A tale of two plasmids: contributions of plasmid associated phenotypes to epidemiological success among Shigella.

Dissemination of antimicrobial resistance (AMR) genes by horizontal gene transfer (HGT) mediated through plasmids is a major global concern. Genomic epidemiology studies have shown varying success of different AMR plasmids during outbreaks, but the underlying reasons for these differences are unclear. Here, we investigated two Shigella plasmids (pKSR100 and pAPR100) that circulated in the same transmission network but had starkly contrasting epidemiological outcomes to identify plasmid features that may have contributed to the differences. We used plasmid comparative genomics to reveal divergence between the two plasmids in genes encoding AMR, SOS response alleviation and conjugation. Experimental analyses revealed that these genomic differences corresponded with reduced conjugation efficiencies for the epidemiologically successful pKSR100, but more extensive AMR, reduced fitness costs, and a reduced SOS response in the presence of antimicrobials, compared with the less successful pAPR100. The discrepant phenotypes between the two plasmids are consistent with the hypothesis that plasmid-associated phenotypes contribute to determining the epidemiological outcome of AMR HGT and suggest that phenotypes relevant in responding to antimicrobial pressure and fitness impact may be more important than those around conjugation in this setting. Plasmid phenotypes could thus be valuable tools in conjunction with genomic epidemiology for predicting AMR dissemination.

Plasmids Increase the Competitive Ability of Plasmid-Bearing Cells Even When Transconjugants Are Poor Donors, as Shown by Computer Simulations.

Bacterial cells often suffer a fitness cost after conjugative plasmids' entry because these cells replicate slower than plasmid-free cells. Compensatory mutations may appear after tens of or a few hundred generations, reducing or eliminating this cost. A previous work based on a mathematical model and computer simulations has shown that plasmid-bearing cells already adapted to the plasmid may gain a fitness advantage when plasmids transfer into neighboring plasmid-free cells because these cells are still unadapted to the plasmid. These slow-growing transconjugants use fewer resources, which can benefit donor cells. However, opportunities for compensatory mutations in transconjugants increase if these cells become numerous (through replication or conjugation). Moreover, transconjugants also gain an advantage when transferring the plasmid, but the original donors may be too distant from conjugation events to gain an advantage. To understand which consequence prevails, we performed further computer simulations allowing versus banning transfer from transconjugants. The advantage to donors is higher if transconjugants do not transfer plasmids, mainly when donors are rare and when the plasmid transfer rate (from donors) is high. These results show that conjugative plasmids are efficient biological weapons even if the transconjugant cells are poor plasmid donors. After some time, conjugative plasmids gain other host-benefit genes, such as virulence and drug-resistance.

Combining Modules for Versatile and Optimal Labeling of Lactic Acid Bacteria: Two pMV158-Family Promiscuous Replicons, a Pneumococcal System for Constitutive or Inducible Gene Expression, and Two Fluorescent Proteins.

Labeling of bacterial cells with fluorescent proteins allows tracking the bacteria in competition and interactomic in vivo and in vitro studies. During the last years, a few plasmid vectors have been developed aimed at the fluorescent labeling of specific members of the lactic acid bacteria (LAB), a heterogeneous group that includes microorganisms used in the food industry, as probiotics, or as live vectors for mucosal vaccines. Successful and versatile labeling of a broad range of LAB not only requires a vector containing a promiscuous replicon and a widely recognized expression system for the constitutive or regulated expression of the fluorescence determinant, but also the knowledge of the main features of the entire plasmid/host/fluorescent protein ensemble. By using the LAB model species Lactococcus lactis, we have compared the utility properties of a set of labeling vectors constructed by combining a promiscuous replicon (pMV158 or pSH71) of the pMV158 plasmid family with the gene encoding either the EGFP or the mCherry fluorescent protein placed under control of promoter PX or PM from the pneumococcal mal gene cluster for maltosaccharide uptake and utilization, respectively. Some vectors carrying PM also harbor the malR gene, whose product represses transcription from this promoter, thus enabling maltose-inducible synthesis of the fluorescent proteins. We have determined the plasmid copy number (PCN) and segregational stability of the different constructs, as well as the effect of these features on the fitness and fluorescence intensity of the lactococcal host. Constructs based on the pSH71 replicon had a high copy number (∼115) and were segregationally stable. The copy number of vectors based on the pMV158 replicon was lower (∼8-45) and varied substantially depending on the genetic context of the plasmid and on the bacterial growth conditions; as a consequence, inheritance of these vectors was less stable. Synthesis of the fluorescent proteins encoded by these plasmids did not significantly decrease the host fitness. By employing inducible expression vectors, the fluorescent proteins were shown to be very stable in this bacterium. Importantly, conditions for accurate quantification of the emitted fluorescence were established based on the maturation times of the fluorescent proteins.