Rapid, label‐free antibiotic susceptibility determined directly from positive blood culture

Abstract

Bacterial bloodstream infections are a significant cause of global morbidity and mortality. Constrained by low bacterial burdens of 1-100 colony-forming-units per ml blood (CFU/ml), clinical diagnosis relies on lengthy culture amplification and isolation steps prior to identification and antibiotic susceptibility testing (AST). The resulting >60-h time to actionable treatment not only negatively impacts patient outcomes, but also increases the misuse and overuse of broad-spectrum antibiotics that accelerates the rise in multidrug resistant infections. Consequently, the development of novel technologies capable of rapidly recovering bacteria from blood-derived samples is crucial to human health. To address this need, we report a novel bacterial recovery technology from positive blood cultures that couples selective hemolysis with centrifugation through a sucrose cushion to perform rapid, background-free cytometric ASTs without long subculturing steps. Demonstrated on the most common bloodstream infection-causing bacteria: Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus, near-pure bacteria are rapidly recovered (≤15 min) with minimal user intervention. Susceptibilities of recovered bacteria are readily performed via high throughput flow cytometry with excellent agreement with much slower, standard microbroth dilution assays. Altogether, this novel direct-from-positive blood culture AST technology enables susceptibility determinations within as little as 5 h, post blood culture positivity.

Leading Pathogens Involved in Co-Infection and Super-Infection with COVID-19: Forensic Medicine Considerations after a Systematic Review and Meta-Analysis

“…Evidence suggests that the overuse of broad-spectrum antibiotics may adversely affect the sensitivity of bacterial culture methods and the ability to detect co-infections [ 54 ]. Therefore, for the patients included in this review, the administration of broad-spectrum antibiotic therapy may have decreased the sensitivity of bacterial culture methods, resulting in an underestimation of the number of co-infections.…”

Current and Future Flow Cytometry Applications Contributing to Antimicrobial Resistance Control

“…Table 1 provides examples of FCM-based AST approaches developed for the detection of AMR in pathogenic bacteria. FCM assays were demonstrated to accurately and rapidly detect AMR profiles of pathogens directly in clinical samples [ 37 , 38 , 42 , 47 , 48 , 49 , 50 , 51 , 52 , 53 ]. Most of the developed AST methods based on FCM used fluorescent dyes to assess the viability of microbial cells after exposure to antibiotics.…”

“…Metabolic changes following exposure to a range of antibiotics are detected in less than two hours using FCM in conjunction with fluorescent dyes [ 39 , 40 , 41 ]. To overcome the nonspecific dye-bacteria interactions, Filbrun et al (2022) [ 53 ] developed a label-free FCM approach for the rapid assessment of bacterial antibiotic susceptibilities directly from positive blood culture. The FCM method was used to determine antibiotic-induced changes in count rate, taking into account the scatter position.…”

Integrated Microfluidic Chip for Rapid Antimicrobial Susceptibility Testing Directly from Positive Blood Cultures

“…For the low concentrations of infecting bacteria in blood, about 1–100 colony-forming-units (CFU) per mL, which is 10 7 to 10 9 times lower than that of mammalian blood cells, blood culturing is still the gold standard for BSI diagnosis . The traditional AST methods widely used in clinical microbiology laboratories, including broth microdilution, disk diffusion, E-test, and automated systems, still rely heavily on further overnight subcultures after blood cultures, which are laborious and time-consuming. – The whole procedure often lasts over 3 days because of the three overnight culture steps involved: blood culture, subculture, and AST culture .…”