Qualitative and quantitative 16S rRNA gene-based real-time PCR and direct sequencing were applied for rapid detection and identification of bacterial DNA (bactDNA) in 356 ascites samples. bactDNA was detected in 35% of samples, with a mean of 3.24 log copies ml ؊1 . Direct sequencing of PCR products revealed 62% mixed chromatograms predominantly belonging to Grampositive bacteria. Terminal restriction fragment length polymorphism (T-RFLP) results of a sample subset confirmed sequence data showing polymicrobial DNA contents in 67% of bactDNA-positive ascites samples. Spontaneous bacterial peritonitis (SBP) is the most severe bacterial infection in patients with decompensated liver disease (1). As evidenced by culture-based diagnosis, Gram-negative bacteria of the genera Escherichia and Klebsiella are the most frequent cause for SBP (2, 3). However, recent data reveal that also Grampositive bacteria such as Staphylococcus and Enterococcus species may be responsible for bacterial peritonitis, especially in hospitalized patients (4, 5). Since detection rates of classical culture techniques are low in the routine setting, the causative agent remains unknown in many cases of SBP (3).Application of culture-independent PCR-based methods for the detection of bacterial DNA (bactDNA) in ascites has been proposed as a suitable tool to improve pathogen identification in patients at risk for SBP (6) and has indicated that bactDNA derives in most cases from a single pathogen (7-9). However, recent studies using 16S rRNA-based fingerprinting analyses have shown that ascites may also be polymicrobial (10,11) and that the bacterial spectrum is broader than that previously reported in context with SBP (11).Based on these findings, we analyzed a set of ascites samples using primers targeting the 16S rRNA gene for qualitative and quantitative PCR and further characterized bactDNA-positive samples by direct sequencing and terminal restriction fragment length polymorphism (T-RFLP) analysis in order to determine the frequency of detectable bactDNA in ascites from patients with end-stage liver disease and to identify the corresponding bacterial agents.A total of 356 ascites samples from 174 patients with liver cirrhosis (77.6% alcoholic, 11.5% cryptogenic, 2.9% viral hepatitis, 1.7% genetic disorders or metabolic diseases, 1.7% autoimmune hepatitis, 0.6% primary biliary cirrhosis, 3.5% nonalcoholic fatty liver disease, and 0.6% cardiac) were obtained at the University Hospital Leipzig, between February 2011 and December 2012. Thirty-seven percent of the patients (n ϭ 61) underwent several diagnostic paracenteses (mean number of paracenteses, 3.9; range, 2 to 11). All patients gave full written informed consent to the study protocol, which was approved by the local ethics committee.Ascitic fluid samples were routinely analyzed for total leukocyte count using an automated blood cell counter and for the presence of bacterial and fungal pathogens by routine microbiological culture with direct inoculation of agar plates.For culture-independe...

BactDNA quantification in ascitic fluid samples using culture-independent 16S rRNA gene-based methods seems to be an interesting approach to identify patients at risk of reduced survival. Our study warrants further evaluation of antibiotic treatment in patients with molecular bacterascites.

Background: As culture-dependent bacterial identification methods are still limited to capture colonisation or infection of ascites fluid we established a culture-independent PCR-based method for the detection, quantification and differentiation of bacterial DNA (bactDNA). This report aims to characterize risk factors for bactDNA identification in non-leukocytic ascites fluid (nlAF) and for progression to SBP.

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