# Targeting relaxase genes for classification of the predominant plasmids in Enterobacteriaceae

Fabrice Compain, Agathe Poisson, Simon Le Hello, Catherine Branger, François-Xavier Weill, Guillaume Arlet, Dominique Decré

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Fabrice Compain, Agathe Poisson, Simon Le Hello, Catherine Branger, François-Xavier Weill, et al.. Targeting relaxase genes for classification of the predominant plasmids in Enterobacteriaceae. International Journal of Medical Microbiology Supplements, 2014, 304 (3-4), pp.236 - 242. 10.1016/j.ijmm.2013.09.009. pasteur-01108896

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Targeting relaxase genes for classification of the predominant plasmids in

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Enterobacteriaceae

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Fabrice Compain¹, Agathe Poisson¹, Simon Le Hello², Catherine Branger³, François-

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Xavier Weill², Guillaume Arlet¹, ⁴, Dominique Decré¹, ⁴*

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¹Université Pierre et Marie Curie-Paris 6, Faculté de Médecine, Site Saint-Antoine,

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Laboratoire de Bactériologie, ER8, Paris, France; ²Institut Pasteur, Unité des Bactéries

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Pathogènes Entériques, Centre National de Référence des *Escherichia coli*, *Shigella* et

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*Salmonella*, WHO Collaborating Centre for Reference and Research on Salmonella, Paris,

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France; ³Université Paris Diderot, UMR-S 722, Faculté de Médecine, Site Xavier Bichat, F-75018

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Paris, France; ⁴Laboratoire de Bactériologie, Hôpital Saint-Antoine, Assistance Publique des

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Hôpitaux de Paris, Paris, France

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*Corresponding author

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Email addresses:

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FC: [compain.fabrice@wanadoo.fr](mailto:compain.fabrice@wanadoo.fr)

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AP: [agatepoisson@gmail.com](mailto:agatepoisson@gmail.com)

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SLH: [slehello@pasteur.fr](mailto:slehello@pasteur.fr)

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CB: [catherine.branger@lmr.aphp.fr](mailto:catherine.branger@lmr.aphp.fr)

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FXW: [fxweill@pasteur.fr](mailto:fxweill@pasteur.fr)

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GA: [guillaume.arlet@upmc.fr](mailto:guillaume.arlet@upmc.fr)

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DD: [dominique.decre@upmc.fr](mailto:dominique.decre@upmc.fr); Tel.: +33 1 40 01 14 46; Fax +33 1 49 28 24 72
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**Abstract**

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**Keywords**

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Plasmid; Classification; Replicon typing; Relaxase; Multiplex PCR
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# Introduction

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Plasmids are important agents of gene flux and have found to be responsible for the dissemination of multiple antibiotic resistance genes. Identification and classification of plasmids is essential for analysis of their distribution, their genetic relatedness and evolution, as well as for study of horizontal gene transfer. A classification scheme should be based on genetic traits that are universally present and constant. It should be robust and the corresponding experimental procedure should be easy. The basic replicon locus, which is always present on plasmids, has been used historically for classification. Plasmids were initially classified according to their incompatibility, which is directly related to replication.

Incompatibility (Inc) was defined as the inability of two plasmids sharing common replication control (same Inc group) to be maintained in the subsequent lineage during conjugation (Datta and Hedges, 1971 and Novick, 1987). This method which requires plasmid transfer to the same host for testing is time-consuming and not practical for large-scale studies. Couturier et al. tested a method using hybridization with cloned replication regions as probes but with this method plasmid diversity is underestimated due to cross-hybridization (Couturier et al., 1988). In 2005, Carattoli et al. developed a PCR scheme of targeting replicons called PCR-based replicon typing (PBRT) (Carattoli et al., 2005). Eighteen pairs of primers were designed in order to perform 5 multiplex and 3 simplex PCRs recognizing the most frequently encountered plasmid incompatibility groups among Enterobacteriaceae. With this method, 27 Inc groups are currently recognized (Carattoli, 2009). It has been widely used to study plasmid spread and diversity in Enterobacteriaceae. However, PBRT has several drawbacks: (i) plasmids may carry multiple replicons and/or mosaic replicons, and new replicon types may escape classification with this technique; (ii) false-negative results with some Inc groups (e.g. L/M) have been reported (Carattoli et al., 2005) and (iii) PBRT targets multiple sites
such as the replication initiation protein gene (*rep*), the active segregation partitioning system (*par*), replication control systems (iterons, antisense RNA), and recently also the relaxase gene (Carattoli et al., 2005 and Johnson et al., 2012), which may cause confusion. Plasmid multilocus sequence typing were also developed to refine classification of plasmid subgroups (García-Fernández and Carattoli, 2010, García-Fernández et al., 2008, García-Fernández et al., 2011 and Phan et al., 2009).

Bacterial conjugation represents a unique process allowing transfer of plasmid DNA from a donor to a recipient bacterium through cell-to-cell contact. In this process relaxase is a key protein encoded by all transmissible plasmids, i.e. mobilizable and conjugative plasmids involved in horizontal gene transfer (reviewed by Smillie et al., 2010 and Wong et al., 2012).

A classification scheme based on the mobilization region of transmissible plasmids has recently been developed (Francia et al., 2004 and Garcillán-Barcia et al., 2009). The scheme classified relaxases in six protein families and 31 subfamilies, depending on their phylogeny; subsequently, 19 degenerate primer pairs targeting the relaxase genes of γ-proteobacterial plasmids were designed (Alvarado et al., 2012). This degenerate primer MOB typing (DPMT) method has been used with success in previous studies (Curiao et al., 2011, Mata et al., 2012, Mata et al., 2010 and Valverde et al., 2009). However, this set of primers was not designed for screening purposes in clinical practice, but rather for experimental purposes in order to discover new relaxases (Alvarado et al., 2012).

The aim of the present study was to design a multiplex PCR method, called "plasmid relaxase gene typing" (PRaseT), including novel oligonucleotide primers targeting relaxase genes of the plasmids most frequently encountered in Enterobacteriaceae in clinical practice. These multiplex PCRs were carried out under maximum consensus thermal cycling conditions, and applied to various plasmids present in clinical isolates from several collections.
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# Materials and methods

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## Database search and primer design

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An *in silico* analysis was carried out using GenBank BLAST ([http://blast.ncbi.nlm.nih.gov/](http://blast.ncbi.nlm.nih.gov/)).

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For each Inc group, the relaxase/helicase gene was used as template; the presence of relaxase-specific multidomains was checked using CD-Search

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([http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi/](http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi/)). Multiple alignments were performed

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with ClustalW2 software ([http://www.ebi.ac.uk/Tools/msa/clustalw2/](http://www.ebi.ac.uk/Tools/msa/clustalw2/)). Primer pairs covering

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most sequences in each family were designed using FastPCR software

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([http://primerdigital.com/fastpcr.html](http://primerdigital.com/fastpcr.html)), while minimizing codon degeneracy (Table 1A).

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## *In silico* primer assay

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Oligonucleotide primers were tested *in silico* for hybridization with plasmids of the

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Enterobacteriaceae referenced in GenBank. Some primers were refined to cover a maximum

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of reported sequences.

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## Bacterial strains

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For validation of the PCR assays, experiments were conducted with 60 *Escherichia coli*

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transconjugants or transformants of Enterobacteriaceae (Table 2). All strains carried replicons

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of various types that encoded diverse β-lactamases conferring resistance to third-generation

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cephalosporins or carbapenems. They were part of three collections of, respectively, (i) *E. coli*

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strains isolated between 1997 and 2002 in various French university hospitals (for further
117 details see Marcadé et al., 2009 and Branger et al., 2005), (ii) *Klebsiella pneumoniae* strains
118 from various geographical regions collected since the 1980s (D. Decré and G. Arlet, personal
collection) and (iii) *Salmonella enterica* subsp. *enterica* strains representing various serovars
119 (collection of the French National Reference Center for *E. coli*, *Shigella*, and *Salmonella*,
120 Institut Pasteur). Assays with transconjugants and transformants for multiplex PCR
121 optimization were carried out in triplicates.
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123 All transconjugants, transformants and clinical strains used in this study were analyzed in
124 parallel with PRaseT and the PBRT method applied previously (Carattoli et al., 2005, García-
Fernández et al., 2009, Götz et al., 1996, Osborn et al., 2000 and Villa et al., 2010). The
125 transconjugants used as positive controls in PRaseT reactions are given in Table 2. Other
126 controls used in this study included three strains of the ECOR collection (Ochman and
127 Selander, 1984) (ECOR 6, ECOR 10 and ECOR 19 harboring, respectively, pTPqnrS-1a-like-
128 pcolE1-like- and IncX2-plasmids), as well as reference plasmids pFBAOT6 (IncU) (Rhodes
129 et al., 2004) and RP4 (IncP1α) (Datta et al., 1971).
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131 After optimization on transconjugants or transformants carrying replicon of various types
132 according to PBRT, we applied the PRaseT method to a panel of 39 clinical strains (21 *E. coli*, 16 *K. pneumoniae* and 2 *S. enterica*) carrying replicons of one to seven different types
133 (Table 3), and 17 transconjugants or transformants that were non-typeable with PBRT (Table
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137 DNA extraction and PCR conditions

138 InstaGene matrix (Biorad, Marnes la Coquette, France) or lysis by boiling were used for total
139 DNA extraction as previously described (Dallenne et al., 2010). Multiplex PCR was carried
140 out using the Qiagen Multiplex PCR Kit (Qiagen, Courtaboeuf, France). The master mix
contained pre-optimized concentrations of HotStarTaq DNA polymerase and MgCl₂,
deoxynucleotide triphosphate and PCR buffer. To all multiplex PCRs, solution Q (Qiagen)
that facilitates the reaction with difficult-to-amplify templates by modifying DNA melting
behavior was added. Total DNA in 5µl of bacterial lysate was subjected to multiplex PCR in a
50 µl volume. The conditions for multiplex PCR were optimized to ensure that all targets
were sufficiently amplified for amplicons to be easily visible on 1.5% agarose gels. The
optimal primer concentrations are reported in Table 1A. PCR conditions consisted in initial
activation at 95°C for 15 min, followed by 30 cycles at 94°C for 30 s, 60°C for 90 s and 72°C
for 90 s with a final extension at 72°C for 10 min; for Multiplex IV and V the annealing
temperature was elevated to 65°C for 90 sec. Simplex PCR were performed in a 50 µL mix
with 2 U of Taq DNA polymerase (Roche Diagnostics), 10x PCR buffer/MgCl₂ (Roche
Diagnostics), 200 µM of each deoxynucleotide triphosphate (dNTP Mix Eurobio), 0.2
pmol/µL of each primer, 40 µL of sterile water and 2 µL of total DNA extract. PCR
conditions consisted in 30 cycles [94°C for 1 min, 55°C for 40 sec, 72°C for 1 min], preceded
by one cycle at 94°C for 5 min followed by one cycle at 72°C for 5 min. PCR products were
separated at 100 V for 90 minutes in 1.5% agarose gel containing ethidium bromide and
visualized using GelDoc (Biorad). PCR products were purified using the Exosap purification
kit (illustra ExoStar 1-Step, Dutscher, Brumath, France) and subjected to bidirectional DNA
sequencing using the BigDye terminator v3.1 cycle sequencing kit (Applied Biosystems,
Foster City, CA, USA) and an Applied Biosystems 3730 XL capillary sequencer. Sequence
analysis was carried out using BLAST and GenBank sequences.

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**Results and discussion**

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***In silico*** ***analysis***
We mainly focused on plasmid families previously found to be involved in the spread of resistance genes in Enterobacteriaceae (Carattoli, 2013, 2011 and 2009). Since no complete sequence of IncFIII, IncFIV, IncFVI, IncFVII and IncY plasmids have been reported and their sequenced segments do not contain relaxase genes, these plasmids were excluded from this study. So were those of the IncR family which is known not to contain any relaxase gene (Alvarado et al., 2012) and those of the IncJ family and R391-like elements that are part of integrative and conjugative elements (Burrus et al., 2006) the analysis of which is beyond the scope of this study. Also not considered here were some relatively rare groups (e.g. IncI2 plasmids) or poorly resolved groups (e.g. IncQ3 or IncT plasmids) (Alvarado et al., 2012).

The majority of plasmids had a single putative relaxase locus, which is consistent with the review by Smillie *et al.* (Smillie et al., 2010). In contrast, complete GenBank sequences of IncHI1 and IncHI2 plasmids had one to three putative relaxase/helicase loci on each reported plasmid (Supplementary Table S1). CD-Search results for each protein confirmed the presence of relaxase- or helicase-specific multidomains. We finally used five relaxase clades (arbitrarily designed HIα, HIβ, HIγ, HIδ and HIε) present among the various IncHI1 and/or IncHI2 plasmids (Supplementary Table S1). HIβ and HIδ relaxases were found to be encoded only on IncHI1 plasmids and HIγ and HIε relaxases only on IncHI2 plasmids while HIα relaxases were encoded on both IncHI1 and IncHI2 plasmids. We therefore designed three oligonucleotide primers covering the HIα, HIβ and HIγ relaxase, respectively.

*In silico* analysis led to the design of 24 pairs of primers for six multiplex PCRs (targeting the relaxase genes of plasmids belonging to Inc groups A/C, colE, FIA, FIB, FIC, FII, FIIk, FV, HI1, HI2, I1, L/M, N, P1α, Q1, U, W, X1, X2, X3 and X4) and one simplex PCR (targeting the relaxase genes of plasmids belonging to Inc groups B/O and K) (Table 1A).
189 Primer evaluation using transconjugants and transformants

190 In order to assess the sensitivity and specificity of each PCR, primers were tested using a
191 collection of 60 recipient cells, with PBRT as the reference method (Table 2). Each primer
192 pair was validated using all recipient cells, first in a simplex and then a multiplex PCR and
193 target DNA of either single cells or cell mixtures was used. PCR conditions were optimized
194 and all amplicons were sequenced. E. coli strain J53 was used as negative control in PCR
195 experiments to test for possible cross-hybridization with chromosomal DNA. No non-specific
196 amplification was observed.

197 All PRaseT results were consistent with the PBRT results, except for two strains which
198 carried an IncR (K. pneumoniae strain S51) or an IncFIB/FII replicon (E. coli strain 81), that
199 were undetected by the PRaseT method. For the first strain the result was not unexpected as
200 IncR plasmids do not encode relaxases. For the second strain, the result was more surprising
201 as IncF plasmids are known to be conjugative in most cases (Smillie et al., 2010). As it has
202 been previously reported that Mob regions and mating pair formation (MPF) systems were in
203 general of the same type (Smillie et al., 2010), we designed new primers targeting other
204 conserved genes of the type IV secretion system (T4SS), i.e. traB (encoding a secretin-like
205 protein) and traC (encoding an ATPase) (Supplementary Table S2). The eighteen PRaseT-
206 positive IncF plasmids reported in Table 2 tested positive for traB and traC while the PRaseT-
207 negative IncFIB/FII plasmid also tested negative with T4SS typing (data not shown).
208 Interestingly, no transconjugant but only transformant was obtained from the parental strain of
209 the later plasmid. We considered three possibilities: (i) a very divergent IncF relaxase gene
210 that could not hybridize with our primers was present, (ii) the relaxase gene was truncated or
211 (iii) the gene was absent. Complete sequencing of the plasmid will be performed to confirm
212 one of these possibilities.
For IncHI plasmids, three primer sets were mandatory to differentiate IncHI1 from IncHI2. As noted above, in most cases, the identification of relaxases from IncHI1 and IncHI2 plasmids was obtained with positive results for HIβ- and HIγ-primers respectively (e.g. transconjugants S01477 and 102, Table 2). However, when Hiα PCR was the only positive result (e.g. IncHI2-containing Salmonella S09118), we used T4SS-typing as a complement to PRaseT (Supplementary Table S2). The positive result of PCR targeting the T4SS from IncHI2 plasmids confirmed the presence of a HI2 relaxase in S09118.

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## 222 Evaluation of relaxase gene typing using clinical strains

To further confirm the specificity of the designed primer set, 39 clinical strains, each carrying from one to seven different replicon types, were submitted to PRaseT (Table 3). An example of the results is shown in Fig. 1. For 31 strains (79.5%) there was a perfect correlation between the results obtained with PRaseT and PBRT.

Five strains were positive with PBRT but negative with PRaseT. Among these, two (E. coli strains 19 and 34, Table 3) carried IncFIA/FIB/FII replicons. PCR targeting the T4SS genes *traB* and *traC* was negative, and the plasmid from neither strain could be transferred to a recipient cell by conjugation. Similarly, for the IncU plasmid of strain KpS15 tested negative with PRaseT, primers targeting *virB4*, the gene coding for the ATPase of the T4SS of IncU plasmids (Supplementary Table 2), were used. The result was negative. The two remaining strains (i.e. *E. coli* 33 and 101) carried an IncP plasmid which could not be typed by PRaseT. Our primer pair was designed using the reference plasmid RP4 as template. However, this plasmid belongs to the IncP1α subgroup, while the IncP1 group consists of at least six
236 divergent subgroups (IncP1α, IncP1β, IncP1γ, IncP1δ, IncP1ε and IncP1ζ), many of which are
237 antibiotic resistance vectors in the environment (Alvarado et al., 2012, Bahl et al., 2009 and
238 Heuer et al., 2012). We think that our IncP1α relaxase primers were unable to classify all
239 IncP1 group replicons because of too great a divergence in gene sequences. PRaseT of IncP
240 group plasmids should be improved in further studies.

241 Finally, three strains (K. pneumoniae KpS63 and FM10, and E. coli 105) which were negative
242 with PBRT (after multiple PCR assays) were found to contain IncFIIk, IncHI and IncI1
243 plasmids, respectively, when PRaseT and T4SS typing was used (Table 3). The presence of
244 sequence divergence or mosaic replicons may explain these results that will be clarified by
245 sequencing. These observations underscore the complementarity between the PBRT and
246 PRaseT methods.

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248 Relaxase gene typing in recipient cells non-typeable with PBRT

249 Seventeen recipient cells whose plasmids were found to be non-typeable with PBRT were
250 subjected to typing with PRaseT; parental strains included 14 strains of E. coli and 3 of K.
251 pneumoniae. PCR results are given in Table 4. In the 17 recipient cells, 11 (64.7%) plasmids,
252 mainly pHUSEC41-4-like mobilizable replicons, could be typed with PRaseT. The complete
253 sequence determination of plasmids from 6 recipient cells (i.e. E. coli strains 65, 66, 70, 71,
254 72 and 99) confirmed these results (unpublished data). The majority of blaSHV ESBL genes
255 (six out of eight) were localized on pHUSEC41-4-like plasmids. Such colE-like plasmids
256 have been already reported as resistance vectors in Enterobacteriaceae (García-Fernández et
257 al., 2009).
Six plasmids could be typed neither with PBRT nor with PRaseT (Table 4). Complete sequence is available for 3 of them, i.e. plasmids from *K. pneumoniae* transformant S77 (pKpS77, SHV-12), *E. coli* transconjugant 76 (RCS47v1_pI, SHV-2) and *E. coli* transformant 93 (RCS63v1_p, CTX-M-3) (unpublished data). Plasmids pKpS77, RCS47v1_pI and RCS63v1_p were, respectively, < 50,000 bp, 117,001 bp and 22,308 bp long. In the sequences of two of them (pKpS77 and RCS63v1_p), no transfer region could be identified.

The third plasmid (RCS47v1_pI, *E. coli* transconjugant 76) carried a new relaxase gene which presented 100% query cover and 99% maximum identity with those on plasmids pO111_2 (GenBank accession no. AP010962.1) and p12579_1 (GenBank accession no. CP003110.1); its replicase gene also presented 100% query cover and 99% maximum identity with its counterparts on plasmids pO111_2 and p12579_1. The three other non-typeable plasmids (contained in transformants from *K. pneumoniae* S33, *E. coli* 55 and *E. coli* 86) could not be transferred by conjugation, which is in favor of non-transmissible plasmids; this should, however, be confirmed by complete plasmid sequencing.

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## 273 Conclusions

Our set of six multiplex PCRs and one simplex PCR allowed classification of the most frequently encountered transmissible plasmids in Enterobacteriaceae by targeting their relaxase gene. It stands as a promising complement to the widely used PBRT method in understanding plasmid spread and evolution, and can be applied to epidemiological surveys as well. Considering the complexity of constant plasmid evolution, the combined use of two complementary and practical classification tools should be advantageous and reduce the need for systematic full-length plasmid sequencing.
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**Competing interest**

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The authors declare that they have no competing interests.

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**Acknowledgements**

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We are grateful to Etienne Carbonnelle (Hôpital Européen Georges Pompidou, Paris, France)

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for providing the three control strains from the ECOR collection, and to Glenn Rhodes

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(Lancaster Environment Centre, Lancaster, United Kingdom) for providing the IncU-positive

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control strain.
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<table><caption>Table 1 - Primers used in this study</caption><thead><tr><th>PCR name</th><th>Plasmid type detected</th><th>Primer name</th><th>Sequence (5'-3')</th><th>Plasmid prototype</th><th>EMBL accession No.</th><th>Target site</th><th>Amplicon size (bp)</th><th>Primer concentration (pmol/µL)</th></tr></thead><tbody><tr><td rowspan="4">Multiplex I</td><td rowspan="4">IncFII, IncFIA, IncFIB, IncFII</td><td>MRxeFII-K_for</td><td>CGATATTCTKGAACCCCGTA</td><td>pKPN3</td><td>CP000648.1</td><td>tral</td><td>297</td><td>0.2</td></tr><tr><td>MRxeFII-K_rev</td><td>TCATTGCCCATATAYCGTCC</td><td></td><td></td><td></td><td></td><td>0.2</td></tr><tr><td>MRxeF-tot_for</td><td>ATCAGGAMCCACAGTTACAC</td><td>R100</td><td>AP000342.1</td><td>tral</td><td>753</td><td>0.2</td></tr><tr><td>MRxeF-tot_rev</td><td>GTTTCATGATRTCRCGACTGAG</td><td></td><td></td><td></td><td></td><td>0.2</td></tr><tr><td rowspan="4">Multiplex II</td><td rowspan="4">IncFV</td><td>MRxeFV_for</td><td>CATCAAAGCGAARGAGYAGTTCAC</td><td>pED208</td><td>AF411480.1</td><td>tral</td><td>642</td><td>0.2</td></tr><tr><td>MRxeFV_rev</td><td>TTACCCTCWGCAATATGRCGRA</td><td></td><td></td><td></td><td></td><td>0.2</td></tr><tr><td>IncN</td><td>CCAGTTTAGAAACCCGATCA</td><td>R46</td><td>AY046276.1</td><td>tral</td><td>332</td><td>0.2</td></tr><tr><td>IncI1</td><td>CGTAATCGTCGTAAATGCTG</td><td></td><td></td><td></td><td></td><td>0.2</td></tr><tr><td rowspan="6">Multiplex III</td><td rowspan="2">IncL/M</td><td>MRxeI1_for</td><td>TTTAATCATGAGCAAACGCC</td><td>R64</td><td>AB027308.1</td><td>nikB</td><td>764</td><td>0.2</td></tr><tr><td>MRxeI1_rev</td><td>ACAACYCTGTCACCATAATC</td><td></td><td></td><td></td><td>0.2</td></tr><tr><td rowspan="2">IncA/C</td><td>MRxeLM_for</td><td>TCGCTATATCGTTGGTGATG</td><td>pCTX-M3</td><td>AF550415.2</td><td>nikB</td><td>491</td><td>0.2</td></tr><tr><td>MRxeLM_rev</td><td>GGGTATGTTGGGCAATTTTC</td><td></td><td></td><td></td><td>0.2</td></tr><tr><td rowspan="2">IncA/C</td><td>MRxeAC_for</td><td>TGATTGAGAAAGTGCGGAACTC</td><td>pRA1</td><td>FJ705807.1</td><td>tral</td><td>659</td><td>0.2</td></tr><tr><td>MRxeAC_rev</td><td>CACACCATAGGAGAACTCGT</td><td></td><td></td><td></td><td>0.2</td></tr><tr><td rowspan="4">Multiplex IV</td><td rowspan="2">IncHI1, IncHI2</td><td>MRxeHIα_for</td><td>CCAAAAGACTGACTTGGGAGCA</td><td>pMAK1</td><td>AB366440.1</td><td>helicase</td><td>873</td><td>0.2</td></tr><tr><td>MRxeHIα_rev</td><td>CCGAGTTAGCCATCAAGGAA</td><td></td><td></td><td></td><td>0.2</td></tr><tr><td>MRxeHIβ_for</td><td>GATGGTATGTGGATAGTCCG</td><td>pNDM-CIT</td><td>JX182975.1</td><td>tral</td><td>368</td><td>0.2</td></tr><tr><td>MRxeHIβ_rev</td><td>GAATATGCCTCACYATCGCT</td><td></td><td></td><td></td><td>0.2</td></tr><tr><td rowspan="2">Multiplex V</td><td rowspan="2">IncX1</td><td>MRxeX1_for</td><td>AATGACGTGGTTGAATACGA</td><td>R478</td><td>BX664015.1</td><td>tral</td><td>225</td><td>0.2</td></tr><tr><td>MRxeX1_rev</td><td>AGAATAGAAATCACGCGGTCC</td><td></td><td></td><td></td><td>0.2</td></tr><tr><td rowspan="6">Multiplex V</td><td rowspan="2">IncX2</td><td>MRxeX2_for</td><td>GAAAATGGTCGTAAAGTCAGCT</td><td>pOLA52</td><td>EU370913.1</td><td>taxC</td><td>845</td><td>0.2</td></tr><tr><td>MRxeX2_rev</td><td>TTCAGYCTGACAAGATCACCTGC</td><td></td><td></td><td></td><td>0.2</td></tr><tr><td rowspan="2">IncX3</td><td>MRxeX3_for</td><td>CGAAAATGGTCGTAAGTCTGCA</td><td>R6K</td><td>X95535.1</td><td>taxC</td><td>560</td><td>0.2</td></tr><tr><td>MRxeX3_rev</td><td>CATTACAAAAGCCCGTCCTG</td><td></td><td></td><td></td><td>0.2</td></tr><tr><td rowspan="2">IncX4</td><td>MRxeX4_for</td><td>TGTTGACAAGGAATTCAGGGT</td><td>pIncX-SHV</td><td>JN247852.1</td><td>taxC</td><td>736</td><td>0.2</td></tr><tr><td>MRxeX4_rev</td><td>ATGGTCGTAGCCAACATCAA</td><td></td><td></td><td></td><td>0.2</td></tr><tr><td rowspan="4">Multiplex V</td><td rowspan="2">Non typeable</td><td>MRxeMobC11_for</td><td>ACGGATTCGCTTRCCTGGMAGTG</td><td>pColEST258</td><td>JN247853.1</td><td>mobB</td><td>997</td><td>0.2</td></tr><tr><td>MRxeMobC11_rev</td><td>TTCAGSGCGTACTGGTCGAGATCG</td><td></td><td></td><td></td><td>0.2</td></tr><tr><td>Non typeable</td><td>MRxeMobQu_for</td><td>TCTCCAGAARCAGCGCTACAG</td><td>pIGWZ12</td><td>DQ311641.1</td><td>mob</td><td>481</td><td>0.04</td></tr><tr><td>colE, Non typeable</td><td>MRxeMobQu_rev</td><td>ATGGTCAGCCCATGATGCACGCCA</td><td></td><td></td><td></td><td>0.04</td></tr><tr><td rowspan="2">Multiplex V</td><td rowspan="2">colE, Non typeable</td><td>MRxeMobP5-1_for</td><td>AGTCCGGGARCTYATYGATGCCAC</td><td>pCOLE1</td><td>J01566.1</td><td>mob3</td><td>367</td><td>0.04</td></tr><tr><td>MRxeMobP5-1_rev</td><td>TATCAGRGAGWGTTCAGYRTCCCKGC</td><td></td><td></td><td></td><td>0.04</td></tr><tr><td rowspan="2">Multiplex V</td><td rowspan="2">colE, Non typeable</td><td>MRxeMobP5-2_for</td><td>TCCGGTGGAYTATCTKCTGGGKA</td><td>pTPqnrS-1a</td><td>AM746977.1</td><td>mobA</td><td>656</td><td>0.2</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>
<table><thead><tr><th></th><th>colE, Non typeable</th><th>MRxeMobP5-2_rev</th><th>AAGACTGYTCATARATRGCTCC</th><th>pHUSEC41-4</th><th>HE603113.1</th><th>mob</th><th>147</th><th>0.2</th></tr></thead><tbody><tr><td></td><td></td><td>MRxeMobP5-3_for</td><td>GCTATCAGTCGCCCTGTTCGTC</td><td></td><td></td><td></td><td></td><td>0.04</td></tr><tr><td></td><td></td><td>MRxeMobP5-3_rev</td><td>AGTACCCGATCATCAGTCAGTG</td><td></td><td></td><td></td><td></td><td>0.04</td></tr><tr><td rowspan="5">Multiplex VI</td><td>IncQ1</td><td>MRxeQ1_for</td><td>GATTTATCACCTTACGGCGAA</td><td>RSF1010</td><td>M28829.1</td><td>mobA</td><td>962</td><td>0.2</td></tr><tr><td></td><td>MRxeQ1_rev</td><td>TACCTCCCGCAACTCTTTGG</td><td></td><td></td><td></td><td></td><td>0.2</td></tr><tr><td>IncU</td><td>MRxeU_for</td><td>TATGACACTCTAGCCGGGTT</td><td>pRA3</td><td>DQ401103.1</td><td>nic</td><td>743</td><td>0.2</td></tr><tr><td></td><td>MRxeU_rev</td><td>TGGATCTTGTCCTTCCCACGGTT</td><td></td><td></td><td></td><td></td><td>0.2</td></tr><tr><td>IncP1α</td><td>MRxeP1α_for</td><td>TGAAGTACATCACCGACGAG</td><td>RP4</td><td>X54459.1</td><td>tral</td><td>424</td><td>0.2</td></tr><tr><td></td><td></td><td>MRxeP1α_rev</td><td>GTTTCGTGATTGTCACGCTC</td><td></td><td></td><td></td><td></td><td>0.2</td></tr><tr><td></td><td>IncW</td><td>MRxeW_for</td><td>CATCGAGTTCGATACCCTTG</td><td>R388</td><td>BR000038.1</td><td>trwC</td><td>531</td><td>0.2</td></tr><tr><td></td><td></td><td>MRxeW_rev</td><td>GCCACATAGTAAACGTCCTT</td><td></td><td></td><td></td><td></td><td>0.2</td></tr><tr><td rowspan="2">Simplex I</td><td>IncB/O, IncK</td><td>MRxeBO/K_for</td><td>GAATGCCATYATTCCGMAMAA</td><td>pO113</td><td>AY258503.2</td><td>nikB</td><td>800</td><td>0.2</td></tr><tr><td></td><td>MRxeBO/K_rev</td><td>GTGATATACAGACCATCACTGG</td><td></td><td></td><td></td><td></td><td>0.2</td></tr></tbody></table>
<table><caption>Table 2 - Recipient cells used in validation experiments*</caption><thead><tr><th rowspan="2">No.</th><th rowspan="2">Parental strain</th><th rowspan="2">Tc/Tf</th><th rowspan="2">β-Lactamase</th><th colspan="2">Plasmid classification</th></tr><tr><th>PBRT</th><th>PRaseT</th></tr></thead><tbody><tr><td>6†</td><td>E. coli</td><td>Tc</td><td>TEM-10</td><td>L/M</td><td>L/M</td></tr><tr><td>40†</td><td>E. coli</td><td>Tc</td><td>TEM-24</td><td>A/C</td><td>A/C</td></tr><tr><td>44†</td><td>E. coli</td><td>Tc</td><td>TEM-52</td><td>I1</td><td>I1</td></tr><tr><td>48†</td><td>E. coli</td><td>Tc</td><td>TEM-52</td><td>X1</td><td>X1</td></tr><tr><td>50</td><td>E. coli</td><td>Tc</td><td>TEM-52</td><td>I1</td><td>I1</td></tr><tr><td>52</td><td>E. coli</td><td>Tf</td><td>TEM-52</td><td>X1</td><td>X1</td></tr><tr><td>51</td><td>E. coli</td><td>Tc</td><td>TEM-3</td><td>L/M</td><td>L/M</td></tr><tr><td>57†</td><td>E. coli</td><td>Tc</td><td>SHV-12</td><td>F</td><td>F</td></tr><tr><td>62</td><td>E. coli</td><td>Tc</td><td>SHV-2</td><td>FIB</td><td>F</td></tr><tr><td>64†</td><td>E. coli</td><td>Tc</td><td>SHV-5</td><td>X4</td><td>X4</td></tr><tr><td>73</td><td>E. coli</td><td>Tc</td><td>SHV-4</td><td>FIIK</td><td>FIIK</td></tr><tr><td>81</td><td>E. coli</td><td>Tf</td><td>CTX-M-3</td><td>FIB, FII</td><td>-</td></tr><tr><td>85</td><td>E. coli</td><td>Tf</td><td>CTX-M-1</td><td>FIA</td><td>F</td></tr><tr><td>91†</td><td>E. coli</td><td>Tc</td><td>CTX-M-3</td><td>N</td><td>N</td></tr><tr><td>98</td><td>E. coli</td><td>Tc</td><td>CTX-M-1</td><td>X1</td><td>X1</td></tr><tr><td>100</td><td>E. coli</td><td>Tc</td><td>CTX-M-1</td><td>FIA, FII</td><td>F</td></tr><tr><td>102†</td><td>E. coli</td><td>Tc</td><td>CTX-M-1</td><td>HI2</td><td>HIα, HIγ</td></tr><tr><td>104</td><td>E. coli</td><td>Tf</td><td>CTX-M-1</td><td>FIA, FIB, FII</td><td>F</td></tr><tr><td>105</td><td>E. coli</td><td>Tc</td><td>CTX-M-1</td><td>L/M</td><td>L/M</td></tr><tr><td>108</td><td>E. coli</td><td>Tf</td><td>CTX-M-1</td><td>FIA</td><td>F</td></tr><tr><td>110</td><td>E. coli</td><td>Tc</td><td>CTX-M-1</td><td>X4</td><td>X4</td></tr><tr><td>111</td><td>E. coli</td><td>Tc</td><td>CTX-M-1</td><td>FIA</td><td>F</td></tr><tr><td>114</td><td>E. coli</td><td>Tc</td><td>CTX-M-1</td><td>L/M</td><td>L/M</td></tr><tr><td>118</td><td>E. coli</td><td>Tc</td><td>CTX-M-1</td><td>FIA, FIB</td><td>F</td></tr><tr><td>120</td><td>E. coli</td><td>Tc</td><td>CTX-M-3</td><td>FIB</td><td>F</td></tr><tr><td>125</td><td>E. coli</td><td>Tc</td><td>CTX-M-1</td><td>FII</td><td>F</td></tr><tr><td>126</td><td>E. coli</td><td>Tc</td><td>CTX-M-1</td><td>N</td><td>N</td></tr><tr><td>127</td><td>E. coli</td><td>Tc</td><td>CTX-M-3</td><td>FIB</td><td>F</td></tr><tr><td>S6</td><td>K. pneumoniae</td><td>Tc</td><td>SHV-5</td><td>A/C</td><td>A/C</td></tr><tr><td>S9†</td><td>K. pneumoniae</td><td>Tc</td><td>SHV-4</td><td>FIIK</td><td>FIIK</td></tr><tr><td>S16</td><td>K. pneumoniae</td><td>Tc</td><td>CTX-M-3</td><td>A/C</td><td>A/C</td></tr><tr><td>S19</td><td>K. pneumoniae</td><td>Tc</td><td>CTX-M-15/OXA-1</td><td>N</td><td>N</td></tr><tr><td>S23</td><td>K. pneumoniae</td><td>Tc</td><td>CTX-M-3</td><td>N, A/C</td><td>N, A/C</td></tr><tr><td>S24</td><td>K. pneumoniae</td><td>Tc</td><td>TEM-3</td><td>A/C</td><td>A/C</td></tr><tr><td>S36</td><td>K. pneumoniae</td><td>Tc</td><td>CTX-M-3/OXA-1</td><td>FII</td><td>F</td></tr><tr><td>S43</td><td>K. pneumoniae</td><td>Tc</td><td>CTX-M-3</td><td>N</td><td>N</td></tr><tr><td>S46</td><td>K. pneumoniae</td><td>Tc</td><td>CTX-M-15</td><td>L/M</td><td>L/M</td></tr><tr><td>S51</td><td>K. pneumoniae</td><td>Tc</td><td>SHV-12</td><td>FIIK, R</td><td>FIIK</td></tr><tr><td>S59</td><td>K. pneumoniae</td><td>Tc</td><td>CTX-M-15</td><td>FIIK</td><td>FIIK</td></tr><tr><td>S75</td><td>K. pneumoniae</td><td>Tc</td><td>CTX-M-15/OXA-1</td><td>FIIK</td><td>FIIK</td></tr><tr><td>S82</td><td>K. pneumoniae</td><td>Tc</td><td>CTX-M-15/OXA-1</td><td>FII</td><td>F</td></tr><tr><td>S88</td><td>K. pneumoniae</td><td>Tc</td><td>SHV-2a</td><td>FIIK</td><td>FIIK</td></tr><tr><td>S90†</td><td>K. pneumoniae</td><td>Tf</td><td>SHV-12, KPC-2</td><td>X3</td><td>X3</td></tr><tr><td>S00056</td><td>S. enterica Typhimurium</td><td>Tc</td><td>CTX-M-2</td><td>HI2</td><td>HIγ</td></tr><tr><td>S00319</td><td>S. enterica Havana</td><td>Tc</td><td>CTX-M-15</td><td>HI2</td><td>HIγ</td></tr><tr><td>S01106</td><td>S. enterica Virchow</td><td>Tc</td><td>SHV-12</td><td>I1</td><td>I1</td></tr><tr><td>S01331</td><td>S. enterica Tel el kebir</td><td>Tc</td><td>CTX-M-15</td><td>HI2</td><td>HIγ</td></tr><tr><td>S01477†</td><td>S. enterica Typhimurium</td><td>Tc</td><td>CTX-M-1/CMY-2</td><td>HI1, I1</td><td>I1, HIα, HIβ</td></tr><tr><td>S01650</td><td>S. enterica Brandeburg</td><td>Tc</td><td>CTX-M-14</td><td>FrepB</td><td>F</td></tr><tr><td>S03207</td><td>S. enterica Typhimurium</td><td>Tf</td><td>CTX-M-15</td><td>FIA, FIB</td><td>F</td></tr><tr><td>S03663</td><td>S. enterica Grumpensis</td><td>Tc</td><td>CTX-M-15</td><td>HI2</td><td>HIγ</td></tr><tr><td>S03664</td><td>S. enterica Typhimurium</td><td>Tc</td><td>CTX-M-15</td><td>N</td><td>N</td></tr><tr><td>S04662</td><td>S. enterica Virchow</td><td>Tc</td><td>CTX-M-32</td><td>N</td><td>N</td></tr><tr><td>S05343</td><td>S. enterica Concord</td><td>Tc</td><td>CTX-M-15</td><td>HI2</td><td>HIγ</td></tr><tr><td>S07364</td><td>S. enterica Miami</td><td>Tc</td><td>SHV-2</td><td>N</td><td>N</td></tr><tr><td>S09118</td><td>S. enterica Keurmassar</td><td>Tc</td><td>SHV-12</td><td>HI2, FI</td><td>F, HIα</td></tr><tr><td>S1922†</td><td>S. enterica Kentucky</td><td>Tc</td><td>VIM-2</td><td>W</td><td>W</td></tr><tr><td>S1923</td><td>S. enterica Kentucky</td><td>Tc</td><td>VIM-2</td><td>W</td><td>W</td></tr><tr><td>S27078</td><td>S. enterica Carmel</td><td>Tc</td><td>CTX-M-15</td><td>FrepB</td><td>F</td></tr><tr><td>S7981</td><td>S. enterica Saintpaul</td><td>Tc</td><td>OXA-48</td><td>L/M</td><td>L/M</td></tr></tbody></table>

<footnote>*Tc, transconjugant, Tf, transformant, PBRT, PCR-based replicon typing, PRaseT, plasmid relaxase typing, T4SS, type IV secretion system, ND, no data; †, Tc used as a positive control in relaxase gene typing.</footnote>
<table><caption>387 Table 3 - Application of relaxase gene typing in 39 clinical strains of *Enterobacteriaceae*\*</caption><thead><tr><th rowspan="3">No.</th><th rowspan="3">Species</th><th rowspan="3">B-Lactamase</th><th rowspan="3">PBRT</th><th colspan="5">Plasmid classification</th><th rowspan="3">Simplex I</th></tr><tr><th colspan="5">Plasmid relaxase gene typing</th></tr><tr><th>Multiplex I</th><th>Multiplex II</th><th>Multiplex III</th><th>Multiplex IV</th><th>Multiplex VI</th></tr></thead><tbody><tr><td>3</td><td>E. coli</td><td>TEM-24</td><td>A/C, B/O</td><td></td><td>A/C</td><td></td><td></td><td></td><td></td><td>K-B/O</td></tr><tr><td>15</td><td>E. coli</td><td>TEM-24</td><td>A/C, II</td><td></td><td>A/C, II</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>17</td><td>E. coli</td><td>TEM-24</td><td>A/C, FIB, FII, HI2, X4</td><td>F</td><td>A/C</td><td>H1α, H1γ</td><td>X4</td><td></td><td></td><td></td></tr><tr><td>19</td><td>E. coli</td><td>TEM-21</td><td>A/C, FIA, FIB, FII, K, N, X1</td><td></td><td>A/C, N</td><td></td><td>X1</td><td></td><td></td><td>K-B/O</td></tr><tr><td>23</td><td>E. coli</td><td>TEM-24</td><td>A/C, FIB, FII, II</td><td>F</td><td>A/C, II</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>26</td><td>E. coli</td><td>TEM-24</td><td>A/C, FIB, HI2</td><td>F</td><td>A/C</td><td>H1α, H1γ</td><td></td><td>X1</td><td></td><td>K-B/O</td></tr><tr><td>28</td><td>E. coli</td><td>TEM-21</td><td>A/C, K, X1</td><td></td><td>A/C</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>33</td><td>E. coli</td><td>TEM-24</td><td>A/C, P</td><td></td><td>A/C</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>34</td><td>E. coli</td><td>TEM-24</td><td>A/C, FIA, FIB, FII, K, N, X1</td><td></td><td>A/C, N</td><td></td><td>X1</td><td></td><td></td><td>K-B/O</td></tr><tr><td>40</td><td>E. coli</td><td>TEM-24</td><td>A/C, B/O, FIA</td><td>F</td><td>A/C</td><td></td><td></td><td></td><td></td><td>K-B/O</td></tr><tr><td>50</td><td>E. coli</td><td>TEM-52</td><td>II</td><td></td><td>II</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>53</td><td>E. coli</td><td>TEM-3</td><td>A/C, B/O, FrepB</td><td>FV</td><td>A/C</td><td></td><td></td><td></td><td></td><td>K-B/O</td></tr><tr><td>66</td><td>E. coli</td><td>SHV-12</td><td>FIB, K</td><td>F</td><td></td><td></td><td></td><td></td><td></td><td>K-B/O</td></tr><tr><td>84</td><td>E. coli</td><td>CTX-M-1</td><td>FIB, HI1, N</td><td>F</td><td>N</td><td>H1β</td><td></td><td></td><td></td><td></td></tr><tr><td>88</td><td>E. coli</td><td>CTX-M-1</td><td>FIB, FII, HI1, N</td><td>F</td><td>N</td><td>H1β</td><td></td><td></td><td></td><td></td></tr><tr><td>101</td><td>E. coli</td><td>CTX-M-2</td><td>HI2, P</td><td></td><td></td><td>H1α, H1γ</td><td></td><td></td><td></td><td></td></tr><tr><td>105</td><td>E. coli</td><td>CTX-M-1</td><td>K, L/M</td><td></td><td>L/M, II</td><td></td><td></td><td></td><td></td><td>K-B/O</td></tr><tr><td>106</td><td>E. coli</td><td>CTX-M-1</td><td>FIB, FII, HI1, N</td><td>F</td><td>N</td><td>H1α, H1β</td><td></td><td></td><td></td><td>K-B/O</td></tr><tr><td>112</td><td>E. coli</td><td>CTX-M-3</td><td>FIB, FII, K</td><td>F</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Ec37040</td><td>E. coli</td><td>ND</td><td>A/C, FIB, FII</td><td>F</td><td>A/C</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Ec43681</td><td>E. coli</td><td>ND</td><td>FIA, FIB, FII, Q</td><td>F</td><td>A/C</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>KpS3</td><td>K. pneumoniae</td><td>FOX-3</td><td>A/C, FIIK, X4</td><td>FIIK</td><td>A/C</td><td></td><td>X4</td><td></td><td></td><td></td></tr><tr><td>KpS5</td><td>K. pneumoniae</td><td>DHA-1</td><td>FIIK, L/M, R</td><td>FIIK</td><td>L/M</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>KpS15</td><td>K. pneumoniae</td><td>GES-9</td><td>FIIK, R, U</td><td>FIIK</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>KpS19</td><td>K. pneumoniae</td><td>CTX-M-15, DHA-1</td><td>FII, FIIK, L/M, N</td><td>FIIK, F</td><td>L/M, N</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>KpS20</td><td>K. pneumoniae</td><td>CTX-M-15, SHV-2a</td><td>FII, X4</td><td>F</td><td></td><td></td><td>X4</td><td></td><td></td><td></td></tr><tr><td>KpS26</td><td>K. pneumoniae</td><td>SHV-12</td><td>FIIK, N, R</td><td>FIIK</td><td>N</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>KpS47</td><td>K. pneumoniae</td><td>CTX-M-3</td><td>HI2, L/M</td><td></td><td>L/M</td><td>H1γ</td><td></td><td></td><td></td><td></td></tr><tr><td>KpS63</td><td>K. pneumoniae</td><td>DHA-1</td><td>L/M, R</td><td>FIIK</td><td>L/M</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>KpS83</td><td>K. pneumoniae</td><td>TEM-3</td><td>A/C, FIB, N</td><td>F</td><td>A/C, N</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>KpS88</td><td>K. pneumoniae</td><td>SHV-2a</td><td>FIIK</td><td>FIIK</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>KpS91</td><td>K. pneumoniae</td><td>KPC-2</td><td>A/C, FIIK, X3</td><td>FIIK</td><td>A/C</td><td></td><td>X3</td><td></td><td></td><td></td></tr><tr><td>KpS92</td><td>K. pneumoniae</td><td>CTX-M-14, VIM-1</td><td>FII, FIIK, II</td><td>FIIK, F</td><td>II</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>KpS93</td><td>K. pneumoniae</td><td>CTX-M-15, OXA-48</td><td>II, L/M</td><td></td><td>II, L/M</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>FM-2</td><td>K. pneumoniae</td><td>OXA-48</td><td>FIIK, L/M, N</td><td>FIIK</td><td>L/M, N</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>FM-10</td><td>K. pneumoniae</td><td>OXA-48</td><td>A/C, L/M</td><td></td><td>A/C, L/M</td><td>H1α</td><td></td><td></td><td></td><td></td></tr><tr><td>Kp29537</td><td>K. pneumoniae</td><td>ND</td><td>A/C, FIIK</td><td>FIIK</td><td>A/C</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>S10-1477</td><td>S. enterica Typhimurium</td><td>CTX-M-1, CMY-2</td><td>HI1, II</td><td></td><td>II</td><td>H1α, H1β</td><td></td><td></td><td></td><td></td></tr><tr><td>S10-1526</td><td>S. enterica Typhimurium</td><td>CTX-M-1, CMY-2</td><td>HI1, II</td><td></td><td>II</td><td>H1α, H1β</td><td></td><td></td><td></td><td></td></tr></tbody></table>

<footnote n="388">388 *PBRT, PCR-based replicon typing, ND, not determined.</footnote>
<table><caption>389 Table 4 - Results of relaxase gene typing in 17 recipient cells non-typeable with PBRT*</caption><thead><tr><th>No.</th><th>Parental species</th><th>Tc/Tf</th><th>β-lactamase</th><th>Plasmid relaxase gene typing</th></tr></thead><tbody><tr><td>16</td><td><i>E. coli</i></td><td>Tc</td><td>TEM-21</td><td>C11</td></tr><tr><td>31</td><td><i>E. coli</i></td><td>Tc</td><td>TEM-24</td><td>P5-3</td></tr><tr><td>37</td><td><i>E. coli</i></td><td>Tf</td><td>TEM-24</td><td>P5-3</td></tr><tr><td>55</td><td><i>E. coli</i></td><td>Tf</td><td>TEM-52</td><td>NT</td></tr><tr><td>65</td><td><i>E. coli</i></td><td>Tc</td><td>SHV-4</td><td>P5-3</td></tr><tr><td>66</td><td><i>E. coli</i></td><td>Tc</td><td>SHV-12</td><td>P5-3</td></tr><tr><td>67</td><td><i>E. coli</i></td><td>Tc</td><td>SHV-12</td><td>P5-3</td></tr><tr><td>70</td><td><i>E. coli</i></td><td>Tc</td><td>SHV-12</td><td>P5-3</td></tr><tr><td>71</td><td><i>E. coli</i></td><td>Tc</td><td>SHV-2</td><td>P5-3</td></tr><tr><td>72</td><td><i>E. coli</i></td><td>Tc</td><td>SHV-2</td><td>P5-3</td></tr><tr><td>76</td><td><i>E. coli</i></td><td>Tc</td><td>SHV-2</td><td>NT, P5-3†</td></tr><tr><td>86</td><td><i>E. coli</i></td><td>Tf</td><td>CTX-M-3</td><td>NT</td></tr><tr><td>93</td><td><i>E. coli</i></td><td>Tf</td><td>CTX-M-3</td><td>NT</td></tr><tr><td>99</td><td><i>E. coli</i></td><td>Tc</td><td>CTX-M-1</td><td>Qu, P5-3†</td></tr><tr><td>S33</td><td><i>K. pneumoniae</i></td><td>Tf</td><td>DHA-1</td><td>NT</td></tr><tr><td>S55</td><td><i>K. pneumoniae</i></td><td>Tc</td><td>CTX-M-15, OXA-1</td><td>FIIK</td></tr><tr><td>S77</td><td><i>K. pneumoniae</i></td><td>Tf</td><td>SHV-12</td><td>NT</td></tr></tbody></table>

390

391

392

393

<footnote>*PBRT, PCR based replicon typing, Tc, transconjugant, Tf, transformant, NT, not typeable; †: recipient cells containing two different plasmids (ESBL was carried by plasmids NT and Qu in strains 76 and 99 respectively).</footnote>
# Appendix A. Supplementary data

## Supplementary Table S1 – Relaxase and T4SS-ATPase gene sequences of completely sequenced IncHI plasmids stored in GenBank

## Supplementary Table S2 – Primers used in this study for T4SS-typing

## Supplementary Figure – Relaxase gene typing assays

All PCR products were separated in 1.5% agarose gels. M, molecular size marker (in bp). (a)

Multiplex PCR assay of the IncF, IncFIIK and IncFV relaxase genes. Lanes: 1, K.

*pneumoniae* KpS19; 2, *E. coli* 84; 3, *E. coli* 53; 4, *K. pneumoniae* KpS83; 5, *K.*

*pneumoniae* KpS19+ *E. coli* 53; 6, *E. coli* J53 (negative control). (b)

Multiplex PCR assay of the IncA/C, IncI1, IncL/M and IncN relaxase genes. Lanes: 1, *E. coli* 15; 2, *E. coli* 34; 3, *K.*

*pneumoniae* KpS19; 4, *K. pneumoniae* FM10; 5, *E. coli* 15+ *E. coli* 34+ *K. pneumoniae*

KpS19; 6, *E. coli* J53 (negative control). (c)

Multiplex PCR assay of the Hiα, Hiβ and Hiγ relaxase genes. Lanes: 1, *K. pneumoniae* FM10; 2, *E. coli* 26; 3, *E. coli* 88; 4, *E. coli* 106;

5, *E. Coli* 17+*E. Coli* 106; 6, *E. coli* J53 (negative control). (d)

Multiplex PCR assay of the IncX1, IncX2, IncX3 and IncX4 relaxase genes. Lanes: 1, *E. coli* 17; 2, *K. pneumoniae* S90

recipient cell; 3, *E. coli* ECOR19; 4, *E. coli* 19; 5, *E. coli* J53 (negative control). (e)

Multiplex PCR assay of the MobP5-1, MobP5-2, MobP5-3, MobC11 and MobQu relaxase genes. Lanes

: 1, *E. coli* ECOR6; 2, *E. coli* ECOR10; 3, *E. Coli* 16 recipient cell; 4, *E. Coli* 99 recipient

cell; 5, *E. coli* ECOR6+ *E. coli* ECOR10+*E. Coli* 16 recipient cell+*E. Coli* 70 recipient cell+

*E. Coli* 99 recipient cell; 6, *E. coli* J53 (negative control). (f)

Multiplex PCR assay of the IncP1α, IncQ1, IncU and IncW relaxase genes. Lanes: 1, *E. coli* Ec43681; 2, *S. enterica*

S1922 recipient cell; 3, pFBAOT6 reference plasmid; 4, RP4 reference plasmid; 5, *E. coli*

Ec43681+ pFBAOT6 reference plasmid+ RP4 reference plasmid; 6, *E. coli* J53 (negative

control). (g) Simplex PCR assay of the IncB/O and IncK relaxase genes. Lanes: 1, *E. coli* 34;

2, *E. coli* 66; 3, *E. coli* 40; 4, *E. Coli* 112; 5, *E. coli* 53; 6, *E. coli* J53 (negative control).