still blood culture, this method is incapable of providing immediate results to clinicians [1] and often leads to false-negative outcomes. [2] The rapid detection and identification of bacteria in blood is vitally important because the delayed administration of antibiotics results in increased mortality [3,4] and prompt treatment of infection is frequently required for patients, particularly in emergency departments and intensive care units of hospitals. In addition, given that pathogen load is frequently proportional to patient mortality, [5] it is critical to gain quantitative information regarding the bacteria present in blood, which is still achieved largely by blood culture. While time-to-positivity (TTP) of blood cultures has been considered to be a prognostic tool for a limited range of bacterial species, such as Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), and Streptococcus pneumoniae (S. pneumoniae), [6][7][8][9][10] it has not been applied more widely because correlations between TTP data for certain bacterial species and the mortality of patients was obscure in several clinical studies. [11] Despite these significant drawbacks, blood culture is still the clinical laboratory method most widely used to identify pathogenic bacteria in the blood due to the lack of appropriate, alternative approaches.Mass spectrometry (MS), [12,13] polymerase chain reaction (PCR) [14,15] and loop-mediated isothermal amplification [16] methods have all been incorporated into diagnostic procedures used for the identification of pathogenic bacteria in blood. However, they are often valid only after significant growth, in vitro, of the bacteria present in biological samples. This is often time-consuming and may not be possible, at all, for certain bacterial species due to their insufficient growth rates in standard culture conditions. Although some commercially available MS-and PCR-based diagnostic systems work directly on whole blood, eliminating the most time-consuming step of bacterial culture, [17,18] their clinical impact is still contentious due to inconsistent performance in the presence of inhibitors (e.g., human DNA, iron and heparin) and complex background signals in blood. [19] Fluorescence in situ hybridization (FISH) has permitted the unique capability in a cytogenetic analysis by using fluorescentThe current diagnosis of bacteremia mainly relies on blood culture, which is inadequate for the rapid and quantitative determination of most bacteria in blood. Here, a quantitative, multiplex, microfluidic fluorescence in situ hybridization method (μFISH) is developed, which enables early and rapid (3-h) diagnosis of bacteremia without the need for prior blood culture. This novel technology employs mannose-binding lectin-coated magnetic nanoparticles, which effectively opsonize a broad range of pathogens, magnetically sequestering them in a microfluidic device. Therein, μFISH probes, based on unique 16S rRNA sequences, enable the identification and quantification of sequestered pathogens both in saline a...

Recent studies have suggested the existence of a blood microbiome in the healthy host. However, changes in the blood microbiome upon bloodstream infection are not known. Here, we analyzed the dynamics of the blood microbiome in a porcine model of polymicrobial bacteremia induced by fecal peritonitis. Surprisingly, we detected bacterial populations in the bloodstream even before the infection, and these populations were maintained over time. The native blood microbiome was notably taxonomically different from the fecal microbiome that was used to induce peritonitis, reflecting microbial tropism for the blood. Although the population composition after the infection was similar to that of the native blood microbiome, new bacterial strains entered the bloodstream upon peritonitis induction as clinical symptoms relevant to sepsis developed. This indicates that the bacteria detected in the blood before peritonitis induction were derived from the blood rather than a contamination. Comparison of the functional pathways enriched in the blood and fecal microbiomes revealed that communication and stress management pathways are essential for the survival of the blood microbiome.

Recent studies have proposed the existence of a blood microbiome, even in the healthy host. However, we do not know how the blood microbiome changes when a bloodstream infection (BSI) occurs. Here, we analyzed the dynamics of the blood microbiome in a porcine model of polymicrobial bacteremia induced by fecal peritonitis. Serial blood samples were taken over 12 hours post-induction of fecal peritonitis, and BSI was validated by conventional blood culture and assessment of clinical symptoms. The bacterial populations in the blood microbiome were retained throughout the experimental period. However, there were significant taxonomic differences between the profile in the fecal and blood microbiomes, reflecting tropism for the blood environment. We also confirmed that the microbiota we detected was not contaminated by low mass bacteria in the bloodstream. However, at the same time, we noted a slight increase in Bacteroidetes, which is a major component of the gut microbiome, as sepsis developed. Comparison of the functional pathways in the blood and fecal microbiomes revealed upregulation of pathways involved in environmental interactions, and downregulation of those related to cell proliferation, in the former. Based on the enriched biological pathways, we concluded that communication and stress management pathways are essential for the survival of the blood microbiome under harsh conditions. This study suggests that the microbiota can be stably retained in the bloodstream over time. Although further investigation in humans is required, we suggest that the blood microbiome may be another factor to be considered in the context of BSI and subsequent sepsis.

The current diagnosis of bacteremia mainly uses blood culture, which is insufficient to offer rapid and quantitative determination of pathogens in blood. Here, we report a quantitative and sequential multiplexed fluorescence in situ hybridization in a microfluidic device (μFISH) that enables early and rapid (2-hour) diagnosis of bacteremia without prior blood culture. Mannose-binding lectin-coated magnetic nanoparticles enrich a broad range of pathogens, and μFISH enables identification and quantification of the magnetically confined bacteria. We detect Escherichia coli (E. coli) and measure their relative proportions to universal bacteria levels in the bacteremic blood of a porcine model and human whole blood collected from E. coli-infected patients, which was elusive with the conventional bacteremia diagnosis methods. Thus, μFISH can be used as a versatile tool to rapidly identify pathogens and further assess the number of both culturable and non-culturable bacteria in biological and environmental samples.