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Veterinary Research Institute, Hudcova 70, 621 00 Brno, Czech Republic
Correspondence
Ivan Rychlik
rychlik{at}vri.cz
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). To differentiate Salmonella strains originating from outbreaks, phage typing, antibiotic-resistance typing, plasmid-profile analysis and PFGE are the most frequently used methods. Out of the DNA-based typing methods, PGFE is the method of choice for Salmonella, and also for other bacterial species, despite the fact that the reasons for the variability in the size of the restriction fragments are not known.
The genome of S. Typhimurium strain LT2 has been known since 2001 (McClelland et al., 2001), and this has allowed in silico identification of restriction sites, and prediction of PFGE profiles. Although not annotated in GenBank, two additional S. Typhimurium genomes, specifically those of SL1344 and the pentadrug-resistant DT104 strain, are publicly available on the website of the institution that performed the sequencing. When we searched for the XbaI sites in the sequenced genomes to verify the PFGE profiles (Van Immerseel et al., 2004; Hradecka et al., 2006; Matiasovicova et al., 2007), we observed that the XbaI sites frequently overlapped with the prophage sequences. In other cases, the presence of a prophage was shown to have led to an increase in the size of DNA fragments generated by XbaI restriction endonuclease. When we realized this, we assumed that a rational design of PCRs targeted to various mobile DNA sequences, mostly of prophage origin, might be a very sensitive tool for differentiation of S. Typhimurium. Also, since the positive and negative results of individual PCRs can be easily shared among laboratories, such a typing system might be simple to perform worldwide. In this study, we showed that this assumption was correct, and that a combination of four triplex PCRs enabled differentiation of S. Typhimurium field strains, with essentially the same sensitivity as their differentiation by PFGE.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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were characterized by the triplex PCRs and PFGE for comparison. In addition, the original host of each strain was known (except for two control laboratory strains, LT2 and SL1344), and the phage type according to Anderson et al. (1977) was available (except for 13 strains, three of which were control laboratory strains LT2, SL1344 and F98).
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PCR design.
Based on the identification of variable DNA sequences in the genomes of three strains, and data originating from microarray genomotyping (Porwollik et al., 2004; Reen et al., 2005; Matiasovicova et al., 2007), 22 individual PCRs specific for the potentially variable DNA sequences were designed. These included the STM0517-0529 deletion, the left junction of SGI1, and STM908, STM2619, STM2697, STM2705, gipA, artA, sodCI, bim2, eac, sieB, g8, gtrA, hldD, sopE, Fels Sop, gp36, rrtT, spvB, oafA and fljB (for primer sequences and the results of these PCRs, see supplementary material, available with the online version of this paper). If possible, sequences of the PCR primers were designed according to previously published data (Faldynova et al., 2003; Matiasovicova et al., 2003; Mikasova et al., 2005; Hermans et al., 2005; Ross & Heuzenroeder, 2005). If a PCR primer pair was not available, we designed it using GeneCompar software (Applied Maths). After evaluation of the results obtained by individual PCRs, 12 target genes that allowed maximal strain discrimination were selected. To simplify the assay, the PCRs were assembled into four triplex PCRs (Table 2).
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Results of each PCR were first converted into a binary code: a positive PCR was assigned the value 1, and a negative PCR was given the value 0. Since four triplex PCRs were used for strain characterization, each strain was characterized by a raw 12-digit code, such as 101 111 000 001. The raw binary code was subsequently transformed into a four-digit code using the following calculation. The first position in each triplex was given the value 4, the second position was given the value 2, and the third position was given the value 1. Each of these numbers was multiplied by the result of the PCR, and all the values were summed. The first three positions of the above-mentioned triplex PCR should therefore calculate as (1x4)+(0x2)+(1x1), which is equal to 5. Performing identical calculations with the remaining triplex PCRs gives a PCR code of 5701 in this particular case.
Pulsed-field gel electrophoresis.
DNA purification for macrorestriction analysis, restriction enzyme digestion and PFGE were performed essentially as described elsewhere (Hunter et al., 2005). PGFE of XbaI-digested DNA was performed using the CHEF-DRIII system (Bio-Rad) in 0.5x TBE (25 mM Tris/25 mM borate/0.5 mM EDTA). After the electrophoresis, the gels were stained with ethidium bromide, and DNA was visualized under UV light.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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To test this hypothesis, we subjected 58 field strains to PCR using 22 different primer pairs. These PCRs resulted in efficient differentiation between the strains. However, when we analysed the results more carefully, we found that several PCRs did not bring additional value to the system. Typical examples were the fljB- and oafA-specific PCRs, which yielded positive results in all the strains, and STM908- and STM2705-specific PCRs, which detected Fels1 and Fels2 prophages of the LT2 strain only (for additional information, see supplementary material, available with the online version of this paper). Based on this evaluation, we simplified the PCR typing into 12 PCRs, which were subsequently grouped into four triplex PCRs. The first triplex PCR amplified allB, the left junction of SGI1, and rrtT. The second triplex PCR was targeted to gipA, spvB and bim2. gipA is encoded by the terminal part of Gifsy1LT2, spvB is encoded by the serovar-specific virulence plasmid, and bim2 is encoded by the ST64B phage. The third multiplex PCR detected gp36, which is encoded by phage 186 of SL1344, sopE, which is localized on the Fels2sopE phage, and artA, which is localized at the terminal part of the Gifsy1DT104. The fourth triplex PCR amplified STM2697, which is encoded by both Fels2 and FelssopE, hldD, which is characteristic for the cryptic prophage found in the DT104 genome, and gtrA, which is localized on the ST104 phage. The order of the target genes in each triplex PCR was also the order in which the data on positivity or negativity were recorded. The order also corresponded to the size of the PCR products: the first PCR in each triplex resulted in the PCR product with the highest molecular mass, and the last resulted in the smallest PCR product.
Using the multiplex PCR and the whole set of 102 field strains (Fig. 1), 22 multiplex PCR profiles were observed. Using PFGE, 25 profiles were detected. This shows that the system developed is essentially as sensitive as PFGE. Multiplex PCR typing was especially useful in characterization of the DT104 strains and their multidrug-resistant variants. These strains dominated in our collection because of the current epidemiological situation in the Czech Republic, and, although these strains were usually of the same PFGE profile, 11 multiplex PCR types could be identified within the group. Furthermore, among the strains classified as belonging to the phage type DT104, those with a multiplex PCR code starting with 1XXX, 2XXX and 3XXX formed the DT104-like clone. Strains with the PCR code 5XXX, despite being the same phage type as those of the DT104-like clone, did not belong to this clone; this was also confirmed by their different PFGE profiles. Finally, strains of the phage type DT2 were confirmed to form a relatively uniform cluster of strains, by both PCR (Table 3) and PFGE.
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and 4; see also supplementary material, available with the online version of this paper).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Reen et al., 2005; Matiasovicova et al., 2007); these studies have indicated that Fels1 and Fels2 prophages are relatively specific for the LT2 strain. Other studies, using conventional PCR, have found that sopE, encoded by a Fels-like prophage, is quite uncommon to S. Typhimurium (Mirold et al., 1999). This information has led to studies that have used the detection of prophage sequences for strain characterization (Mikasova et al., 2005; Hermans et al., 2005; Ross & Heuzenroeder, 2005); however, the approach in those studies was rather arbitrary. In this study, we used accumulated genomic data, either from genome sequencing (McClelland et al., 2001) or from microarray genomotyping (Porwollik et al., 2004; Reen et al., 2005; Matiasovicova et al., 2007), for rational design of a PCR-typing system based on the presence or absence of mobile DNA sequences. Multiplexing of the specific PCR further simplified the protocol, and made it user friendly. When compared with the PFGE, the system was essentially of the same discriminatory power. However, unlike PFGE, the multiplex PCR typing system can be easily adopted by any laboratory capable of running PCR, thus allowing simple data comparison. A further advantage of the proposed system is that it remains open for the introduction of additional PCRs when new sequence data become available. In such cases, a new target sequence can be added to the current sequences, provided that the order of the PCRs is not changed.
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ACKNOWLEDGEMENTS |
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Edited by: D. W. Ussery
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 16 November 2007;
revised 15 December 2007;
accepted 22 February 2008.
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