ColE1 plasmids are small mobilizable replicons that play an important role in the spread of antibiotic resistance in Pasteurellaceae. In this study, we describe how a natural single nucleotide polymorphism (SNP) near the origin of replication of the ColE1-type plasmid pB1000 found in a Pasteurella multocida clinical isolate generates two independent plasmid variants able to coexist in the same cell simultaneously. Using the Haemophilus influenzae Rd KW20 strain as a model system, we combined antibiotic susceptibility tests, quantitative PCRs, competition assays, and experimental evolution to characterize the consequences of the coexistence of the pB1000 plasmid variants. This coexistence produced an increase of the total plasmid copy number (PCN) in the host bacteria, leading to a rise in both the antibiotic resistance level and the metabolic burden produced by pB1000. Using experimental evolution, we showed that in the presence of ampicillin, the bacteria maintained both plasmid variants for 300 generations. In the absence of antibiotics, on the other hand, the bacteria are capable of reverting to the single-plasmid genotype via the loss of one of the plasmid variants. Our results revealed how a single mutation in plasmid pB1000 provides the bacterial host with a mechanism to increase the PCN and, consequently, the ampicillin resistance level. Crucially, this mechanism can be rapidly reversed to avoid the extra cost entailed by the increased PCN in the absence of antibiotics. KEYWORDS ColE1, experimental evolution, Haemophilus influenzae, plasmid stability pB1000 is a small ColE1-like plasmid carrying the bla ROB-1 ␤-lactamase gene and conferring high-level resistance to ␤-lactams. pB1000 has been described in multiple pathogens from Pasteurellaceae, including Haemophilus influenzae (1-7).The replication of ColE1 plasmids is under the control of a preprimer RNA (RNAII) which is negatively regulated by a small antisense RNA (RNAI) (8). Mutations affecting these RNAs encoded near the origin of replication (oriV) of the plasmid alter both the plasmid copy number (PCN) and the compatibility of ColE1 plasmids. Single mutations can give rise to new compatible plasmids that are able to coexist in the same cell, increasing the global PCN (7,8). These mutations have been widely described in vitro (9) and arise naturally by chance due to the high plasmid copy number exhibited by ColE1-like plasmids (10), but to the best of our knowledge, there are no natural examples of cohabiting ColE1 plasmids differentiated by only a single nucleotide polymorphism (SNP).In this work we analyze a heterozygous pB1000 differentiated only by an SNP near the oriV and isolated from a high-level ampicillin-resistant clinical isolate of Pasteurella multocida (5). We show that this SNP creates a new variant of the plasmid, which

Plasmids can be transferred between cells by conjugation, thereby driving bacterial evolution by horizontal gene transfer. Yet, we ignore the molecular mechanisms of transfer for many plasmids because they lack all protein-coding genes required for conjugation. We solved this conundrum by identifying hundreds of plasmids and chromosomes with conjugative origins of transfer in Escherichia coli and Staphylococcus aureus. These plasmids (pOriT) hijack the relaxases of conjugative or mobilizable elements, but not both. The functional dependencies between pOriT and other plasmids explain their co-occurrence: pOriT are abundant in cells with many plasmids, whereas conjugative plasmids are the most common in the others. We systematically characterized plasmid mobility in relation to conjugation and alternative mechanisms of transfer and can now propose a putative mechanism of transfer for ∼90% of them. In most cases, plasmid mobility seems to involve conjugation. Interestingly, the mechanisms of mobility are important determinants of plasmid-encoded accessory traits, since pOriTs have the highest densities of antimicrobial resistance genes, whereas plasmids lacking putative mechanisms of transfer have the lowest. We illuminate the evolutionary relationships between plasmids and suggest that many pOriT may have arisen by gene deletions in other types of plasmids. These results suggest that most plasmids can be transferred by conjugation.

Objectives To investigate the relevance of multicopy plasmids in antimicrobial resistance and assess their mobilization mediated by phage particles Methods Several databases with complete sequences of plasmids and annotated genes were analysed. The 16S methyltransferase gene armA conferring high-level aminoglycoside resistance was used as a marker in eight different plasmids, from different incompatibility groups, and with differing sizes and plasmid copy numbers. All plasmids were transformed into Escherichia coli bearing one of four different lysogenic phages. Upon induction, encapsidation of armA in phage particles was evaluated using qRT–PCR and Southern blotting. Results Multicopy plasmids carry a vast set of emerging clinically important antimicrobial resistance genes. However, 60% of these plasmids do not bear mobility (MOB) genes. When carried on these multicopy plasmids, mobilization of a marker gene armA into phage capsids was up to 10 000 times more frequent than when it was encoded by a large plasmid with a low copy number. Conclusions Multicopy plasmids and phages, two major mobile genetic elements (MGE) in bacteria, represent a novel high-efficiency transmission route of antimicrobial resistance genes that deserves further investigation.

ColE1 plasmids are important vehicles for the spread of antibiotic resistance in the Enterobacteriaceae and Pasteurellaceae families of bacteria. Their monitoring is essential, as they harbor important resistant determinants in humans, animals and the environment. In this work, we have analyzed ColE1 replicons using bioinformatic and experimental approaches. First, we carried out a computational study examining the structure of different ColE1 plasmids deposited in databases. Bioinformatic analysis of these ColE1 replicons revealed a mosaic genetic structure consisting of a host-adapted conserved region responsible for the housekeeping functions of the plasmid, and a variable region encoding a wide variety of genes, including multiple antibiotic resistance determinants. From this exhaustive computational analysis we developed a new PCR-based technique, targeting a specific sequence in the conserved region, for the screening, capture and sequencing of these small plasmids, either specific for Enterobacteriaceae or specific for Pasteurellaceae. To validate this PCR-based system, we tested various collections of isolates from both bacterial families, finding that ColE1 replicons were not only highly prevalent in antibiotic-resistant isolates, but also present in susceptible bacteria. In Pasteurellaceae, ColE1 plasmids carried almost exclusively antibiotic resistance genes. In Enterobacteriaceae, these plasmids encoded a large range of traits, including not only antibiotic resistance determinants, but also a wide variety of genes, showing the huge genetic plasticity of these small replicons. Finally, we also used a metagenomic approach in order to validate this technique, performing this PCR system using total DNA extractions from fecal samples from poultry, turkeys, pigs and humans. Using Illumina sequencing of the PCR products we identified a great diversity of genes encoded by ColE1 replicons, including different antibiotic resistance determinants, supporting the previous results achieved with the collections of bacterial isolates. In addition, we detected cryptic ColE1 plasmids in both families with no known genes in their variable region, which we have named sentinel plasmids. In conclusion, in this work we present a useful genetic tool for the detection and analysis of ColE1 plasmids, and confirm their important role in the dissemination of antibiotic resistance, especially in the Pasteurellaceae family of bacteria.