Targeting relaxase genes for classification of the predominant plasmids in Enterobacteriaceae

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Plasmids are important agents of gene flux and have found to be responsible for the 48 dissemination of multiple antibiotic resistance genes. Identification and classification of 49 plasmids is essential for analysis of their distribution, their genetic relatedness and evolution, 50 as well as for study of horizontal gene transfer. A classification scheme should be based on 51 genetic traits that are universally present and constant. It should be robust and the 52 corresponding experimental procedure should be easy. The basic replicon locus, which is 53 always present on plasmids, has been used historically for classification. Plasmids were 54 initially classified according to their incompatibility, which is directly related to replication. 55 Incompatibility (Inc) was defined as the inability of two plasmids sharing common replication 56 control (same Inc group) to be maintained in the subsequent lineage during conjugation (Datta 57 and Novick, 1987). This method which requires plasmid transfer to the Inc groups are currently recognized (Carattoli, 2009). It has been widely used to study 66 plasmid spread and diversity in Enterobacteriaceae. However, PBRT has several drawbacks:   The aim of the present study was to design a multiplex PCR method, called "plasmid relaxase   (Table 1A).

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In silico primer assay 106 Oligonucleotide primers were tested in silico for hybridization with plasmids of the 107 Enterobacteriaceae referenced in GenBank. Some primers were refined to cover a maximum 108 of reported sequences.      Table S1). HIβ and HIδ relaxases were found to be encoded  (Table 1A).

Primer evaluation using transconjugants and transformants 189
In order to assess the sensitivity and specificity of each PCR, primers were tested using a 190 collection of 60 recipient cells, with PBRT as the reference method (Table 2) Table S2). The eighteen PRaseT-205 positive IncF plasmids reported in Table 2 tested positive for traB and traC while the PRaseT-206 negative IncFIB/FII plasmid also tested negative with T4SS typing (data not shown).

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Interestingly, no transconjugant but only transformant was obtained from the parental strain of 208 the later plasmid. We considered three possibilities: (i) a very divergent IncF relaxase gene 209 that could not hybridize with our primers was present, (ii) the relaxase gene was truncated or 210 (iii) the gene was absent. Complete sequencing of the plasmid will be performed to confirm 211 one of these possibilities.

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For IncHI plasmids, three primer sets were mandatory to differentiate IncHI1 from IncHI2. As 213 noted above, in most cases, the identification of relaxases from IncHI1 and IncHI2 plasmids 214 was obtained with positive results for HIβ-and HIγ-primers respectively (e.g. transconjugants 215 S01477 and 102,  To further confirm the specificity of the designed primer set, 39 clinical strains, each carrying 223 from one to seven different replicon types, were submitted to PRaseT (Table 3). An example  with PBRT (after multiple PCR assays) were found to contain IncFIIk, IncHI and IncI1 242 plasmids, respectively, when PRaseT and T4SS typing was used ( Table 3). The presence of 243 sequence divergence or mosaic replicons may explain these results that will be clarified by

Relaxase gene typing in recipient cells non-typeable with PBRT 248
Seventeen recipient cells whose plasmids were found to be non-typeable with PBRT were 249 subjected to typing with PRaseT; parental strains included 14 strains of E. coli and 3 of K.  Six plasmids could be typed neither with PBRT nor with PRaseT (Table 4)