Study On Plasmid-mediated AmpC β-lactamases In Local Enterobacteriaceae

Background.Many species of Enterobacteriaceae have chromosomal ampC genes that confer resistance to mostβ-lactam antibiotics.These genes have also been acquired by plasmids,allowing them to move into other species such as Escherichia coli and Klebsiella pneumoniae.The CMY-2(originating from Citrobacter) and DHA-1(from Morganella morganii)β-lactamases appear to be the most common AmpC enzymes worldwide.While bla_CMY-2-like genes appear to have been transferred to plasmids by ISEcp1 and bla_DHA-1-like genes by ISCR1,relatively few sequenced examples provide information about the wider genetic contexts of these genes.Little was known about the distribution of ampC genes in clinical isolates from Australia,so we have screened selected isolates from the Sydney area for ampC genes,and have begun to examine the wider genetic contexts and plasmid associations of these genes.Methods.E.coli (n=52) and K.pneumoniae clinical isolates(n=31) collected from different patients at four hospitals in western Sydney between Feb 2005 and Jan 2008 for which the cefoxitin MIC was≥16ug/ml were subjected to a multiplex PCR to detect different groups of ampC genes.Complete ampC genes were sequenced and conjugative plasmids carrying these genes were obtained by mating.Isolates were typed by pulse-field gel electrophoresis and plasmids by PCR-based replicon typing.PCR was used to screen for associated qnr genes and to map the contexts of ampC genes.Results.Only two different types of ampC genes were detected,bla_DnA-1-like(2 E.coli,4 K.pneumoniae) and bla_CMY-2-like(6 E.coli,2 K.pneumoniae).One bla_DHA gene had 1 change from an ampC gene found in a Morganella morganii strain and 9 nucleotides changes from plasmid-borne bla_DHA-1 but the proteins differed at only one position(N152H).One bla_CMY-2-like gene had 2 adjacent nucleotide changes from bla_CMY-2 that both affected the same amino acid(V240S).A qnrB4 gene was detected in all but one isolate with a bla_DHA-1-like gene.Several different types conjugative plasmids were obtained,bla_DHA-1 genes were associated with ISCR1 or IS26 in different genetic structures related to those seen previous in France and China,may be derived from one another as a result of rearrangement,insertions and deletions. Conclusions.Two different types of plasmid-borne ampC genes,including novel variants,were found in local E.coli and K.pneumoniae isolates,but apparently explained only a small proportion(14/83=17%) of cefoxitin resistant phenotypes,pJIE092 may precede pJIE142 and pRDDHA.One full article has been published in the journal Pathology(Paradoxical effect of Klebsiella pneumoniae OmpK porin deficiency).A more substantial part of this work included the detailed characterization of the genetic contexts of bla_DHA-1 and bla_CMY-2-like genes was presented as a poster at the prestigious 48^th Interscience Conference on Antimicrobial Agents and Chemotherapy in Washington DC in October 2008 and two manuscripts are in preparation.