Identification of bla(CMY-7) and associated plasmid-mediated resistance genes in multidrug-resistant Escherichia coli isolated from dogs at a veterinary teaching hospital in Australia

Objectives: To determine clonality and identify plasmid-mediated resistance genes in 11 multidrug-resistant Escherichia coli (MDREC) isolates associated with opportunistic infections in hospitalized dogs in Australia. Methods: Phenotypic (MIC determinations, modified double-disc diffusion and isoelectric focusing) and genotypic methods (PFGE, plasmid analysis, PCR, sequencing, Southern hybridization, bacterial conjugation and transformation) were used to characterize, investigate the genetic relatedness of, and identify selected plasmid-mediated antimicrobial resistance genes, in the canine MDREC. Results: Canine MDRECs were divided into two clonal groups (CG 1 and 2) with distinct restriction endonuclease digestion and plasmid profiles. All isolates possessed bla(CMY-7) on an similar to 93 kb plasmid. In CG 1 isolates, bla(TEM), catA1 and class 1 integron-associated dfrA17-aadA5 genes were located on an similar to 170 kb plasmid. In CG 2 isolates, a second similar to 93 kb plasmid contained bla(TEM) and unidentified class 1 integron genes, although a single CG 2 strain carried dfrA5. Antimicrobial susceptibility profiling of E. coli K12 transformed with CG 2 large plasmids confirmed that the bla(CMY-7)-carrying plasmid did not carry any other antimicrobial resistance genes, whereas the bla(TEM)/class 1 integron-carrying plasmid carried genes conferring resistance to tetracycline and streptomycin also. Conclusions: This is the first report on the detection of plasmid-mediated bla(CMY-7) in animal isolates in Australia. MDREC isolated from extraintestinal infections in dogs may be an important reservoir of plasmid-mediated resistance genes.

The in vivo expression of actin/salt-resistant hyperactive DNase I inhibits the development of anti-ssDNA and anti-histone autoantibodies in a murine model of systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is characterised by the production of autoantibodies against ubiquitous antigens, especially nuclear components. Evidence makes it clear that the development of these autoantibodies is an antigen-driven process and that immune complexes involving DNA-containing antigens play a key role in the disease process. In rodents, DNase I is the major endonuclease present in saliva, urine and plasma, where it catalyses the hydrolysis of DNA, and impaired DNase function has been implicated in the pathogenesis of SLE. In this study we have evaluated the effects of transgenic overexpression of murine DNase I endonucleases in vivo in a mouse model of lupus. We generated transgenic mice having T-cells that express either wild-type DNase I (wt. DNase I) or a mutant DNase I ( ash. DNase I), engineered for three new properties - resistance to inhibition by G-actin, resistance to inhibition by physiological saline and hyperactivity compared to wild type. By crossing these transgenic mice with a murine strain that develops SLE we found that, compared to control nontransgenic littermates or wt. DNase I transgenic mice, the ash. DNase I mutant provided significant protection from the development of anti-single-stranded DNA and anti-histone antibodies, but not of renal disease. In summary, this is the first study in vivo to directly test the effects of long-term increased expression of DNase I on the development of SLE. Our results are in line with previous reports on the possible clinical benefits of recombinant DNase I treatment in SLE, and extend them further to the use of engineered DNase I variants with increased activity and resistance to physiological inhibitors.

RNA interference-inducing hairpin RNAs in plants act through the viral defence pathway

RNA interference (RNAi) is widely used to silence genes in plants and animals. it operates through the degradation of target mRNA by endonuclease complexes guided by approximately 21 nucleotide (nt) short interfering RNAs (siRNAs). A similar process regulates the expression of some developmental genes through approximately 21 nt microRNAs. Plants have four types of Dicer-like (DCL) enzyme, each producing small RNAs with different functions. Here, we show that DCL2, DCL3 and DCL4 in Arabidopsis process both replicating viral RNAs and RNAi-inducing hairpin RNAs (hpRNAs) into 22-, 24- and 21 nt siRNAs, respectively, and that loss of both DCL2 and DCL4 activities is required to negate RNAi and to release the plant's repression of viral replication. We also show that hpRNAs, similar to viral infection, can engender long-distance silencing signals and that hpRNA-induced silencing is suppressed by the expression of a virus-derived suppressor protein. These findings indicate that hpRNA-mediated RNAi in plants operates through the viral defence pathway.