Analysis of large CTX-M plasmids in E. coli using antisense silencing

1. Analysis of large CTX-M plasmids in E. coli using antisense silencing Abigail R. Edwards(1) (2), Liam Good (1) , Roberto M. La Ragione (2) (1) Pathology and Infectious Diseases, Royal Veterinary College, North Mymms, Hertfordshire, AL9 7TA (2) Department of Bacteriology, Animal Health and Veterinary Laboratories Agency, New Haw, Addlestone, Surrey, KT15 3NB 35% 39.5% 80.5% Methods Introduction • Extended spectrum beta-lactamases, including the CTX-M family, pose serious problems for human and animal health as they substantially reduce the number of effective therapeutic antibiotics. In enteric bacteria, ESBLs are carried on large (>60kb) promiscuous plasmids, which are of interest because, apart from contributing resistance traits, little is known about how they benefit or burden the host bacteria. Two ESBL E. coli strains were isolated from a cattle farm for study. Due to difficulty in studying genes on large plasmids by traditional means, antisense silencing is an attractive alternative for functional analyses, and it can give a clearer picture of gene contribution in comparison to other techniques which may distort whole cell processes. • The project objectives are: • 1. Sequence and annotate the two ESBL plasmids; • 2. Establish RNA silencing of large plasmid encoded genes; • 3. Determine the fitness contribution of the plasmid and certain plasmid-borne genes in relation to the host. • Two ESBL E. coli strains were isolated from an outbreak on a cattle farm • O1 strain • O26 Strain • Plasmids were named pARE01 and pARE02 respectively • Transformants into K12 derivative DH10B were created by electroporation • Strains were sequenced and are currently being annotated • A nitrocefin assay was used to look at relative enzyme activity • Two methods of silencing were used to target ftsZ and ctx-m • PNA oligomer • Antisense RNA silencer inducibly expressed from a plasmid • Antimicrobial resistance phenotypic effect of silencing was tested with growth curves at different CTX concentration and disc diffusions assays Results Figure 3: Elongation of E. coli cells by progressive ftsZ silencing (XAS method) visualised with DAPI stain 50μM IPTG No IPTG Figure 1: Illustrations of the plasmids as created by the BaSYS server 100μM IPTG 70μM IPTG Figure 5: Standard curve of CTX-M producing O1 cells with red star and arrow marking rate of silenced cells (PNA method) Figure 4: Cleavage of Nitrocefin by ESBL expressed on large plasmids in DH10B Figure 6: Standard curve of CTX-M producing cells with red star and arrow marking rate of silenced cells (XAS method) IPTG, CTX disc IPTG, other discs No IPTG, other discs No IPTG, CTX disc Figure 7: Zones of Inhibition from disc diffusion assay with O1 cells (XAS) Figure 2: Table of pARE01 plasmid genes Figure 8: Growth differences between silenced and unsilenced cells in the presence of different concentrations of cefotaxime. • Automated and manual sequence annotation is ongoing. (Fig. 1&2) • The ftsZ gene, essential for cell division, was silenced to different degrees causing elongation visible under fluorescence microscopy using DAPI stain. (Fig 3) • The nitrocefin assay before silencing showed a statistically significant difference (p=0.0026) in the rates of cleavage between the beta-lactamase from pARE01 and the beta-lactamase from pARE02 despite carrying the same ESBL gene and a very high whole plasmid sequence similarity. The rate at which plasmid pARE01 cleaves nitrocefin is 58% of the rate of plasmid pARE02. (Fig. 4) • The ctx-m gene was targeted by synthetic oligomer PNA and XAS methods and amount of silencing measured against a standard curve (Fig. 5&6) • This silencing was also tested to determine if it could cause an increase in susceptibility to cefotaxime (fig 7&8) Conclusions and Future Work • The plasmids have 99% homology as determined by nucleotide BLAST • Although the plasmids isolated from the two strains appear to be the same or very similar there may be a difference in expression or activity of the enzyme • The environmental O1 strain appears to be more susceptible to silencing with ftsZ silencing however the opposite with anti-ctx-m silencers. • The ctx-m gene can be reduced in activity in comparison to the activity of untreated cells however this is not enough to cause a phenotypic difference. • Future studies will include further analysis of the sequence data and silencing of the interesting targets found by this analysis. Phenotypic testing e.g. Biolog will also be carried out to assess metabolic effects of the silencing. Together these studies will help to determine how plasmid carriage influences bacterial fitness. AREdwards@rvc.ac.uk