Re-incubation of positive anaerobic blood culture bottles for detecting additional anaerobes in polymicrobial bacteremia

ABSTRACT Anaerobes are important causes of bloodstream infections, but may be overlooked in routine blood culture workflows because of their slow growth and frequent involvement in polymicrobial infections. We hypothesized that in polymicrobial bacteremia, rapidly growing organisms may trigger early blood culture positivity before slow-growing anaerobes have sufficiently proliferated, potentially leading to underdetection during routine subculture. In this observational study, we evaluated whether re-incubation of positive anaerobic blood culture bottles improves the detection of anaerobic bacteria. All blood samples were cultured using the BacT/Alert Virtuo automated blood culture system. Adult patients with at least one positive anaerobic blood culture bottle were included. Positive anaerobic bottles were subcultured using the conventional method and then re-incubated for an additional 4 days before repeat subculture to detect additional anaerobes. Species were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, supplemented by 16S rRNA gene sequencing. Of 310 patients with positive anaerobic blood cultures, 29 (9.4%) had an additional 36 bacterial isolates identified after re-incubation, of which only 3 were judged to be contaminants. Of 27 cases with two positive anaerobic bottles, additional organisms were detected in both bottles in 23 cases (85%), and concordant organisms were detected from the suspected infection focus in 9 cases (31%). Additional species identified following repeat incubation included Bacteroides fragilis , Veillonella parvula , Parvimonas micra , Dysgonomonas capnocytophagoides , and Eggerthella lenta . This re-incubation approach provides microbiological evidence that slow-growing anaerobes may be underdetected in routine blood culture workflows for polymicrobial bloodstream infections. IMPORTANCE Anaerobes are important causes of bloodstream infections but may be overlooked in routine blood culture workflows. In polymicrobial infections, rapidly growing microorganisms may trigger early blood culture positivity before slow-growing anaerobes have had adequate time to proliferate, leading to missed detection of slower-growing anaerobes during routine subculture. In this study, we re-incubated positive anaerobic blood culture bottles for an additional 4 days after they became positive to provide more time for slower-growing anaerobes to grow. This led to the detection of 36 additional bacterial isolates that would otherwise have remained undetected, of which only 3 were judged to be contaminants. Some of these slow-growing microorganisms are rarely reported in bloodstream infections and some were concordant with anaerobic microorganisms isolated from the suspected infection focus. These findings provide microbiological evidence that slow-growing anaerobes may be underdetected in routine workflows for polymicrobial bloodstream infections, although prolonged turnaround time limits routine implementation.