The effect of human serum DNAases on the ability to detect antibiotic-killed Escherichia coli in blood by PCR

PCR has proved superior to conventional blood culture for diagnosing bacter aemia in the presence of antibiotics. Nevertheless, even PCR might yield fa lse-negative results if the template DNA were to be cleaved by serum DNAase s after antibiotics had induced bacterial death. To evaluate the cleavage o f bacterial template DNA by human serum DNAase I, serum samples inoculated with purified Escherichia coli DNA were incubated with increasing amounts o f recombinant human DNAase (rhDNAase) and then examined by a PCR specific f or E, coli, As a prerequisite of potential DNAase attack, the release of E, coli DNA after antibiotic-induced bacterial death was quantified by fluore scence microscopy and flow cytometry, Finally, the influence of rhDNAase on the PCR-based detection of antibiotic-killed E, coli in serum was assessed . The results indicated that purified E, roll DNA is remarkably stable in h uman serum; positive PCR results did not decrease significantly until the r atio of recombinant human DNAase I:E, coli rose to 10(6):1. As only 14.8-28 .4% of the total E, roll DNA was released after antibiotic killing, the PCR -based detection of E, coli fell by only 10% when cefotaxime-killed E, coli were incubated,vith rhDNAase, It was concluded that human serum DNAases an d antibiotic killing do not compromise the reliability of PCR examinations for bacteraemia.