Universal Method for detecting and capturing ColE1 plasmids reveals high prevalence of small plasmids in multiresistant clinical and Pasteurellaceae Santos-Lopez, A.1, Ortega-Huedo, R. 1, San Millan, A. 1, Bernabe-Balas, C. 1, Gutierrez, B. 1 , Carrilero, L. 1, Ovejero, C.M. 1, Prasad, K.N.2, Gonzalez-Zorn, B. 1

1 Universidad Complutense de Madrid, Spain 2 Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, India

[email protected]

Introduction Results In the last years the importance of ColE1-like plasmids in antimicrobial resistance In silico analysis reveled that our system could detect all ColE1 plasmids described Finally, we tested 50 multiresistant Enterobacteriaceae isolates from the Lucknow has increased (1, 2, 3). ColE1 like plasmids have been found both in Pasteurellaceae to date. Evidence for the importance of ColE1-like replicons is that 91% of Hospital in India. 74% of the isolates bore ColE1 plasmid resulting in positive to the and Enterobacteriaceae carrying genes that confer resistance to key antibiotics for Pasteurellaceae ColE1 plasmids deposited in the databases bear at least one detection PCR. Capture PCR generated up to 4 different amplicons per cell clinical practice such as TEM-1, CMY beta-lactamases, aac(6’)-Ib-cr and QnrB19. resistance gene. In addition the 29% of Enterobacteriaceae ColE1 plasmids corresponding to ColE1 replicons coexisiting in the same isolate. 6 plasmids were There replicons are capable of cohabiting in the same cell, conferring multidrug carry resistance determinants, most of them beta-lactamases or aminoglycosides completely sequenced (figure 3), proving the utility of our detection and capture phenotype to the and can be mobilized disseminating resistance resistance genes. method. determinants among different bacteria (1). We have designed a PCR-based screening system capable of detecting and capturing all known ColE1-like plasmids. Methods

Two PCR-based screening system were designed for both families. The first one was used to detect the presence of ColE1-like replicon in the cell. The “detection” PCR, using a universal primers for each family, was designed to amplify a fragment of 450 bp in Enterobacteriaceae and In order to test our system, we confirmed by analyzing Pasteurellaceae strains 250 bp in Pasteurellaceae. Conclusions bearing more than one replicon that our screening system was able to capture all (figures 1A and 2). The ColE1 plasmids cohabiting in a single cell (figure 2). We analyzed 200 susceptible second PCR, was based in • We have developed a universal method to detect and amplify Pasteurellaceae strains. Detection PCR resulted positive in two isolates ( a “capture” system, ColE1 plasmids in Enterobacteriaceae and Pasteurellaceae. stomatis and Taxon Bisgaard 16). Capture PCR, here gave rise to two amplicons of consisting of the same pair 2,1 and 2,7 kb. The plasmids have a size of 2,3 and 2,9 kb and are the smallest • We have shown high prevalence of ColE1 plasmids in of primers but amplifying ColE1 plasmids described to date (figures 2 and 3A). multiresistant isolates. outwards, thus allowing the amplification of the entire plasmid (figure 1B and 2). References

1. San Millán, A. et al. Multiresistance in Is Mediated by Coexistence of Small Plasmids. Antimicrob. Agents Chemother. 53: 339-404 2. San Millán, A. et al. influenzae Clinical Isolates with Plasmid pB1000 Bearing blaROB-1: Fitness Cost and Interspecies Dissemination. Antimicrob. Agents Chemother. 54: 1506-1511. 3. Pallechi, L. et al. Characterization Of Small ColE-like Plasmids Mediating Widespread Dissemination of the qnrB19 Gene In Commensal enterobacteria. Antimicrob. Agents Chemother. 54:678-682.