Search

Bloodstream infections are a significant cause of morbidity and mortality worldwide. The rapid initiation of effective antibiotic treatment is critical for patients with bloodstream infections. However, the diagnosis of bloodborne pathogens is largely complicated by the matrix effect of blood and the lengthy blood tube culture procedure. Here we report a culture-free workflow for the rapid isolation and enrichment of bacterial pathogens from whole blood for single-cell antimicrobial susceptibility testing (AST). A dextran sedimentation step reduces the concentration of blood cells by 4 orders of magnitude in 20-30 min while maintaining the effective concentration of bacteria in the sample. Red blood cell depletion facilitates the downstream centrifugation-based enrichment step at a sepsis-relevant bacteria concentration. The workflow is compatible with common antibiotic-resistant bacteria and does not influence the minimum inhibitory concentrations. By applying a microfluidic single-cell trapping device, we demonstrate the workflow for the rapid determination of bacterial infection and antimicrobial susceptibility testing at the single-cell level. The entire workflow from blood to categorical AST result can be completed in less than two hours.

Rapid pathogen detection and antimicrobial susceptibility testing (AST) are required in diagnosis of acute bacterial infections to determine the appropriate antibiotic treatment. Molecular approaches for AST are often based on the detection of known antibiotic resistance genes. Phenotypic culture analysis requires several days from sample collection to result reporting. Toward rapid diagnosis of bacterial infection in non-traditional healthcare settings, we have developed a rapid AST approach that combines phenotypic culture of bacterial pathogens in physiological samples and electrochemical sensing of bacterial 16S rRNA. The assay determines the susceptibility of pathogens by detecting bacterial growth under various antibiotic conditions. AC electrokinetic fluid motion and Joule heating induced temperature elevation are optimized to enhance the sensor signal and minimize the matrix effect, which improve the overall sensitivity of the assay. The electrokinetics enhanced biosensor directly detects the bacterial pathogens in blood culture without prior purification. Rapid determination of the antibiotic resistance profile of Escherichia coli clinical isolates is demonstrated.

This study demonstrates a low-cost, portable diagnostic system for rapid antimicrobial susceptibility testing in resource-limited settings. To determine the antimicrobial resistance phenotypically, the growth of pathogens in microwell arrays is detected under different antibiotic conditions. The use of a colorimetric cell viability reagent is shown to significantly improve the sensitivity of the assay compared with standard absorbance spectroscopy. Gas-permeable microwell arrays are incorporated for facilitating rapid bacterial growth and eliminating the requirement of bulky supporting equipment. Antibiotics can also be precoated in the microwell array to simplify the assay protocol toward point-of-care applications. Furthermore, a low-cost cell phone-based microphotometric system is developed for detecting the bacterial growth in the microwell array. By optimizing the operating conditions, the system allows antimicrobial susceptibility testing for samples with initial concentrations from 10(1) to 10(6) cfu/mL. Using urinary tract infection as the model system, we demonstrate rapid antimicrobial resistance profiling for uropathogens in both culture media and urine. With its simplicity and cost-effectiveness, the cell phone-based microphotometric system is anticipated to have broad applicability in resource-limited settings toward the management of infectious diseases caused by multidrug-resistant pathogens., (© 2013 Society for Laboratory Automation and Screening.)

Multidrug-resistant pathogens are an emerging global health problem. In addition to the need of developing new antibiotics in the pipeline, the ability to rapidly determine the antibiotic resistance profiles of bacteria represents one of the most crucial steps toward the management of infectious diseases and the prevention of multidrug-resistant pathogens. Here, we report a single cell antimicrobial susceptibility testing (AST) approach for rapid determination of the antibiotic resistance of bacterial pathogens. By confining individual bacteria in gas permeable microchannels with dimensions comparable to a single bacterium, the antibiotic resistance of the bacteria can be monitored in real-time at the single cell level. To facilitate the dynamic loading of the bacteria into the confined microchannels for observation, AC electrokinetics is demonstrated for capturing bacteria to defined locations in high-conductivity AST buffer. The electrokinetic technique achieves a loading efficiency of about 75% with a negligible effect on the bacterial growth rate. To optimize the protocol for single cell AST, the bacterial growth rate of individual bacteria under different antibiotic conditions has been determined systematically. The applicability of single cell AST is demonstrated by the rapid determination of the antimicrobial resistant profiles of uropathogenic clinical isolates in Mueller-Hinton media and in urine. The antibiotic resistance profiles of bacteria can be determined in less than 1 h compared to days in standard culture-based AST techniques.

Urinary tract infection (UTI) is among the most common bacterial infections and poses a significant healthcare burden. The standard culture-based diagnosis of UTI has a typical delay of two to three days. In the absence of definitive microbiological diagnosis at the point of care, physicians frequently initiate empirical broad-spectrum antibiotic treatment, and this has contributed to the emergence of resistant pathogens. Biosensors are emerging as a powerful diagnostic platform for infectious diseases. Paralleling how blood glucose sensors revolutionized the management of diabetes, and how pregnancy tests are now conducted in the home, biosensors are poised to improve UTI diagnosis significantly. Biosensors are amenable to integration with microfluidic technology for point-of-care (POC) applications. This review focuses on promising biosensor technology for UTI diagnosis, including pathogen identification and antimicrobial susceptibility testing, and hurdles to be surpassed in the translation of biosensor technology from bench to bedside., (Published by Elsevier Ltd.)

Purpose: A significant barrier to efficient antibiotic management of infection is that the standard diagnostic methodologies do not provide results at the point of care. The delays between sample collection and bacterial culture and antibiotic susceptibility reporting have led to empirical use of antibiotics, contributing to the emergence of drug resistant pathogens. As a key step toward the development of a point of care device for determining the antibiotic susceptibility of urinary tract pathogens, we report on a biosensor based antimicrobial susceptibility test., Materials and Methods: For assay development bacteria were cultured with or without antibiotics, and growth was quantitated by determining viable counts and electrochemical biosensor measurement of bacterial 16S rRNA. To determine antibiotic susceptibility directly from patient samples, urine was cultured on antibiotic plates for 2.5 hours and growth was determined by electrochemical measurement of bacterial 16S rRNA. For assay validation 252 urine samples were collected from patients at the Spinal Cord Injury Service at Veterans Affairs Palo Alto Health Care System. The biosensor based antimicrobial susceptibility test was completed for samples containing gram-negative organisms. Pathogen identification and antibiotic susceptibility results were compared between our assay and standard microbiological analysis., Results: A direct biosensor quantitation of bacterial 16S rRNA can be used to monitor bacterial growth for a biosensor based antimicrobial susceptibility test. Clinical validation of a biosensor based antimicrobial susceptibility test with patient urine samples demonstrated that this test was 94% accurate in 368 pathogen-antibiotic tests compared to standard microbiological analysis., Conclusions: This biosensor based antimicrobial susceptibility test, in concert with our previously described pathogen identification assay, can provide culture and susceptibility information directly from a urine sample within 3.5 hours., (Copyright © 2011 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

This study reports the use of microfluidics, which intrinsically has a large surface-to-volume ratio, toward rapid antimicrobial susceptibility testing at the point of care. By observing the growth of uropathogenic Escherichia coli in gas permeable polymeric microchannels with different dimensions, we demonstrate that the large surface-to-volume ratio of microfluidic systems facilitates rapid growth of bacteria. For microchannels with 250 microm or less in depth, the effective oxygenation can sustain the growth of E. coli to over 10(9) cfu/mL without external agitation or oxygenation, which eliminates the requirement of bulky instrumentation and facilitates rapid bacterial growth for antimicrobial susceptibility testing at the point of care. The applicability of microfluidic rapid antimicrobial susceptibility testing is demonstrated in culture media and in urine with clinical bacterial isolates that have different antimicrobial resistance profiles. The antimicrobial resistance pattern can be determined as rapidly as 2 h compared to days in standard clinical procedures facilitating diagnostics at the point of care.

Bloodstream infections (BSI) are a leading cause of death worldwide. The lack of timely and reliable diagnostic practices is an ongoing issue for managing BSI. The current gold standard blood culture practice for pathogen identification and antibiotic susceptibility testing is time-consuming. Delayed diagnosis warrants the use of empirical antibiotics, which could lead to poor patient outcomes, and risks the development of antibiotic resistance. Hence, novel techniques that could offer accurate and timely diagnosis and susceptibility testing are urgently needed. This review focuses on BSI and highlights both the progress and shortcomings of its current diagnosis. We surveyed clinical workflows that employ recently approved technologies and showed that, while offering improved sensitivity and selectivity, these techniques are still unable to deliver a timely result. We then discuss a number of emerging technologies that have the potential to shorten the overall turnaround time of BSI diagnosis through direct testing from whole blood—while maintaining, if not improving—the current assay's sensitivity and pathogen coverage. We concluded by providing our assessment of potential future directions for accelerating BSI pathogen identification and the antibiotic susceptibility test. While engineering solutions have enabled faster assay turnaround, further progress is still needed to supplant blood culture practice and guide appropriate antibiotic administration for BSI patients. [ABSTRACT FROM AUTHOR]

Single-molecule and single-cell analysis techniques have opened new opportunities for characterizing and analyzing heterogeneity within biological samples. These detection methods are often referred to as digital assays because the biological sample is partitioned into many small compartments and each compartment contains a discrete number of targets (e.g. cells). Using digital assays, researchers can precisely detect and quantify individual targets, and this capability has made digital techniques the basis for many modern bioanalytical tools (including digital PCR, single cell RNA sequencing, and digital ELISA). However, digital assays are dominated by optical analysis systems that typically utilize microscopy to analyze partitioned samples. The utility of digital assays may be dramatically enhanced by implementing cost-efficient and portable electrical detection capabilities. Herein, we describe a digital electrical impedance sensing platform that enables direct multiplexed measurement of single cell bacterial cells. We outline our solutions to the challenge of multiplexing impedance sensing across many culture compartments and demonstrate the potential for rapidly differentiating antimicrobial resistant versus susceptible strains of bacteria. [ABSTRACT FROM AUTHOR]

A nanotube assisted microwave electroporation (NAME) technique is demonstrated for delivering molecular biosensors into viable bacteria for multiplex single cell pathogen identification to advance rapid diagnostics in clinical microbiology. Due to the small volume of a bacterial cell (~femtoliter), the intracellular concentration of the target molecule is high, which results in a strong signal for single cell detection without amplification. The NAME procedure can be completed in as little as 30 minutes and can achieve over 90% transformation efficiency. We demonstrate the feasibility of NAME for identifying clinical isolates of bloodborne and uropathogenic pathogens and detecting bacterial pathogens directly from patient's samples. In conjunction with a microfluidic single cell trapping technique, NAME allows single cell pathogen identification and antimicrobial susceptibility testing concurrently. Using this approach, the time for microbiological analysis reduces from days to hours, which will have a significant impact on the clinical management of bacterial infections. A nanotube assisted microwave electroporation (NAME) technique is demonstrated for rapid microbiological analysis of pathogenic bacteria. NAME delivers molecular biosensors into viable bacteria for multiplex single cell pathogen identification and antimicrobial susceptibility testing. The technique determines the species in as little as 30 minutes and perform antimicrobial susceptibility testing in less than 3 hours. Unlabelled Image [ABSTRACT FROM AUTHOR]