Approximately 50 million people suffer from sepsis yearly, and 13 million die from it. For every hour a patient with septic shock is untreated, their survival rate decreases by 8%. Therefore, rapid detection and antibiotic susceptibility profiling of bacterial agents in the blood of sepsis patients are crucial for determining appropriate treatment. Here, we introduce a method to isolate bacteria from whole blood with high separation efficiency through Smart centrifugation , followed by microfluidic trapping and subsequent detection using deep learning applied to microscopy images. We detected, within 2 h, E. coli , K. pneumoniae , or E. faecalis from spiked samples of healthy human donor blood at clinically relevant concentrations as low as 9, 7 and 32 colony-forming units per ml of blood, respectively. However, the detection of S. aureus remains a challenge. This rapid isolation and detection represents a significant advancement towards culture-free detection of bloodstream infections.

Sepsis has an incidence of 50 million cases per year and represents a significant cause of morbidity and mortality worldwide. Current diagnostic methods rely on blood drawn directly into blood culture media in hemoculture bottles, followed by culturing, often taking days to yield results and failing to meet urgent clinical needs. We present here a protocol for isolating and identifying bacteria from blood within 12 h after sampling, bypassing prior hemocultures. Starting from blood added into blood culture media, according to standard hospital sampling practice, we isolated up to 85% of bacteria at clinically-relevant concentrations in less than 15 min using an optimized centrifugation protocol. Subsequent overnight culture of the isolated bacteria on chromogenic agar plates enabled species identification of five of the most prevalent sepsis-causing bacteria. The rapidity and simplicity of the protocol may accelerate the diagnostic pipeline for sepsis patients. Moreover, the use of standard laboratory equipment may enable direct translation to clinical praxis and concatenation with downstream assays for antibiotic susceptibility testing.

Sepsis is a time‐critical condition causing over 13 million deaths annually, with each hour of treatment delay in patients with septic shock increasing mortality by 8%. Rapid pathogen identification is crucial, yet current workflows depend on multiple culture steps that delay pathogen identification and targeted treatment by days. A plug‐and‐play, fully automated centrifuge tube is presented that isolates and concentrates bacteria directly from blood or blood culture using only conventional lab centrifuges. Each tube can process 7.5 ml of sample and yields, within 40 min, a 0.7 mL clear suspension with greater than threefold enhanced bacteria concentration and 99.9% blood cell rejection, ready for downstream detection. It is demonstrated that this approach supports key diagnostic workflows, including 1) a novel isolate‐then‐culture strategy detecting bacterial concentrations as low as 10 CFU/mL; 2) direct matrix‐assisted laser desorption ionization time‐of‐flight (MALDI‐TOF) identification, bypassing subculturing, and; 3) microfluidic single‐cell detection. This fully automated platform is compatible with existing centrifuges, is anticipated to facilitate broader adoption in routine clinical practice, while its ability to enable rapid, same‐workshift bacterial enhancement can reduce diagnostic time by about one day in the context of time‐critical sepsis diagnostics.

Sepsis is a life-threatening condition with high mortality, claiming over 13 million lives annually. In patients with septic shock, each hour of delayed treatment increases the risk of death by 8%. Current diagnostic methods rely heavily on blood culture, a time-consuming process involving multiple culture steps that can take several days, delaying targeted antibiotic therapy. This underscores the urgent need for faster, culture-free diagnostic approaches.

In this work, we developed methods to detect bacteria directly from blood at low, clinically relevant concentrations ( 10 CFU/mL), enabling the potential to bypass the traditional culture process entirely. We further established technologies for direct bacterial identification and antimicrobial susceptibility testing (AST) from blood, bypassing subculture or solid culture steps, thereby facilitating targeted antibiotic therapy.

For detection at clinically relevant concentrations, we combined a centrifugation-based method for efficient bacterial isolation and concentration from blood with microfluidic trapping and automated bacterial detection using deep learning applied to microscopy images. We also designed two novel centrifugal devices to automate the above sample preparation in a single step: (1) a One-step device and (2) an inclined filter device. Both are fully automated, plug-and-play, centrifuge-only systems that isolate and concentrate bacteria while selectively lysing blood cells—without manual intervention and using only standard laboratory centrifuges. The One-step device employs trapped air to control fluid movement during centrifugation, integrating seamlessly into clinical workflows and supporting three key applications: (a) direct subculturing from low bacterial concentrations, eliminating the need for initial blood culture; (b) species identification via matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS); and (c) microfluidic single-cell detection from moderate bacterial loads, along with in-device colorimetric AST. The inclined filter device uses size-based filtration during centrifugation to isolate and concentrate bacteria, lyse blood cells, and enable subculture-based detection.

We further demonstrated AST from positive blood cultures using the One-step device in combination with an impedance-based cytometer, delivering results within 2 hours. Additionally, we adapted the inclined filter device for stool sample preparation, enabling bacterial detection using Optical DNA Mapping and demonstrating the platform’s versatility. 

Together, these technologies provide a rapid, comprehensive, and automated solution for pathogen detection and antibiotic profiling, laying the foundation for same-shift, culture-free sepsis diagnostics.

Sepsis är ett livshotande tillstånd med hög dödlighet som orsakar över 13 miljoner dödsfall årligen. Hos patienter med septisk chock ökar risken för dödlig utgång med 8 % varje timme som behandlingen fördröjs. Nuvarande diagnostiska metoder är starkt beroende av blododling – en tidskrävande process som innefattar flera odlingssteg och kan ta flera dagar, vilket fördröjer riktad antibiotikabehandling. Detta understryker det akuta behovet av snabbare, odlingsfria diagnostiska metoder.

I detta arbete har vi utvecklat metoder för att direkt detektera bakterier i blod vid låga, kliniskt relevanta koncentrationer ( 10 CFU/mL), vilket möjliggör att helt kringgå den traditionella odlingsprocessen. Vi har dessutom utvecklat teknologier för direkt bakteriell identifiering och resistensbestämning (AST) från blod, utan behov av subkultivering eller fasta odlingssteg, vilket underlättar riktad antibiotikabehandling.

För detektion vid kliniskt relevanta koncentrationer kombinerade vi en centrifugeringsbaserad metod för effektiv isolering och koncentrering av bakterier från blod med mikrofluidisk infångning och automatiserad bakteriedetektering med hjälp av djupinlärning applicerad på mikroskopibilder. Vi utvecklade även två nya centrifu genheter för att automatisera ovanstående provberedning i ett enda steg: (1) en One-step-enhet och (2) en lutande filterenhet. Båda är fullt automatiserade, plug-and-play-system som enbart använder centrifugering och som isolerar och koncentrerar bakterier samtidigt som blodceller selektivt lyseras – helt utan manuella moment och med endast standard-laboratoriecentrifuger. One-step-enheten använder luft instängd i enheten för att styra vätskeflödet under centrifugering och kan sömlöst integreras i kliniska arbetsflöden. Enheten har tre huvudsakliga tillämpningar: (a) direkt subkultur från låga bakteriekoncentrationer, vilket eliminerar behovet av initial blododling; (b) artidentifiering via matrix-assisted laser desorption ionization time-of-flight masspektrometri (MALDI-TOF-MS); och (c) mikrofluidisk detektion av enskilda celler vid måttliga bakterielaster, samt färgmetrisk AST i enheten. Den lutande filterenheten använder storleksbaserad filtrering under centrifugering för att isolera och koncentrera bakterier, lysera blodceller och möjliggöra subkultur-baserad detektion.

Vi har även demonstrerat AST från positiva blododlingar med hjälp av One-step-enheten i kombination med en impedansbaserad cytometer, vilket ger resultat inom 2 timmar. Dessutom anpassade vi den lutande filterenheten för provberedning av avföringsprover, vilket möjliggjorde bakteriedetektering med Optical DNA Mapping och visade plattformens mångsidighet.

Tillsammans skapar dessa teknologier en snabb, heltäckande och automatiserad lösning för patogendetektion och antibiotikaprofilering, och lägger grunden för odlingsfri sepsisdiagnostik.

Separating bacteria from infected blood is an important step in preparing samples for downstream bacteria detection and analysis. However, the extremely low bacteria concentration and extremely high blood cell count make efficient separation challenging. In this study, we introduce a method for separating bacteria from blood in a single centrifugation step, which involves sedimentation velocity-based differentiation followed by size-based cross-flow filtration over an inclined filter. Starting from 1 mL spiked whole blood, we recovered 32 ± 4% of the bacteria (Escherichia coli, Klebsiella pneumonia, or Staphylococcus aureus) within one hour while removing 99.4 ± 0.1% of the red blood cells, 98.4 ± 1.4% of the white blood cells, and 90.0 ± 2.6% of the platelets. Changing the device material could further increase bacteria recovery to >50%. We demonstrated bacterial recovery from blood spiked with 10 CFU mL⁻¹. Our simple hands-off efficient separation of low-abundant bacteria approaches clinical expectations, making the new method a promising candidate for future clinical use.