Identification of plasmids by PCR-based replicon typing
Introduction
Plasmids are extra-chromosomal circular fragments of DNA that replicate autonomously in a host cell. They are present in nearly all bacterial species and vary in size from a few to more than several hundred kilobase pairs (kb) (Waters, 1999). Plasmids appear to increase bacterial genetic diversity, acquiring and losing genes, and can be horizontally exchanged among bacterial populations by conjugation or mobilization (Francia et al., 2004). They contain genes essential for initiation and control of replication and accessory genes that may be useful to their bacterial host such as antimicrobial resistance or virulence genes (Amabile-Cuevas and Chicurel, 1992, Bergstrom et al., 2000, Thomas, 1973).
A formal scheme of plasmid classification is based on incompatibility (Inc) groups (Novick, 1987). The procedure for incompatibility grouping is based on the introduction, by conjugation or transformation, of a plasmid of an “unknown” Inc group into a strain carrying a plasmid of a known Inc group. If the resident plasmid is eliminated in the progeny, the incoming plasmid is assigned to its same Inc group (Datta and Hedges, 1971). Plasmids with the same replication control are “incompatible”, whereas plasmids with different replication controls are “compatible”. On this basis two plasmids belonging to the same Inc group cannot be propagated in the same cell line (Datta and Hughes, 1983, Couturier et al., 1988). Inc group identification has been frequently used to classify plasmids. The method has been an important tool to trace the diffusion of plasmids conferring antimicrobial resistance and also to follow the evolution and spread of emerging plasmids (Anderson et al., 1977).
In 1988 Couturier et al. developed a new method for the identification of the major replicons of plasmids circulating among the Enterobacteriaceae. This method was based on hybridization with 19 DNA probes that recognize different basic replicons (Couturier et al., 1988). The conjugation- and hybridization-based methodologies cannot be easily applied to a large number of strains and their application has been limited by the laborious and time-consuming work required. A PCR-based detection of plasmids was previously devised on the basis of published sequences but it was limited to the IncP, IncN, IncW and IncQ plasmids (Gotz et al., 1996).
The necessity of tracing plasmids conferring drug resistance prompted us to develop an inc/rep PCR-based typing method. In this method, 18 pairs of primers were designed to perform 5 multiplex- and 3 simplex-PCRs, recognizing the FIA, FIB, FIC, HI1, HI2, I1-Iγ, L/M, N, P, W, T, A/C, K, B/O, X, Y, F, and FIIA replicons, representative of the major plasmid incompatibility groups circulating among the Enterobacteriaceae (Couturier et al., 1988).
The specificity of the method was tested on 61 reference plasmids. Plasmids in a collection of 20 previously characterized multi-drug resistant Salmonella enterica isolates of different serotypes (Pezzella et al., 2004) have then been typed by PCR-based inc/rep typing.
Section snippets
Bacterial strains and plasmids
Sixty-one Escherichia coli and S. enterica strains carrying plasmids belonging to 22 different incompatibility (Inc) groups, were available from the collections of the Istituto Superiore di Sanità in Rome and the UK Health Protection Agency. Thirty-nine plasmids analyzed in this study were reference plasmids of known Inc groups (Anderson et al., 1977, Carattoli et al., 2001, Couturier et al., 1988, Frydman and Meynell, 1969, Grindley et al., 1972, Hedges and Datta, 1971, Villa et al., 2000); 22
Specificity of the PCR-based inc/rep typing method on reference plasmids
Eighteen specific primer pairs were designed, on the basis of the multiple comparative analysis of nucleotide sequence on the EMBL Gene Databank, for HI1, HI2, I1-Iγ, X, L/M, N, FIA, FIB, W, Y, P, FIC, A/C, T, FIIAS, F, K and B/O replicons. The specificity of each primer pair, listed in Table 1, was initially performed on bacterial genomic DNA extracted from the plasmid-free E. coli DH5α strain (Invitrogen-Life Technologies). None of the primer pairs gave positive amplification results on this
Acknowledgments
We are grateful to V. Miriagou, L.S. Tzouvelekis, E. Tzelepi and G.M. Rossolini for helpful discussion and continuous encouragement. We thank Fabio Riccobono for the DNA sequencing. This work was supported by grants from the Istituto Superiore di Sanità (Art. 502, project no. 2012/RI; Art. 524, project no. C3MD).
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