (Jalava et al., 2004). In the data set that was randomly selected (except the 17 outbreak strains from the year 2008) for our study, 11 O:1 isolates and three O:3 isolates represented this outbreak in 2001. There were four distinct MLVA profiles amongst the outbreak isolates. One serotype O:1 MLVA profile (4-7-7-3-2-2-3) differed considerably from the profiles of 11
ACCEPTED MANUSCRIPT the other 10 serotype O:1 outbreak isolates. The other outbreaks caused by Y. pseudotuberculosis in Finland from 2003 to 2008 were all associated with serotype O:1 (Jalava et al., 2006; Kangas et al.,
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2008; R Rimhanen-Finne et al., 2009; Ruska Rimhanen-Finne and L. Sihvonen, 2010; Vasala et al.,
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2013). The isolates from 2003 to 2006 outbreaks represented two MLVA profiles that were single
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locus variants. The 17 isolates from the outbreak at the North-Eastern part of Finland in 2008 had identical MLVA profiles as expected.
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Overall, the serotype O:1 MLVA profile 4-7-3-5-5-5-2 was the most common (52 isolates) among all 63 O:1 isolates tested and it was present during several years. This dominant
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serotype O:1 MLVA profile was associated with all of the O:1 outbreaks. Out of the total 63 serotype O:1 isolates, 14 were clearly sporadic. The most common serotype O:3 MLVA profile 4-6-
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9-2-1-3-2 was observed in 18 of the 44 isolates tested. In 2001, three O:3 isolates were associated
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with several small infection clusters (Jalava et al., 2004). Two MLVA profiles were obtained from these three outbreak isolates, which differed in two loci from the most common O:3 MLVA profile.
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The remaining 41 O:3 isolates studied were considered sporadic. This finding that the vehicle in five of the outbreaks from 2003 to 2008 was carrots (Kangas et al., 2008; Rimhanen-Finne et al.,
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2009; Rimhanen-Finne and Sihvonen, 2010) could easily lead to an assumption that the reservoir for all mentioned outbreaks are identical or that the strain is widely spread in nature and that the MLVA type has remained stable in that reservoir. Further study is required to elucidate this possibility. This study focused on the MLVA typing of the serotype O:1 and O:3 isolates that have been common in human infections in Finland during the past 15 years. The MLVA method described here is an additional tool for subtyping Y. pseudotuberculosis isolates and it is in line with the recent methodological development for other enteric bacteria. Testing the method with isolates of other serotypes will yield more information on the discriminatory potential and sensitivity of the Y. pseudotuberculosis MLVA scheme. Multiplexing the PCR primers with the same annealing 12
ACCEPTED MANUSCRIPT temperature and labelled with different fluorescent dyes can be done for routine use, which would
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shorten the time necessary to perform the analysis further.
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5. Conclusions
During the recent years, MLVA has been successfully applied for molecular typing of several
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bacterial pathogens. We designed a MLVA scheme based on seven loci, which were able to discriminate clinical isolates of Y. pseudotuberculosis serotypes O:1 and O:3 into several MLVA
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types. In conclusion, the MLVA method developed by us offers a new molecular tool for epidemiological investigation of Y. pseudotuberculosis outbreaks and for comparison of the
Acknowledgements
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sporadic isolates.
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We thank Prof. Mikael Skurnik for providing three Y. pseudotuberculosis reference strains and the staff of the Bacteriology Unit, THL, for their assistance. The work was supported by a grant
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(STM/4608/2010) from the Finnish Ministry of Social Affairs and Health.
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Larsson, J.T., Torpdahl, M., Petersen, R.F., Sorensen, G., Lindstedt, B.A., Nielsen, E.M., 2009. Development of a new nomenclature for Salmonella typhimurium multilocus variable number of tandem repeats analysis (MLVA). Euro surveillance : bulletin européen sur les maladies transmissibles = European communicable disease bulletin 14, 1–5.
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Lindstedt, B.-A., 2005. Multiple-locus variable number tandem repeats analysis for genetic fingerprinting of pathogenic bacteria. Electrophoresis 26, 2567–82.
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Lindstedt, B.-A., Heir, E., Gjernes, E., Kapperud, G., 2003. DNA fingerprinting of Salmonella enterica subsp. enterica serovar typhimurium with emphasis on phage type DT104 based on variable number of tandem repeat loci. Journal of clinical microbiology 41, 1469–79.
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Niskanen, T., Fredriksson-Ahomaa, M., Korkeala, H., 2002. Yersinia pseudotuberculosis with limited genetic diversity is a common finding in tonsils of fattening pigs. Journal of food protection. Niskanen, T., Waldenström, J., Fredriksson-Ahomaa, M., Olsen, B., Korkeala, H., 2003. virF-positive Yersinia pseudotuberculosis and Yersinia enterocolitica found in migratory birds in Sweden. Applied and environmental microbiology 69, 4670–5. Noller, A.C., McEllistrem, M.C., Pacheco, A.G.F., Boxrud, D.J., Harrison, L.H., 2003. Multilocus variablenumber tandem repeat analysis distinguishes outbreak and sporadic Escherichia coli O157:H7 isolates. Journal of clinical microbiology 41, 5389–97. Nuorti, J.P., Niskanen, T., Hallanvuo, S., Mikkola, J., Kela, E., Hatakka, M., Fredriksson-Ahomaa, M., Lyytikainen, O., Siitonen, A., Korkeala, H., Ruutu, P., 2004. A widespread outbreak of Yersinia pseudotuberculosis O:3 infection from iceberg lettuce. The Journal of infectious diseases 189, 766–74. Platonov, M.E., Blouin, Y., Evseeva, V.V., Afanas’ev, M.V., Pourcel, C., Balakhonov, S.V., Vergnaud, G., Anisimov, A.P., 2013. Draft Genome Sequences of Five Yersinia pseudotuberculosis ST19 Isolates and One Isolate Variant. Genome announcements 1, e0012213.
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ACCEPTED MANUSCRIPT Rimhanen-Finne, R., Niskanen, T., Hallanvuo, S., Makary, P., Haukka, K., Pajunen, S., Siitonen, A., Ristolainen, R., Pöyry, H., Ollgren, J., Kuusi, M., 2009. Yersinia pseudotuberculosis causing a large outbreak associated with carrots in Finland, 2006. Epidemiology and infection 137, 342–7.
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Rimhanen-Finne, R., Sihvonen, L., 2010. Yersinia, in: Hulkko, T., Lyytikäinen, O., Kuusi, M., Seppälä, S., Ruutu, P. (Eds.), Infectious Diseases in Finland 1995–2009. National Institute for Health and Welfare, Helsinki, p. 22.
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Rozen, S., Skaletsky, H., 2000. Primer3 on the WWW for general users and for biologist programmers. Methods in molecular biology (Clifton, N.J.) 132, 365–86. SantaLucia, J., 1998. A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. Proceedings of the National Academy of Sciences of the United States of America.
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Sihvonen, L.M., Toivonen, S., Haukka, K., Kuusi, M., Skurnik, M., Siitonen, A., 2011. Multilocus VariableNumber Tandem-Repeat Analysis, Pulsed-Field Gel Electrophoresis, and Antimicrobial Susceptibility Patterns in Discrimination of Sporadic and Outbreak-Related Strains of Yersinia enterocolitica. BMC Microbiology 11, 42. Skuce, R. a, McCorry, T.P., McCarroll, J.F., Roring, S.M.M., Scott, A.N., Brittain, D., Hughes, S.L., Hewinson, R.G., Neill, S.D., 2002. Discrimination of Mycobacterium tuberculosis complex bacteria using novel VNTR-PCR targets. Microbiology (Reading, England) 148, 519–28.
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Skurnik, M., Peippo, A., Ervelä, E., 2000. Characterization of the O-antigen gene clusters of Yersinia pseudotuberculosis and the cryptic O-antigen gene cluster of Yersinia pestis shows that the plague bacillus is most closely related to and has evolved from Y. pseudotuberculosis serotype O:1b. Molecular microbiology 37, 316–30.
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Tenover, F.C., Arbeit, R.D., Goering, R. V, Mickelsen, P.A., Murray, B.E., Persing, D.H., Swaminathan, B., 1995. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. Journal of clinical microbiology 33, 2233–9.
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Tertti, R., Granfors, K., Lehtonen, O.P., Mertsola, J., Mäkelä, A.L., Välimäki, I., Hänninen, P., Toivanen, A., 1984. An outbreak of Yersinia pseudotuberculosis infection. The Journal of infectious diseases 149, 245–50. Tertti, R., Vuento, R., Mikkola, P., Granfors, K., Mäkelä, a L., Toivanen, A., 1989. Clinical manifestations of Yersinia pseudotuberculosis infection in children. European journal of clinical microbiology & infectious diseases : official publication of the European Society of Clinical Microbiology 8, 587–91. Van Belkum, A., Scherer, S., van Leeuwen, W., Willemse, D., van Alphen, L., Verbrugh, H., 1997. Variable number of tandem repeats in clinical strains of Haemophilus influenzae. Infection and immunity 65, 5017–27. Vasala, M., Hallanvuo, S., Ruuska, P., Suokas, R., Siitonen, A., Hakala, M., 2013. High frequency of reactive arthritis in adults after Yersinia pseudotuberculosis O:1 outbreak caused by contaminated grated carrots. Annals of the rheumatic diseases. Vincent, P., Leclercq, A., Martin, L., Duez, J.-M., Simonet, M., Carniel, E., 2008. Sudden onset of pseudotuberculosis in humans, France, 2004-05. Emerging infectious diseases 14, 1119–22.
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ACCEPTED MANUSCRIPT Wobeser, G., Campbell, G.D., Dallaire, A., McBurney, S., 2009. Tularemia, plague, yersiniosis, and Tyzzer’s disease in wild rodents and lagomorphs in Canada: a review. The Canadian veterinary journal. La revue vétérinaire canadienne 50, 1251–6.
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Zhang, Z., Schwartz, S., Wagner, L., Miller, W., 2000. A greedy algorithm for aligning DNA sequences. Journal of computational biology : a journal of computational molecular cell biology 7, 203–14.
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YPb7
YPb8
YPb9
YPb10
YPbF3
5'-6FAM-TGCCTGTTGAGGTACGGC-3'
YPbR3
5'-GCGAAAGCCACGATGATT-3'
YPbF5
5'-6FAM-GGGCCTGACACTGCGTAT-3'
YPbR5
5'-GGCGGATTACCACACCTG-3'
YPbF7
5'-6FAM-CGCTGACTGGCAGGAAAT-3'
YPbR7
5'-GCGAGCAGAGAAACTCGC-3'
YPbF8
5'-6FAM-TGGTCAGAACAAAGCGCA-3'
YPbR8
5'-GGCCATCTGGGTCAACAG-3'
YPbF9
5'-6FAM-CCCAGAGTACCACGACCG-3'
YPbR9
5'-GTCAATCAATCGACGCCC-3'
YPbF10
5'-6FAM-GGAGGGGGTGACACACTG-3'
YPbR10
5'-CGAGACGGTGAGCGAGTT-3'
ORF… 122007-122891 YPTB0107 metF 5,10methylenetetrahydrofolate reductase
from 443723 to 443953
231
(X-[152 or 153])/7
from 1462081 to 1462282
202
ORF... 443899-444408 YPTB0371 - zinc uptake transcriptional repressor (Protein is coded in complementary strand; from 444408 to 443899) ORF… 1461648-1462121 YPTB1227 - hypothetical protein
ATTTCCTG T
(X-143)/9
from 1703649 to 1703845
197
ATCAAC
(X-127)/6
from 2306115 to 2306295
181
TAACGAC
(X-175)/7
from 2538646 to 2538876
231
TGAAGTT
(X-159)/7
from 3322462 to 3322648
187
TGGTGGTT
(X-147)/8
147
TGCTTTT
(X-147)/7
152 or 153
GTTAATA
143
127
175
159
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5'-AACAACCGAACCCGATCA-3'
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Fragment position in Y. pseudotuberculosis IP32953
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YPbR1
from 122802 to 122980
Repeat number
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5'-6FAM-GCGAAGGGGTGAAGGATT-3'
Fragment size (bp)
Repeat
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YPb5
YPbF1
Length of flanking region (bp) 147
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YPb3
Primer sequence
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YPb1
Primer
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Locus
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Table 1. MLVA primers designed for Y. pseudotuberculosis, lengths of the flanking sequences and fragments, repeat sequences and the formula for calculating the repeat number from the size of the amplified fragment. Part of gene(s) amplified in Y. pseudotuberculosis IP32953
ORF… 1703459-1704904 YPTB1422 - hypothetical protein (Protein is coded in complementary strand; from 1704904 to 1703459) ORF… 2304769-2306190 YPTB1958 - GntR family transcriptional regulator ORF… 2537434-2538744 YPTB2158 - major facilitator superfamily tartrate transporter ORF... 2538824-2539822 YPTB2159 - hypothetical protein ORF... 3321396-3322481 YPTB2812 - ABC opine/polyamine transporter, ATP-binding subunit
Table 2. Diversity of the seven VNTR loci in 63 serotype O:1 isolates and 44 serotype O:3 isolates.
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4-6-9-2-1-3-2 4-6-10-2-1-3-2 4-6-12-2-1-3-2 5-6-9-2-1-3-2 5-7-9-2-1-3-2 5-6-10-2-1-3-2 12-8-9-2-1-3-2a 4-6-11-2-1-3-2 4-6-13-2-1-3-2 5-3-9-2-1-3-2 5-5-9-2-1-3-2 5-6-8-2-1-3-2 Unique: 12
18 7 4 4 3 2 1 1 1 1 1 1 Total: 44
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Total: 63
Frequency
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a
Unique: 12 Isolates received from Prof. Skurnik
52 1 1 1 1 1 1 1 1 1 1 1
MLVA Profiles O:3
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4-7-3-5-5-5-2 3-16-4-2-5-9-23 4-10-5-3-3-9-5 4-11-4-6-4-2-8a 4-3-6-3-2-2-5a 4-7-2-5-5-5-2 4-7-7-3-2-2-3 5-11-13-5-3-7-8 6-7-3-2-3-7-12 7-9-3-2-3-7-13 8-6-5-3-9-4-5 9-6-5-3-8-4-5
Frequency
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MLVA Profiles O:1
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5 4 4 4 5 5 4 4 5 5 4 4 4 5 4 4
6 10 16 7 9 7 7 7 7 7 7 7 6 7 11 7 7
-
7 6 6 6 6 6 6 6 3 7 6 6 6 6 6 6
-
5 5 4 3 3 3 7 3 3 3 3 2 5 3 13 3 3
-
9 9 10 9 9 8 10 9 9 9 11 10 9 10 10 10
-
3 3 2 5 2 5 3 5 5 2 5 5 3 5 5 5 5
-
2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
-
9 3 5 5 3 5 2 5 5 3 5 5 8 5 3 5 5
-
4 9 9 5 7 5 2 5 5 7 5 5 4 5 7 5 5
-
5 5 23 2 13 2 3 2 2 12 2 2 5 2 8 2 2
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
-
3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3
-
2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
1 6 1 5 4 1 1 1 1a 2a 1 1 4 2 1 1
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O:3 O:3 O:3 O:3 O:3 O:3 O:3 O:3 O:3 O:3 O:3 O:3 O:3 O:3 O:3 O:3
-
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8 4 3 4 7 4 4 4 4 6 4 4 9 4 5 4 4
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O:1 O:1 O:1 O:1 O:1 O:1 O:1 O:1 O:1 O:1 O:1 O:1 O:1 O:1 O:1 O:1 O:1
no. MLVA profiles 1 1 1 6 1 10a 1a 1 7b 1 6c 1c 1 4d 1 17e 1
CR
MLVA profile
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1997 1998 1998 1999 2000 2000 2000 2000 2001 2001 2001 2001 2001 2001 2002 2006
ST
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Isolation year 1999 1999 1999 2000 2000 2001 2001 2002 2003 2003 2004 2004 2005 2006 2006 2008 2009
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Table 3. The year of isolation for the studied 104 clinical Y. pseudotuberculosis strains, their serotype, MLVA profile and the number of strains with the certain MLVA profile. Isolates FE100991, FE100992 and FE100994 were not included.
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MA N
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CR
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2007 O:3 4 6 10 - 2 1 - 3 2 1 2008 O:3 5 5 9 - 2 1 - 3 2 1 2008 O:3 4 6 9 - 2 1 - 3 2 1 2008 O:3 4 6 10 - 2 1 - 3 2 1 2012 O:3 4 6 12 - 2 1 - 3 2 4 2012 O:3 4 6 13 - 2 1 - 3 2 1 2012 O:3 4 6 9 - 2 1 - 3 2 1 a 11 serotype O:1 isolates and 3 serotype O:3 isolates belonging to the infection clusters in 2001 (Jalava et al., 2004) b 7 serotype O:1 isolates belonging to the infection cluster in 2003 (Jalava et al., 2006) c 7 serotype O:1 isolates belonging to the infection cluster in 2004 (Kangas et al., 2008) d 4 serotype O:1 isolates belonging to the infection cluster in 2006 Rimhanen-Finne and Sihvonen, 2010) e 17 serotype O:1 isolates belonging to the infection cluster in 2008 (Ruska Rimhanen-Finne and L. Sihvonen, 2010; Vasala et al., 2013)
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Yersinia pseudotuberculosis serotypes O:1 and O:3 are most common in Europe We developed a new genotyping method (MLVA) for Yersinia pseudotuberculosis The MLVA scheme discriminated serotypes O:1 and O:3 into several MLVA types All of the obtained MLVA profiles from 107 isolates were serotype-specific
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Highlights
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S0167-7012(13)00290-X doi: 10.1016/j.mimet.2013.09.007 MIMET 4225
To appear in:
Journal of Microbiological Methods
Received date: Revised date: Accepted date:
10 July 2013 9 September 2013 9 September 2013
Please cite this article as: Halkilahti, Jani, Haukka, Kaisa, Siitonen, Anja, Genotyping of outbreak-associated and sporadic Yersinia pseudotuberculosis strains by novel multilocus variable-number tandem repeat analysis (MLVA), Journal of Microbiological Methods (2013), doi: 10.1016/j.mimet.2013.09.007
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REVISED
Genotyping of outbreak-associated and sporadic Yersinia
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pseudotuberculosis strains by novel multilocus variable-number
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tandem repeat analysis (MLVA)
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Jani Halkilahti1#, Kaisa Haukka1,2 and Anja Siitonen1 1 Bacteriology Unit, Department of Infectious Disease Surveillance and Control, National Institute for Health and Welfare (THL), P.O. Box 30, FI-00271 Helsinki, Finland 2 Division of Microbiology, Department of Food and Environmental Sciences, P.O. Box 56, FI00014 University of Helsinki, Finland
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#National Institute for Health and Welfare Bacteriology Unit
P.O. Box 30, 00271 Helsinki, Finland Email: [email protected] Tel: +358 29 52 48172
ACCEPTED MANUSCRIPT Abstract Yersinia pseudotuberculosis human infections caused by serotype O:1 and O:3 isolates have been
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common in Finland and have also caused outbreaks. Epidemiological studies on the outbreaks have
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been limited by the lack of accurate typing methods. During the recent years, multilocus variable-
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number tandem repeat analysis (MLVA) has been successfully applied for molecular typing of several bacterial pathogens. We designed a MLVA scheme based on seven loci for Y. pseudotuberculosis. The method was able to discriminate clinical isolates of serotypes O:1 and O:3
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into several MLVA types. The MLVA profiles were based on the number of 6 to 9 bp long tandem
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repeats in each locus. The number of repeats varied from 1 to 23 depending on the locus. The loci were all located in the bacterial chromosome for stability of the markers. The MLVA method
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developed was serotype-specific and will be a new additional tool for the epidemiological
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investigations of isolates associated with disease outbreaks and for comparison of sporadic isolates. Keywords
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1. Introduction
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Genotyping, MLVA, VNTR, Yersinia pseudotuberculosis
The genus Yersinia contains three species pathogenic for humans: Yersinia pestis, Yersinia enterocolitica and Yersinia pseudotuberculosis (Brubaker, 1991). Y. pseudotuberculosis consists of 15 O-serotypes (O:1-O:15) and 10 subtypes (O:1a-O:1c, O:2a-O:2c, O:4a-O:4b, O:5a-O:5b) according to their varying lipopolysaccharide O-antigen structure (Skurnik et al., 2000). Different serotypes are common in different geographical areas. Serotypes O:1 and O:3 are most common in Europe whereas serotypes O:4 and O:5 are more prevailing in the Far East. Serotypes O:6-O:14 have not been isolated from human infections (Fukushima et al., 2001). Y. pseudotuberculosis infection in humans is associated with fever and abdominal pain due to mesenteric lymphadenitis, which are easily interpreted as symptoms of acute appendicitis 2
ACCEPTED MANUSCRIPT leading to unnecessary surgery. Post-infectious complications may include erythema nodosum and reactive arthritis (Tertti et al., 1989, 1984). In Finland, the annual incidence of yersiniosis caused by
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Y. pseudotuberculosis has varied from 28 to 252 according to the Statistical Database of the
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Infectious Diseases Register (www3.thl.fi/stat/). In addition, several outbreaks caused by strains of
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serotypes O:1 or O:3 have occurred (Jalava et al., 2004; Rimhanen-Finne and Sihvonen, 2010; Vasala et al., 2013). In the outbreaks, various vegetables have been vehicles of the pathogen but the sources of sporadic infections are largely unknown (Hallanvuo et al., 2003; Kangas et al., 2008;
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Nuorti et al., 2004; Rimhanen-Finne et al., 2009).
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Epidemiological studies have been limited by the lack of accurate molecular biology typing methods (Vincent et al., 2008; Wobeser et al., 2009) to compare Y. pseudotuberculosis
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isolates from humans and from the various environmental sources (Fukushima et al., 2001;
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Niskanen et al., 2003). The method that has commonly been applied to study the genotypes of Y. pseudotuberculosis, is pulsed-field gel electrophoresis (PFGE) (Tenover et al., 1995). PFGE has
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traditionally been the golden standard for epidemiological studies of Y. pseudotuberculosis and several other bacteria. However, PFGE is time-consuming, labour-intensive and difficult to equalize
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between laboratories. Furthermore, PFGE does not offer the desired resolution at subtyping level for epidemiological study purposes (Jalava et al., 2006; Niskanen et al., 2002; Nuorti et al., 2004). Therefore, a method that has high level of discrimination, low cost and is suited for epidemiological investigation is required. Bacterial genomes contain variable number tandem repeats (VNTR), which typically are highly polymorphic regions and consequently suited for subtyping of strains. Multilocus variable-number tandem repeat analysis (MLVA) is based on detecting size variations of several VNTR loci. These VNTR regions are utilized in MLVA, which is most commonly done using capillary electrophoresis and fluorescent dye labelled primers (Lindstedt, 2005). VNTR-based methods have already been published for Y. pestis, Y. enterocolitica, Escherichia coli, Francisella 3
ACCEPTED MANUSCRIPT tularensis, Mycobacterium tuberculosis, Haemophilus influenzae, Bacillus anthracis and Salmonella enterica (Gierczyński et al., 2007; Johansson et al., 2001; Keim et al., 2000; Klevytska
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et al., 2001; Lindstedt et al., 2003; Noller et al., 2003; Sihvonen et al., 2011; Skuce et al., 2002; van
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Belkum et al., 1997) among others. Although some studies have utilized the VNTR regions in
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characterization of Y. pseudotuberculosis strains (Platonov et al., 2013), no detailed MLVA method for epidemiological purposes has been described for the species this far. Especially the recent outbreaks in Finland have shown that with the current methods it is not possible to confirm whether
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certain strains belong to an outbreak or not. Therefore, the aim of this study was to develop a
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MLVA method suitable for more comprehensive subtyping of Y. pseudotuberculosis strains of
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2.1. Bacterial strains
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2. Materials and methods
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serotypes O:1 and O:3.
A set of 104 human isolates of Y. pseudotuberculosis serotypes O:1 (n=61) and O:3 (n=43) were selected from the 551 Y. pseudotuberculosis strains (394 O:1 and 127 O:3 strains) isolated in
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Finland in 1997 – 2012. All strains were stored at -70oC in the culture collection of the Bacteriology Unit of the National Institute for Health and Welfare (THL), Helsinki. Only one isolate per patient was permitted. The isolates studied included 17 serotype O:1 isolates associated with an outbreak of 38 laboratory-confirmed cases in Finland in 2008 (Ruska Rimhanen-Finne and Sihvonen, 2010). These 17 isolates were chosen due to a high frequency of reactive arthritis associated with this outbreak (Vasala et al., 2013). In addition, 32 randomly selected isolates (29 O:1, 3 O:3) from four small infection clusters were included (within these clusters the isolates had temporal and spatial linkage between each other). All other 55 isolates were from sporadic human infections and represented dispersed geographical occurrence and the year (between 1997 and 2012) of isolation. Three additional isolates were previously obtained from Prof. Mikael Skurnik (Department of 4
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collection no. 2066) (Skurnik et al., 2000) and used as controls. The isolates were stored in the THL
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culture collection as FE100991, FE100992 and FE100994 bringing the total number of isolates in
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this study to 107. These three isolates and two randomly selected serotype O:1 isolates (FE73488 and FE82911) and one serotype O:3 isolate (FE81617) were used for the VNTR region sequencing.
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2.2. VNTR markers
The four complete genome sequences of Y. pseudotuberculosis available in the GenBank database
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in 2011 were used for primer design: Y. pseudotuberculosis IP32953, Y. pseudotuberculosis IP31758, Y. pseudotuberculosis PB1/+ and Y. pseudotuberculosis YPIII (GenBank accession
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numbers NC_006155.1, NC_009708.1, NC_010634.1 and NC_010465.1, respectively). The
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genomes were analysed to locate candidate VNTR markers using a tool provided by the
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Microsatellites/Tandem Repeats database (Bikandi et al., 2004). A total of 42 markers were found. The potential markers were compared with the Tandem Repeats Finder program (Benson, 1999). The tandem repeats that contained a minimum of four bases, were repeated at least four times in
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tandem and were located in the chromosome of Y. pseudotuberculosis were selected. The selected repeats were present in at least two of the four complete genome sequences examined. In all, 16 VNTR markers were obtained that did not contain any mismatches or gaps using the Tandem Repeats Finder program. The primers for the 16 VNTR markers were designed by using the tool provided by the Microsatellites/Tandem Repeats database. An in silico PCR (Bikandi et al., 2004) was performed to narrow the potential options using Y. pseudotuberculosis IP32953 and Y. pseudotuberculosis PB1/+ complete genome sequences as template. The in silico PCR amplification products were compared with each other (data not shown). Ten VNTR marker candidates that had identical flanking regions per definite locus and differed only in the number of tandem repeats were selected for further analysis. Primer3 software 5
ACCEPTED MANUSCRIPT (Rozen and Skaletsky, 2000) was used to locate suitable binding sites for 10 new primer pairs on the complete genome sequences of Y. pseudotuberculosis IP32953 and Y. pseudotuberculosis
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PB1/+. The number of loci was reduced to seven during the study. It was ensured that the tandem
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repeats would not be located in a plasmid. Theoretical annealing temperatures were calculated for
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the primers (SantaLucia, 1998). The PCR thermal cycling conditions were optimized by increasing the annealing temperature of each primer pair higher, than the calculated values to obtain PCR products with specific, single bands detectable in gel electrophoresis. For capillary electrophoresis,
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the forward primers were labelled with a fluorescent 6-FAM dye (Table 1).
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2.3. DNA purification
The 107 isolates were grown on Drigalski agar plates at 30 °C for 24 h. A loopful of biomass was
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inoculated into Penassay broth and incubated at 30 °C for another 24 h. The genomic DNA
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purification was carried out from 1 ml of the bacterial cell suspension using the GeneJET Genomic
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DNA Purification Kit (Fermentas, St. Leon-Rot, Germany) according to the manufacturer’s instructions. The precipitation phase of the DNA isolation was performed twice to ensure maximum yield of DNA. DNA quality and quantity were assessed with NanoDrop ND-1000
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Spectrophotometer (NanoDrop Technologies Inc., Wilmington, Delaware, USA) using ND-1000 software version 3.1.0. The genomic DNA had an A260/280 ratio of ≥1.8 and a concentration of >10 ng/μL.
2.4. PCR The PCR amplification was performed with a BioRad DNA Engine Dyad Peltier thermal cycler (Hercules, California, USA). The PCR reactions were carried out in the total volume of 25 μl containing 1 U of Taq DNA polymerase (Thermo Scientific, Massachusetts, USA), 1x Taq buffer supplied with the enzyme and 1.5 mM MgCl2, 5 pmol of each primer and approximately 15 ng of
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min final extension step at 72°C. Primer YPb9 had an annealing temperature of 65°C.
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2.5. Capillary electrophoresis
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The PCR products were diluted in a ratio of 1:85 in sterile water and run in capillary electrophoresis at SeqLab of the Institute for Molecular Medicine Finland (FIMM, Helsinki) on an ABI3730xl
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DNA Analyzer (Applied Biosystems, Foster City, CA). The GeneScan™ 600 LIZ ® (Applied Biosystems) was used as an internal size standard. The electropherograms were analysed using the
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Local Southern size calling method in the Peak Scanner 1.0 software (Applied Biosystems).
2.6. DNA sequencing
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Sequencing of the seven VNTR markers was performed for six isolates (FE100991, FE100992, FE10094, FE73488, FE82911 and FE81617) on both strands using a Big Dye Terminator v1.1
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Cycle Sequencing Kit (Applied Biosystems) with an ABI 3730xl DNA Analyzer (Applied Biosystems). The sequencing was done with the MLVA primers without the fluorescent label
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(Table 1). The sequences were aligned by using BioEdit version 7.1.3.0 (Hall, 1999). The location of the fragments on the chromosome of Y. pseudotuberculosis IP32953 were determined by Basic Local Alignment Search Tool (BLAST) (Zhang et al., 2000) search (Table 1). The sizes of the flanking regions of each locus were calculated from the obtained sequencing data and from the genome sequences in the GenBank. In addition, locus YPb10 was sequenced from 20 randomly selected isolates to investigate the stability of the 3’ flanking region of the locus.
2.7. Data analysis Simpson’s diversity index (DI) (Hunter and Gaston, 1988) was calculated for each of the loci by using the PHYLOViZ software (Francisco et al., 2012) version 1.0. All the generated MLVA
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separately.
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3. Results
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Ten VNTR loci fulfilled our criteria for minimum of four bases that were repeated at least four times in a tandem repeat. Three of these VNTR markers were omitted due to their poorly repeatable PCR-results, leaving us with the seven loci suitable for genotyping of the Y. pseudotuberculosis
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isolates (Table 1). The different MLVA profiles were named as a string of seven numbers
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representing the actual number of tandem repeat units in each locus. The profiles were based on the number of 6 to 9 bp long tandem repeats in each locus, and the flanking regions of the loci ranged
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from 127 to 175 bp (Table 1).
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Comparison of the complete Y. pseudotuberculosis genomes available in the GenBank revealed that locus YPb5 had a flanking region of either 152 or 153 bp long (Table 1). Alignment of
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the 3’-flanking region of locus YPb5 of all six partially sequenced isolates revealed a one-base deletion in the same position of the region. This one base difference was observed in 50% of all
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available complete genome sequences of Y. pseudotuberculosis and also in the six partially sequenced isolates.
The fragment sizes obtained from sequencing (EMBL Nucleotide Sequence Database accession numbers HF584708 - HF584747) and from capillary electrophoresis differed from each other in all of the cases (data not shown). However, the differences between the sequencing and capillary electrophoresis results were always less than one tandem repeat in any of the loci with a mean value of 2,1 bp. Isolate FE100992 had a 7 bp deletion in the 3’-flanking sequence at locus YPb10 when compared to the 25 other YPb10 loci sequences (data not shown).
8
ACCEPTED MANUSCRIPT The groups of 63 serotype O:1 isolates and 44 serotype O:3 isolates were both discriminated into 12 unique, serotype-specific MLVA profiles (Table 2). The number of repeats
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varied from 1 to 23 depending on the locus. The most frequent, MLVA profiles were obtained for
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52 serotype O:1 and 18 serotype O:3 isolates. There was no correlation between the MLVA profiles
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and the time of isolation (Table 3).
To study the stability of the VNTR markers, 17 isolates from an outbreak at NorthEastern part of Finland in 2008 were examined. These 17 outbreak isolates represented serotype
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O:1 and all of them had an identical MLVA profile 4-7-3-5-5-5-2 (Table 3). This profile occurred
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previously in 2000 and since then every year. In 2000, it was found among six of the randomly selected seven O:1 isolates. In 2001, one multilocus variant (4-7-7-3-2-2-3) was found among 11 O:1 isolates that were thought to belong to the same cluster. Similarly in 2004, one single locus
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variant (4-7-2-5-5-5-2) was found among seven O:1 isolates. Three of the O:3 isolates previously thought to represent a small outbreak in 2001 belonged to two single locus variants (5-3-9-2-1-3-2
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and 5-7-9-2-1-3-2) (Table 3).
Diversity indices were calculated for individual loci to investigate their discriminatory
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power. The DIs for serotype O:1 isolates for the loci YPb1, YPb3, YPb5, YPb7, YPb8, YPb9 and YPb10 were 0,7727; 0,8788; 0,8939; 0,7576; 0,8485; 0,8636 and 0,8788, respectively. The DIs for serotype O:3 isolates for the loci YPb1, YPb3 and YPb5 were 0,6212; 0,5758; 0,7576, respectively, and 0,0 for the loci YPb7 to YPb10.
4. Discussion We designed a MLVA method based on seven loci in the chromosome of Y. pseudotuberculosis for comparison of the clinical isolates. The method is based on capillary electrophoresis separation of PCR products labelled with a fluorescent dye. In assigning the tandem repeat numbers, we followed the style used for Salmonella Typhimurium (Larsson et al., 2009). A minimum of four bases, that 9
ACCEPTED MANUSCRIPT were repeated at least four times in a tandem repeat were selected. The prokaryotic microsatellites have been proposed to be connected to the slippage of the DNA polymerase, which is required for
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the polymorphism of the VNTR loci and is more frequent with short repeats (Noller et al., 2003).
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Therefore, short tandem repeats were chosen as VNTR markers in this study. In the previous five-
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loci MLVA method designed for Salmonella Typhimurium, one of the VNTR markers is located in a plasmid (STTR10pl) (Lindstedt et al., 2003). The locus in question had the highest percentage (48 %) of missing PCR products in comparison to all the other loci used in the method. The locus
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STTR10pl also had the highest polymorphism rate. However, plasmids are gained and lost quite
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easily by Yersinia (Eppinger et al., 2007), especially in laboratory cultures. Therefore, we did not include any VNTR markers possibly located in Y. pseudotuberculosis plasmids, although they
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might have added to the polymorphism.
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The VNTR loci showed variety of polymorphism within our set of Y. pseudotuberculosis isolates: the analysis of the 107 isolates yielded altogether 24 distinct MLVA
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patterns. The 12 MLVA patterns obtained for both serotypes O:1 and O:3 showed that the method presented here is capable of discriminating within as well as between the isolates of the two
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serotypes; namely, all of the MLVA profiles detected from 107 isolates were serotype-specific. Partial sequencing of six isolates were used to cross-check the theoretical and actual sizes of the flanking regions in each loci. The theoretical sizes of each locus, which were calculated from the complete genome sequences, matched the actual flanking region sizes obtained via sequencing. Thus, it is apparent that the flanking region sizes are conserved throughout the studied isolates. As an exception, the isolate FE100992 (serotype O:1) had a 7 bp deletion in the 3’ flanking sequence at locus YPb10 when compared to the other 25 YPb10 loci sequenced. Also, the occurrence of flanking regions of the two slightly different sizes (1 bp) at locus YPb5 was observed in our study with the partially sequenced isolates. These findings should be accounted for when
10
ACCEPTED MANUSCRIPT analysing the results to avoid incorrect size calling and binning of the capillary electrophoresis results.
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In epidemiological investigation, the attention should always be focused on the
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combination of both pheno- and/or genotypic features of the causative agent as well as on the
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analytical epidemiological data. The possibility that one strain evolves into several genotypes during an outbreak can pose a problem in the use of the MLVA method (Noller et al., 2003) as in
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the use of other genotypic subtyping methods. In theory, the cellular mechanisms that increase or decrease the number of repeats by one in a given loci can also alter the tandem repeat number by
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several steps (Bichara et al., 2006). Therefore, an isolate with a change in the copy number in one locus might still be part of an outbreak. Common guidelines, similar to those described by Tenover
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et al. (1995) for PFGE, aiding the interpretation of the different MLVA profiles in the
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epidemiological investigations should be created as more isolates are analysed.
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Several investigations for epidemiology of Y. pseudotuberculosis have been conducted in Finland. They have shown that the non-outbreak isolates were genetically very closely related to the observed outbreak isolates (Hallanvuo et al., 2002). In the 2000’s, multiple Y.
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pseudotuberculosis outbreaks, mainly caused by O:1 isolates, occurred in Finland (Jalava et al., 2006, 2004; Kangas et al., 2008; Rimhanen-Finne and Sihvonen, 2010). Grated carrot was recognized as a vehicle in some of the outbreaks (Kangas et al., 2008; Rimhanen-Finne et al., 2009; Rimhanen-Finne and Sihvonen, 2010) but in some the vehicle of the pathogen remained unknown (Hallanvuo et al., 2002; Jalava et al., 2006; Rimhanen-Finne and Sihvonen, 2010). For example in 2001, there were altogether 55 cases caused by serotype O:1 isolates and 34 cases caused by serotype O:3 isolates (Jalava et al., 2004). In the data set that was randomly selected (except the 17 outbreak strains from the year 2008) for our study, 11 O:1 isolates and three O:3 isolates represented this outbreak in 2001. There were four distinct MLVA profiles amongst the outbreak isolates. One serotype O:1 MLVA profile (4-7-7-3-2-2-3) differed considerably from the profiles of 11
ACCEPTED MANUSCRIPT the other 10 serotype O:1 outbreak isolates. The other outbreaks caused by Y. pseudotuberculosis in Finland from 2003 to 2008 were all associated with serotype O:1 (Jalava et al., 2006; Kangas et al.,
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2008; R Rimhanen-Finne et al., 2009; Ruska Rimhanen-Finne and L. Sihvonen, 2010; Vasala et al.,
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2013). The isolates from 2003 to 2006 outbreaks represented two MLVA profiles that were single
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locus variants. The 17 isolates from the outbreak at the North-Eastern part of Finland in 2008 had identical MLVA profiles as expected.
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Overall, the serotype O:1 MLVA profile 4-7-3-5-5-5-2 was the most common (52 isolates) among all 63 O:1 isolates tested and it was present during several years. This dominant
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serotype O:1 MLVA profile was associated with all of the O:1 outbreaks. Out of the total 63 serotype O:1 isolates, 14 were clearly sporadic. The most common serotype O:3 MLVA profile 4-6-
D
9-2-1-3-2 was observed in 18 of the 44 isolates tested. In 2001, three O:3 isolates were associated
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with several small infection clusters (Jalava et al., 2004). Two MLVA profiles were obtained from these three outbreak isolates, which differed in two loci from the most common O:3 MLVA profile.
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The remaining 41 O:3 isolates studied were considered sporadic. This finding that the vehicle in five of the outbreaks from 2003 to 2008 was carrots (Kangas et al., 2008; Rimhanen-Finne et al.,
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2009; Rimhanen-Finne and Sihvonen, 2010) could easily lead to an assumption that the reservoir for all mentioned outbreaks are identical or that the strain is widely spread in nature and that the MLVA type has remained stable in that reservoir. Further study is required to elucidate this possibility. This study focused on the MLVA typing of the serotype O:1 and O:3 isolates that have been common in human infections in Finland during the past 15 years. The MLVA method described here is an additional tool for subtyping Y. pseudotuberculosis isolates and it is in line with the recent methodological development for other enteric bacteria. Testing the method with isolates of other serotypes will yield more information on the discriminatory potential and sensitivity of the Y. pseudotuberculosis MLVA scheme. Multiplexing the PCR primers with the same annealing 12
ACCEPTED MANUSCRIPT temperature and labelled with different fluorescent dyes can be done for routine use, which would
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shorten the time necessary to perform the analysis further.
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5. Conclusions
During the recent years, MLVA has been successfully applied for molecular typing of several
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bacterial pathogens. We designed a MLVA scheme based on seven loci, which were able to discriminate clinical isolates of Y. pseudotuberculosis serotypes O:1 and O:3 into several MLVA
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types. In conclusion, the MLVA method developed by us offers a new molecular tool for epidemiological investigation of Y. pseudotuberculosis outbreaks and for comparison of the
Acknowledgements
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sporadic isolates.
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We thank Prof. Mikael Skurnik for providing three Y. pseudotuberculosis reference strains and the staff of the Bacteriology Unit, THL, for their assistance. The work was supported by a grant
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(STM/4608/2010) from the Finnish Ministry of Social Affairs and Health.
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Niskanen, T., Fredriksson-Ahomaa, M., Korkeala, H., 2002. Yersinia pseudotuberculosis with limited genetic diversity is a common finding in tonsils of fattening pigs. Journal of food protection. Niskanen, T., Waldenström, J., Fredriksson-Ahomaa, M., Olsen, B., Korkeala, H., 2003. virF-positive Yersinia pseudotuberculosis and Yersinia enterocolitica found in migratory birds in Sweden. Applied and environmental microbiology 69, 4670–5. Noller, A.C., McEllistrem, M.C., Pacheco, A.G.F., Boxrud, D.J., Harrison, L.H., 2003. Multilocus variablenumber tandem repeat analysis distinguishes outbreak and sporadic Escherichia coli O157:H7 isolates. Journal of clinical microbiology 41, 5389–97. Nuorti, J.P., Niskanen, T., Hallanvuo, S., Mikkola, J., Kela, E., Hatakka, M., Fredriksson-Ahomaa, M., Lyytikainen, O., Siitonen, A., Korkeala, H., Ruutu, P., 2004. A widespread outbreak of Yersinia pseudotuberculosis O:3 infection from iceberg lettuce. The Journal of infectious diseases 189, 766–74. Platonov, M.E., Blouin, Y., Evseeva, V.V., Afanas’ev, M.V., Pourcel, C., Balakhonov, S.V., Vergnaud, G., Anisimov, A.P., 2013. Draft Genome Sequences of Five Yersinia pseudotuberculosis ST19 Isolates and One Isolate Variant. Genome announcements 1, e0012213.
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Zhang, Z., Schwartz, S., Wagner, L., Miller, W., 2000. A greedy algorithm for aligning DNA sequences. Journal of computational biology : a journal of computational molecular cell biology 7, 203–14.
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YPb7
YPb8
YPb9
YPb10
YPbF3
5'-6FAM-TGCCTGTTGAGGTACGGC-3'
YPbR3
5'-GCGAAAGCCACGATGATT-3'
YPbF5
5'-6FAM-GGGCCTGACACTGCGTAT-3'
YPbR5
5'-GGCGGATTACCACACCTG-3'
YPbF7
5'-6FAM-CGCTGACTGGCAGGAAAT-3'
YPbR7
5'-GCGAGCAGAGAAACTCGC-3'
YPbF8
5'-6FAM-TGGTCAGAACAAAGCGCA-3'
YPbR8
5'-GGCCATCTGGGTCAACAG-3'
YPbF9
5'-6FAM-CCCAGAGTACCACGACCG-3'
YPbR9
5'-GTCAATCAATCGACGCCC-3'
YPbF10
5'-6FAM-GGAGGGGGTGACACACTG-3'
YPbR10
5'-CGAGACGGTGAGCGAGTT-3'
ORF… 122007-122891 YPTB0107 metF 5,10methylenetetrahydrofolate reductase
from 443723 to 443953
231
(X-[152 or 153])/7
from 1462081 to 1462282
202
ORF... 443899-444408 YPTB0371 - zinc uptake transcriptional repressor (Protein is coded in complementary strand; from 444408 to 443899) ORF… 1461648-1462121 YPTB1227 - hypothetical protein
ATTTCCTG T
(X-143)/9
from 1703649 to 1703845
197
ATCAAC
(X-127)/6
from 2306115 to 2306295
181
TAACGAC
(X-175)/7
from 2538646 to 2538876
231
TGAAGTT
(X-159)/7
from 3322462 to 3322648
187
TGGTGGTT
(X-147)/8
147
TGCTTTT
(X-147)/7
152 or 153
GTTAATA
143
127
175
159
IP
5'-AACAACCGAACCCGATCA-3'
179
Fragment position in Y. pseudotuberculosis IP32953
CR
YPbR1
from 122802 to 122980
Repeat number
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5'-6FAM-GCGAAGGGGTGAAGGATT-3'
Fragment size (bp)
Repeat
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YPb5
YPbF1
Length of flanking region (bp) 147
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YPb3
Primer sequence
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YPb1
Primer
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Locus
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Table 1. MLVA primers designed for Y. pseudotuberculosis, lengths of the flanking sequences and fragments, repeat sequences and the formula for calculating the repeat number from the size of the amplified fragment. Part of gene(s) amplified in Y. pseudotuberculosis IP32953
ORF… 1703459-1704904 YPTB1422 - hypothetical protein (Protein is coded in complementary strand; from 1704904 to 1703459) ORF… 2304769-2306190 YPTB1958 - GntR family transcriptional regulator ORF… 2537434-2538744 YPTB2158 - major facilitator superfamily tartrate transporter ORF... 2538824-2539822 YPTB2159 - hypothetical protein ORF... 3321396-3322481 YPTB2812 - ABC opine/polyamine transporter, ATP-binding subunit
Table 2. Diversity of the seven VNTR loci in 63 serotype O:1 isolates and 44 serotype O:3 isolates.
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CR
4-6-9-2-1-3-2 4-6-10-2-1-3-2 4-6-12-2-1-3-2 5-6-9-2-1-3-2 5-7-9-2-1-3-2 5-6-10-2-1-3-2 12-8-9-2-1-3-2a 4-6-11-2-1-3-2 4-6-13-2-1-3-2 5-3-9-2-1-3-2 5-5-9-2-1-3-2 5-6-8-2-1-3-2 Unique: 12
18 7 4 4 3 2 1 1 1 1 1 1 Total: 44
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Total: 63
Frequency
AC
a
Unique: 12 Isolates received from Prof. Skurnik
52 1 1 1 1 1 1 1 1 1 1 1
MLVA Profiles O:3
MA N
4-7-3-5-5-5-2 3-16-4-2-5-9-23 4-10-5-3-3-9-5 4-11-4-6-4-2-8a 4-3-6-3-2-2-5a 4-7-2-5-5-5-2 4-7-7-3-2-2-3 5-11-13-5-3-7-8 6-7-3-2-3-7-12 7-9-3-2-3-7-13 8-6-5-3-9-4-5 9-6-5-3-8-4-5
Frequency
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MLVA Profiles O:1
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5 4 4 4 5 5 4 4 5 5 4 4 4 5 4 4
6 10 16 7 9 7 7 7 7 7 7 7 6 7 11 7 7
-
7 6 6 6 6 6 6 6 3 7 6 6 6 6 6 6
-
5 5 4 3 3 3 7 3 3 3 3 2 5 3 13 3 3
-
9 9 10 9 9 8 10 9 9 9 11 10 9 10 10 10
-
3 3 2 5 2 5 3 5 5 2 5 5 3 5 5 5 5
-
2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
-
9 3 5 5 3 5 2 5 5 3 5 5 8 5 3 5 5
-
4 9 9 5 7 5 2 5 5 7 5 5 4 5 7 5 5
-
5 5 23 2 13 2 3 2 2 12 2 2 5 2 8 2 2
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
-
3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3
-
2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
1 6 1 5 4 1 1 1 1a 2a 1 1 4 2 1 1
US
O:3 O:3 O:3 O:3 O:3 O:3 O:3 O:3 O:3 O:3 O:3 O:3 O:3 O:3 O:3 O:3
-
MA N
8 4 3 4 7 4 4 4 4 6 4 4 9 4 5 4 4
TE D
O:1 O:1 O:1 O:1 O:1 O:1 O:1 O:1 O:1 O:1 O:1 O:1 O:1 O:1 O:1 O:1 O:1
no. MLVA profiles 1 1 1 6 1 10a 1a 1 7b 1 6c 1c 1 4d 1 17e 1
CR
MLVA profile
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1997 1998 1998 1999 2000 2000 2000 2000 2001 2001 2001 2001 2001 2001 2002 2006
ST
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Isolation year 1999 1999 1999 2000 2000 2001 2001 2002 2003 2003 2004 2004 2005 2006 2006 2008 2009
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Table 3. The year of isolation for the studied 104 clinical Y. pseudotuberculosis strains, their serotype, MLVA profile and the number of strains with the certain MLVA profile. Isolates FE100991, FE100992 and FE100994 were not included.
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MA N
US
CR
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2007 O:3 4 6 10 - 2 1 - 3 2 1 2008 O:3 5 5 9 - 2 1 - 3 2 1 2008 O:3 4 6 9 - 2 1 - 3 2 1 2008 O:3 4 6 10 - 2 1 - 3 2 1 2012 O:3 4 6 12 - 2 1 - 3 2 4 2012 O:3 4 6 13 - 2 1 - 3 2 1 2012 O:3 4 6 9 - 2 1 - 3 2 1 a 11 serotype O:1 isolates and 3 serotype O:3 isolates belonging to the infection clusters in 2001 (Jalava et al., 2004) b 7 serotype O:1 isolates belonging to the infection cluster in 2003 (Jalava et al., 2006) c 7 serotype O:1 isolates belonging to the infection cluster in 2004 (Kangas et al., 2008) d 4 serotype O:1 isolates belonging to the infection cluster in 2006 Rimhanen-Finne and Sihvonen, 2010) e 17 serotype O:1 isolates belonging to the infection cluster in 2008 (Ruska Rimhanen-Finne and L. Sihvonen, 2010; Vasala et al., 2013)
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Yersinia pseudotuberculosis serotypes O:1 and O:3 are most common in Europe We developed a new genotyping method (MLVA) for Yersinia pseudotuberculosis The MLVA scheme discriminated serotypes O:1 and O:3 into several MLVA types All of the obtained MLVA profiles from 107 isolates were serotype-specific
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