A contemporary pediatric automated blood culture system reliably recovered common neonatal sepsis pathogens at low and ultra-low bacterial concentrations, including inocula at or below one colony-forming unit per milliliter. In an experimental laboratory evaluation modeling low-density neonatal bacteremia, the platform detected six clinically important bacterial pathogens across varying specimen volumes and concentrations, with the vast majority of positive cultures flagging within twenty-four hours of automated incubation. Organism-specific growth characteristics drove the most substantial differences in time to automated detection, while increasing the inoculated suspension volume primarily boosted overall pathogen recovery rather than accelerating detection speeds.
The investigation was designed to address diagnostic challenges surrounding neonatal sepsis, where clinical signs frequently overlap with normal postnatal transition and other neonatal conditions, prompting extensive empiric antimicrobial exposure in uninfected infants. Although blood culture serves as the clinical reference microbiological test for neonatal bacteremia, concerns have long persisted regarding its sensitivity under typical neonatal conditions, which frequently involve low-level bacteremia at or below ten colony-forming units per milliliter. While early foundational work conducted three decades ago established that pathogen recovery depends heavily on inoculated blood volume when bacterial densities are low, limited contemporary data had evaluated modern pediatric blood culture platforms across clinically relevant specimen volumes.
To assess recovery rates and time to positivity under standardized conditions, the researchers evaluated six common neonatal pathogens: Escherichia coli, Haemophilus influenzae, Streptococcus agalactiae, Staphylococcus aureus, Staphylococcus epidermidis, and a clinical isolate of Staphylococcus capitis from a preterm infant. Bacterial suspensions were prepared in sterile normal saline to an initial turbidity equivalent to a 0.5 McFarland standard and subsequently diluted in ten-fold series to target organism concentrations. These suspensions were mixed in equal parts with adult whole blood anticoagulated with lithium heparin, producing a 1:1 blood-saline experimental suspension at final pathogen concentrations of 10 to 20, 5 to 10, 2.5 to 5, 1.25 to 2.5, and 1 or fewer colony-forming units per milliliter.
The experimental protocol utilized BD BACTEC Peds Plus/F culture bottles inoculated with suspension aliquots of 0.25, 0.5, 0.75, 1.0, and 2.0 milliliters. Because the experimental matrix consisted of equal parts blood and saline suspension, these test aliquots corresponded to whole-blood volumes of 0.125, 0.25, 0.375, 0.5, and 1.0 milliliter, respectively. Inoculated bottles were transferred into the automated BD BACTEC FX blood culture system within fifteen to twenty minutes of preparation and continuously monitored for up to five days. To confirm the precise density of viable bacteria introduced into each bottle, quantitative colony counts were performed by plating aliquots of the suspensions onto blood agar or chocolate agar.
Across the complete study, duplicate experimental runs evaluated a total of 312 blood culture bottles, encompassing 50 bottles per pathogen, 60 bottles across each concentration group, 60 bottles across each suspension volume category, and 12 uninoculated negative control bottles. All negative control bottles remained entirely free of microbial growth throughout five days of automated incubation. Across the active testing arms, clinically important neonatal pathogens remained detectable across all five evaluated concentration tiers and across all five inoculated suspension volumes.
Pathogen recovery demonstrated a clear concentration-dependent response across the tested bacterial species. At concentrations of five colony-forming units per milliliter or greater, overall recovery rates reached 90 to 100 percent for all six organisms. At ultra-low bacterial densities of one or fewer colony-forming units per milliliter, recovery varied substantially by pathogen species, ranging from 30 percent for Staphylococcus aureus to 80 percent for both Escherichia coli and Streptococcus agalactiae. Non-staphylococcal pathogens maintained consistently high recovery across most tested conditions, whereas staphylococcal recovery was lower at smaller inoculated suspension volumes, particularly at ultra-low organism densities.
Time to automated positivity, defined as the interval between placing the bottle in the instrument and the system flagging positive, differed markedly among bacterial species. Escherichia coli and Streptococcus agalactiae exhibited the fastest detection times, registering median times to positivity of approximately 10 to 12 hours across concentration ranges, including at ultra-low inocula. In contrast, coagulase-negative staphylococci required substantially longer incubation periods, with Staphylococcus capitis recording median times to positivity of up to 25.7 hours at concentrations of one or fewer colony-forming units per milliliter.
Statistical analyses using the Kruskal-Wallis test with Bonferroni correction revealed that increasing organism concentrations significantly shortened time to positivity for Escherichia coli, Haemophilus influenzae, Streptococcus agalactiae, and Staphylococcus capitis. Conversely, concentration-dependent differences in detection time did not achieve statistical significance for Staphylococcus aureus or Staphylococcus epidermidis. Despite statistical significance for several organisms, the absolute differences in time to positivity associated with varying bacterial concentrations were generally modest when compared to the baseline differences observed between different bacterial species.
Inoculated suspension volume exerted comparatively little influence on detection speed among recovered blood cultures. A statistically significant association between suspension volume and time to positivity appeared solely in Staphylococcus aureus cultures, where a volume of 2.0 milliliters achieved faster detection than 0.25 milliliters, with no significant volume-dependent acceleration detected for the remaining five organisms. In addition, two culture bottles failed to flag positive during automated incubation but subsequently yielded bacterial colonies during terminal subculture, an occurrence consistent with the manufacturer’s established performance parameters for the instrument platform.
The investigators emphasized several methodological boundaries, noting that adult donor whole blood diluted with saline was used to provide a standardized matrix, which does not entirely reproduce undiluted neonatal blood with its distinct immune profile. Furthermore, the analysis was confined to a single automated platform using aerobic pediatric bottles, omitting anaerobic bottles, fungal pathogens, and prior antimicrobial therapy. The authors concluded that because smaller specimen volumes still retained substantial pathogen recovery, sub-milliliter samples should not be viewed as diagnostically uninformative, while reinforcing that measuring and documenting actual inoculated blood volume remains a vital component of neonatal diagnostic and antimicrobial stewardship.
The experimental protocol incorporated rigorous pre-analytical timing and microbiological verification steps to maintain testing consistency across all evaluation arms. Inoculation of the blood culture bottles was completed within thirty to sixty minutes following suspension preparation, and each organism run included negative control bottles containing either 0.5 or 2.0 milliliters of sterile donor whole blood. To verify automated instrument performance, positive bottles were promptly subcultured onto two blood agar plates and one chocolate agar plate, alongside MacConkey agar for gram-negative isolates. Cultures were maintained at 35 degrees Celsius in five to ten percent carbon dioxide, with a second blood agar plate incubated anaerobically at 35 degrees Celsius. Aerobic growth was evaluated at twenty-four hours, while both aerobic and anaerobic cultures underwent formal assessment at forty-eight hours. The identical incubation conditions and assessment timeline were applied during terminal subcultures for bottles that remained negative after five days of automated incubation. Statistical analyses were performed using IBM SPSS Statistics version 29.0.
Detailed stratified analyses underscored that the interaction between pathogen density and inoculated suspension volume was most pronounced among staphylococcal isolates at ultra-low concentrations. In contrast, recovery rates for non-staphylococcal pathogens remained consistently elevated across nearly all evaluated experimental settings. The clinical importance of these distinctions centers on the pathophysiology of late-onset neonatal sepsis, which is frequently characterized by low-density bacteremia involving commensal organisms. In such presentations, delayed automated detection can prolong broad-spectrum empiric antibiotic therapy before microbiological confirmation and targeted susceptibility results become accessible. The authors observed that advancements in automated detection systems, culture media, resin technology, and specialized bottle designs likely contributed to the high recovery frequencies achieved in modern systems despite low initial bacterial densities.
These experimental findings also contextualize recurring challenges in neonatal clinical care, where concerns regarding iatrogenic blood loss, difficult venipuncture, and limited circulating blood volume frequently result in smaller blood culture volumes being obtained from premature infants. Crucially, these same preterm infants face the greatest risk of late-onset sepsis caused by low-density bacteremia, precisely the clinical scenario in which specimen volume proves most consequential. The investigators emphasized that because the experimental inoculum was a 1:1 mixture of blood and saline rather than undiluted whole blood, their results illustrate the influence of inoculated specimen volume and expected organism count rather than establishing an experimental whole-blood threshold. Consequently, current neonatal guidelines recommending the collection of at least one milliliter of blood represent pragmatic clinical targets derived from broader literature rather than an experimentally defined cut-off. Furthermore, at ultra-low bacterial concentrations, stochastic sampling effects mean that smaller inoculated volumes may randomly fail to contain a viable organism, demonstrating that sterile results under trace conditions cannot be attributed solely to automated analytical performance.
Documenting the precise volume of blood inoculated into culture bottles addresses critical diagnostic and antimicrobial stewardship priorities within the neonatal intensive care setting. Unwarranted empiric antibiotic therapy in uninfected infants generates recognized adverse consequences, including disruption of the developing microbiome, selection for antimicrobial resistance, exposure to invasive procedures, prolonged hospitalization, and parent-infant separation. Systematically recording inoculated whole-blood volumes provides essential clinical context for interpreting negative culture results, directly informing clinician confidence when deciding whether empiric therapy can be safely stopped. Quality improvement initiatives demonstrate that target inoculum volumes are achievable with focused hospital programs and should serve as a cornerstone of neonatal stewardship. Building on these observations, the authors concluded that future clinical research must evaluate whether routine measurement and documentation of whole-blood culture volume improves pathogen recovery, aids the interpretation of negative cultures, and strengthens antimicrobial stewardship outcomes in clinical practice.
Subject of Research: Detection and time to positivity of neonatal sepsis pathogens in pediatric automated blood culture
Article Title: Detection of common neonatal sepsis pathogens using a contemporary pediatric blood culture system
Article References: Faugno, A., Strunk, T., MacKay, C., Joubert, I. A., Peck, A., Currie, A., Nathan, E., & Porter, M. (2026). Detection of common neonatal sepsis pathogens using a contemporary pediatric blood culture system. Pediatric Research. https://doi.org/10.1038/s41390-026-05626-9
Image Credits: AI Generated
DOI: 10.1038/s41390-026-05626-9
Keywords: neonatal sepsis, blood culture, pediatric bacteremia, time to positivity, diagnostic stewardship, coagulase-negative staphylococci, Escherichia coli, Streptococcus agalactiae, Detection, common, neonatal, sepsis
