Using the polymerase chain reaction coupled with denaturing gradient gel electrophoresis to investigate the association between bacterial translocation and systemic inflammatory response syndrome in predicted acute severe pancreatitis

Abstract

Aim: To investigate the use of PCR and DGGE to investigate the association between bacterial translocation and systemic inflammatory response syndrome in predicted severe AP.

Methods: Patients with biochemical and clinical evidence of acute pancreatitis and an APACHE II score > or = 8 were enrolled. PCR and DGGE were employed to detect bacterial translocation in blood samples collected on d 1, 3, and 8 after the admission. Standard microbial blood cultures were taken when there was clinical evidence of sepsis or when felt to be clinically indicated by the supervising team.

Results: Six patients were included. Of all the patients investigated, only one developed septic complications; the others had uneventful illness. Bacteria were detected using PCR in 4 of the 17 collected blood samples. The patient with sepsis was PCR-positive in two samples (taken on d 1 and 3), despite three negative blood cultures. Using DGGE and specific primers, the bacteria in all blood specimens which tested positive for the presence of bacterial DNA were identified as E coli.

Conclusion: Our study confirmed that unlike traditional microbiological techniques, PCR can detect the presence of bacteria in the blood of patients with severe AP. Therefore, this latter method in conjunction with DGGE is potentially an extremely useful tool in predicting septic morbidity and evaluating patients with the disease. Further research using increased numbers of patients, in particular those patients with necrosis and sepsis, is required to assess the reliability of PCR and DGGE in the rapid diagnosis of infection in AP.

Figure 1

PCR profiles of total DNA recovered from human blood samples and of E coli DNA (control) obtained using primers BD1 and BD2 specific for bacterial 16S rRNA region. Positive control: Chromosomal DNA from E coli. Lanes 1-11: total DNA extracted from human blood samples.

Figure 2

PCR profile of E coli chromosomal DNA. Lanes 1-3: DNA amplified using primers BG1 and BG4 specific for E coli. Lanes 4-6: DNA amplified using primers BFR1 and BFR2 specific for Bacteroides spp. Lane 7: 1 kb standard DNA ladder.

Figure 3

DGGE profiles of the amplified region of bacterial DNA coding for 16S rRNA using primers F3 and Rev 2. Positive controls: Lane 3. Staphylococcus aureus; Lane 4. Pseudomonas aeruginosa; Lane 5. Bacillus cereus; Lane 6. E coli; Lanes 1-2 and 7-13: bacterial DNA present in human blood samples.