Characterisation of bacteria in ascites—reporting the potential of culture-independent, molecular analysis

Abstract

Spontaneous bacterial peritonitis (SBP) is a severe complication of liver disease. A significant proportion of patients have culture-negative ascites, despite having similar signs, symptoms and mortality to those with SBP. Therefore, empirical antibiotic treatment for infection is often started without knowledge of the causative organisms. Here, we investigated the potential of molecular techniques to provide rapid and accurate characterisation of the bacteria present in ascitic fluid. Ascites samples were obtained from 29 cirrhotic patients undergoing clinically indicated therapeutic paracentesis. Bacterial content was determined by terminal restriction fragment length polymorphism (T-RFLP) analysis, quantitative polymerase chain reaction (PCR) and 16S ribosomal clone sequence analysis. Bacterial signal was detected in all samples, compared to three out of ten using standard methods. Bacterial loads ranged from 5.5 × 10 2 to 5.4 × 10 7 cfu/ml, with a mean value of 1.9 × 10 6 cfu/ml (standard deviation± 9.6 × 10 6 cfu/ml). In all but one instance, bacterial species identified by culture were also confirmed by molecular analyses. Preliminary data presented here suggests that culture-independent, molecular analyses could provide rapid characterisation of the bacterial content of ascites fluid, providing a basis for the investigation of SBP development and allowing early and targeted antibiotic intervention.

Application of Qualitative and Quantitative Real-Time PCR, Direct Sequencing, and Terminal Restriction Fragment Length Polymorphism Analysis for Detection and Identification of Polymicrobial 16S rRNA Genes in Ascites

“…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).…”

Ascitic Microbiota Composition Is Correlated with Clinical Severity in Cirrhosis with Portal Hypertension

“…Following incubation, pellets were exposed to light for 10 min using an LED Active Blue equipment light source (Ingenia Biosystems, Terrassa, Spain). Total DNA extraction was then performed as described previously [13]. Full details of the protocol used are provided as Methods S1.…”

“…This assay was described previously [24], and applied in the analysis of ascites [13]. Full details of the protocol used are provided as Methods S1.…”

“…Here, approaches typically exploit the phylogenetically informative 16S ribosomal RNA gene, and include quantitative (Q) PCR (a means of determining bacterial load) and pyrosequencing (a massive parallel sequencing approach that allows the rapid, high depth characterisation of bacterial composition). Such approaches have been applied across a wide range of clinical contexts, including the analysis of intestinal microbial communities of cirrhotic patients [12], and the identification of bacterial species present in ascites [13] (although the presented study is the first reported use of pyrosequencing in this context). However, whilst having the potential to be greatly informative, such molecular techniques are weakened by their inability to distinguish between DNA present in viable bacterial cells, from that present in dead bacteria or in the extracellular environment.…”

Performance of the LightCycler SeptiFast Test M grade in Detecting Microbial Pathogens in Purulent Fluids

“…Molecular methods based on species-specific PCR or broad- range PCR for detecting the 16S rRNA gene followed by sequencing for species identification are being increasingly used for the etiological diagnosis of purulent infections, including SBP, septic arthritis, or CAPD-related peritonitis (4,7,8,12,18,19). These assays have been shown to improve the diagnostic efficiency of culture methods due to their higher sensitivities, especially with samples obtained from patients who have been exposed to antibiotics prior to specimen sampling.…”