Addressing Diagnostic Challenges in the Detection of Polymicrobial Bacteraemia Using Molecular Microbiological Tools

Mariam Doualeh

Sepsis is a leading cause of morbidity and mortality worldwide, with rapid, accurate diagnosis essential for effective treatment. Polymicrobial bacteraemia, characterised by the coexistence of multiple bacterial species in the bloodstream, poses additional diagnostic challenges, as routine blood cultures may fail to detect co-infecting pathogens. This thesis aimed to evaluate the limitations of conventional blood culture and molecular methods in the context of polymicrobial bacteraemia and to assess how these limitations influence pathogen detection in clinical samples. We first compared the performance of traditional blood culture with an in-house multiplex real-time polymerase chain reaction (PCR) assay using simulated polymicrobial blood cultures. Standard culture detected all pathogens in only 42% of cases, compared to 83% with the multiplex real-time PCR. This discrepancy was most pronounced when slower-growing or low-abundance pathogens coexisted with dominant species, highlighting the limitations of culture-based diagnostics. Notably, Staphylococcus aureus was undetected by routine methods when co-cultured with Escherichia coli at 1:1, 1:10, and 1:25 ratios, only becoming detectable when spiked at 50 times the concentration of E. coli. We then performed a comparative analysis of blood culture and multiplex real-time PCR on clinical samples from adult, paediatric, and neonatal patients at Fiona Stanley Hospital, Perth Children’s Hospital, and King Edward Memorial Hospital to assess whether blood culture underestimates the incidence of polymicrobial bacteraemia. While overall incidence was not significantly underestimated, PCR testing detected pathogens earlier in several cases, whereas culture often required multiple samples collected over time to identify all pathogens, demonstrating the potential of multiplex real-time PCR to enhance diagnostic turnaround time. Additionally, we investigated the impact of bacterial DNAemia on molecular diagnostics by comparing blood samples from 19 healthy adults post-toothbrushing with samples from 10 sepsis patients using 16S rRNA gene PCR. No significant differences in Ct values were observed. This is likely due to the high background of human genomic DNA in whole blood, which can overwhelm low-abundance bacterial DNA by reducing effective primer availability and promoting non-specific primer binding to human sequences, thereby limiting the sensitivity of broad -range bacterial PCR. Further 16S rRNA gene sequencing revealed significant differences in microbial profiles between healthy and sepsis samples but no differences between healthy samples and negative controls. This pattern indicates that the microbial profiles observed in the healthy individuals are more consistent with low-level environmental or reagent-derived contamination than with a genuine endogenous “blood microbiome”. Overall, this thesis supports the integration of molecular techniques with conventional methods to enhance the detection of polymicrobial bacteraemia while highlighting the need for further validation in larger cohorts. Additionally, optimisation of protocols, particularly through host DNA depletion, will be critical for improving the application of universal bacterial molecular tools directly to whole blood in the clinical setting.

Addressing Diagnostic Challenges in the Detection of Polymicrobial Bacteraemia Using Molecular Microbiological Tools

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