Isolation of bacterial DNA followed by sequencing and differing cytokine response in peritoneal dialysis effluent help in identifying bacteria in culture negative peritonitis

Nephrology (Carlton). 2018 Feb;23(2):148-154. doi: 10.1111/nep.12969.

Abstract

Aim: The treatment of peritoneal dialysis related culture negative peritonitis is empirical which increases the cost of therapy and moreover antibiotic resistance. We aimed the study to isolate bacterial DNA from PD effluent and indentify bacteria causing peritonitis in culture negative situations. We have also studied the cytokine response with different bacteria causing peritonitis.

Methods: We have isolated bacterial DNA from PD effluent of culture negative and culture positive peritonitis patients. Bacterial DNA was subjected to polymerase chain reaction using universal bacteria specific primers and subsequently to Gram type specific primers for the differentiation of the etiologic agents into Gram-positive and Gram-negative. The amplified products were sequenced and subjected to blast search to identify agent at genus/ species level.

Results: Of the 30 molecular method positive samples, 16 (53.33%) samples were positive for Gram-negative bacteria and 4 (13.33%) for Gram-positive, while the remaining10 (33.33%) were positive for both Gram-positive and Gram-negative bacteria. We have found organisms that usually do not grow on normal culture methods. TNF-α was significantly associated with Gram-positive peritonitis and regulatory cytokine IL-10 with Gram-negative peritonitis.

Conclusions: The molecular techniques are helpful in detecting and identifying organisms from culture negative PD effluent.

Keywords: Bacterial DNA; Culture negative; Peritonitis; Polymerase chain reaction.

MeSH terms

  • Adult
  • Bacteriological Techniques*
  • Biomarkers / metabolism
  • Cytokines / metabolism*
  • DNA, Bacterial / genetics*
  • DNA, Bacterial / isolation & purification
  • Female
  • Gram-Negative Bacteria / classification
  • Gram-Negative Bacteria / genetics*
  • Gram-Negative Bacteria / immunology
  • Gram-Negative Bacteria / isolation & purification
  • Gram-Negative Bacterial Infections / diagnosis
  • Gram-Negative Bacterial Infections / immunology
  • Gram-Negative Bacterial Infections / microbiology*
  • Gram-Positive Bacteria / classification
  • Gram-Positive Bacteria / genetics*
  • Gram-Positive Bacteria / immunology
  • Gram-Positive Bacteria / isolation & purification
  • Gram-Positive Bacterial Infections / diagnosis
  • Gram-Positive Bacterial Infections / immunology
  • Gram-Positive Bacterial Infections / microbiology*
  • Host-Pathogen Interactions
  • Humans
  • Interleukin-10 / metabolism
  • Male
  • Middle Aged
  • Peritoneal Dialysis / adverse effects*
  • Peritonitis / diagnosis
  • Peritonitis / immunology
  • Peritonitis / microbiology*
  • Polymerase Chain Reaction
  • Predictive Value of Tests
  • Tumor Necrosis Factor-alpha / metabolism

Substances

  • Biomarkers
  • Cytokines
  • DNA, Bacterial
  • IL10 protein, human
  • Tumor Necrosis Factor-alpha
  • Interleukin-10