Enhanced detection of polymicrobial bloodstream infections with 16S rDNA PCR library amplicons and clinical relevance

Abstract

Polymicrobial bloodstream infections (BSIs) are clinically challenging because conventional blood culture may fail to recover all causative microorganisms, particularly in patients with low microbial burden, prior antimicrobial exposure, or infections caused by fastidious organisms. This study evaluated a 16S rDNA PCR library construction strategy based on cloning and Sanger sequencing as a complementary approach to conventional blood culture and compared its microbial detection profile with that of direct 16S rDNA amplicon high-throughput sequencing. We retrospectively analyzed 16,970 blood culture specimens collected at a hospital in northwestern China to characterize the clinical distribution and epidemiological features of BSIs. In a retrospective cohort of 295 patients with culture-confirmed BSI, clinical predictors associated with blood culture-defined polymicrobial BSI were evaluated using multivariable logistic regression and receiver operating characteristic (ROC) curve analyses, with particular emphasis on white blood cell (WBC) count and procalcitonin (PCT) levels. In a separate molecular comparison cohort of 20 patients, cell-free DNA (cfDNA) was analyzed using two 16S rDNA-based workflows: a clone-based approach involving PCR amplification, gel purification, cloning, amplified ribosomal DNA restriction analysis (ARDRA), and Sanger sequencing, and a direct 16S rDNA amplicon high-throughput sequencing approach. Among the 16,970 blood culture specimens, 1,321 were positive, including 45 polymicrobial BSI cases. Escherichia coli was the most frequently identified pathogen, followed by coagulase-negative staphylococci and Brucella spp. Compared with monomicrobial BSI, polymicrobial BSI was associated with higher WBC counts and PCT levels. In multivariable analysis, both WBC count and PCT were independently associated with polymicrobial BSI, and their combination showed good discriminatory performance (AUC = 0.927). In the separate molecular comparison cohort of 20 patients, the three methods showed distinct detection ranges. Blood culture detected 0–1 microbial taxon per patient, with one pathogen identified in 17 patients and negative results in 3 patients. The clone-based method detected a broader microbial spectrum, identifying 0–5 taxa per patient, whereas direct 16S rDNA amplicon sequencing showed the broadest detection range, with more than five taxa detected in 9 patients. In contrast, 16S rDNA high-throughput sequencing generated broader taxonomic profiles but showed lower agreement with blood culture. Compared with conventional blood culture, the 16S rDNA PCR library method showed an overall percent agreement of 80.0%. WBC and PCT may help identify patients at increased risk of polymicrobial BSI and may support the selection of patients for additional molecular testing. The cloning-based 16S rDNA PCR library strategy provides a complementary approach to conventional blood culture and may offer a balance between broader microbial detection and more focused sequence-level interpretation compared with direct amplicon high-throughput sequencing. Its potential utility may be particularly relevant in selected patients with culture-negative or clinically suspected polymicrobial BSI.