Standalone self-compartmentalizing microfluidic chip for digital single-cell antimicrobial susceptibility testing.

Standard, phenotypic antimicrobial susceptibility testing (AST) methods require 16-20 h of incubation and are considered as the bottleneck in providing timely input for appropriate antimicrobial treatment. In this study, a novel adenosine triphosphate (ATP)-bioluminescence-based method which allows rapid AST within 3 h was described. Standard AST was performed for 56 Enterobacterales isolates using EUCAST disk diffusion (DD) methodology. For the bioluminescence-based rapid AST, suspensions of bacteria were prepared using Mueller-Hinton broth to obtain a turbidity of 0.5 McFarland. The suspensions were distributed into 96-well microtiter plates. ATP (20 mM) and fixed concentrations of different antibiotics were added. Following incubation at 37°C for 1 h, a luminescent reaction mixture, including the substrate luciferin and luciferase enzyme solutions, was added. The chemiluminescence was monitored using an imaging system. Light production demonstrated the presence of ATP, indicating that the isolate was susceptible to the antibiotic in the well. Absence or decrease of light intensity, compared with the growth control well, indicated the use of ATP as an indirect measure of bacterial growth, and therefore resistance to the antibiotic in the well. The novel AST method was tested using a total of 348 test wells. Concordance was achieved for 290 (83.3%) of the tests, whereas 52 (14.9%) and 6 (1.7%) tests caused minor and major errors, respectively. In this study, a bioluminescence-based rapid AST was developed based on the consumption of ATP by bacteria. Our method's uniqueness relies on determining ATP consumption by microorganisms in the presence or absence of an antibiotic. The novel AST method described in this study lays the groundwork for obtaining rapid results, which should be considered as a proof of concept. With further optimization studies, this novel method can provide higher accuracy and be introduced into clinical practice as a routine AST method.

BackgroundPatients infected with multi-drug-resistant (MDR) pathogens may experience long delays to targeted therapies due to the incomplete antimicrobial menus and/or breakpoints tested on current commercial antimicrobial susceptibility testing (AST) systems. Detection of carbapenem resistance poses a challenge to rapid, accurate, minimum inhibitory concentrations (MIC) determinations because some resistant organisms may be inhibited by a carbapenem antibiotic until sufficient carbapenemase production has been achieved and traditional AST platforms must wait to make MIC calls. More accurate carbapenem MICs can be determined by implementing a carbapenemase test alongside rapid AST.MethodsWe demonstrate a novel, proprietary test to detect carbapenemase production that enables rapid MIC testing for carbapenem antibiotics. The test is processed in parallel with the Selux next-generation phenotyping (NGP) AST method, enabling rapid, <6-hour, accurate MIC determinations. The carbapenemase assay utilizes high concentrations of intact bacteria. After 3 hours of incubation, a fluorescent pH indicator is read spectroscopically. The solution pH is lowered by carbapenemase-mediated imipenem degradation and is indicative of enzyme activity.ResultsThis assay accurately identifies carabapenemases across multiple enzyme classes and bacterial species. Figure 1 shows the accuracy and speed of NGP AST at determining MICs for representative isolates from the FDA-CDC antimicrobial resistance bank compared with results from overnight broth microdilution (BMD). To date, over 100 challenge strains of Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii have been tested with no very major errors and an average time-to-result of 5.3 hours.ConclusionBy incorporating a rapid, on-board carbapenemase activity assay, the NGP AST platform rapidly delivers accurate carbapenem results. Combined with NGP’s comprehensive antibiotic menus, this platform will therefore ensure prompt delivery of personalized antibiotic therapies for all patients, including those infected with MDROs, and enable streamlined antibiotic stewardship coordination.DisclosuresAll authors: No reported disclosures.

Background: Rapid antimicrobial susceptibility testing (RAST) allows early detection of resistance directly from positive blood cultures, potentially improving outcomes in bloodstream infections (BSIs). We evaluated the performance of EUCAST RAST for Gram-negative ESKAPEEc pathogens and characterized carbapenemase genes in carbapenem-resistant Klebsiella pneumoniae (CRKP). Methods: A total of 354 positive blood cultures were screened, including 51 monomicrobial Gram-negative ESKAPEEc isolates. RAST results at 4, 6, 8, and 16–20 h were compared with standard antimicrobial susceptibility testing (AST) obtained using the BD Phoenix™ system. Categorical agreement (CA) and error frequency were calculated. Multiplex PCR and Sanger sequencing were performed on 15 CRKP isolates to identify carbapenemase genes and allelic variants. Results: 51 Gram-negative ESKAPEEc isolates met the inclusion criteria for RAST (15 E. coli, 19 K. pneumoniae, 11 A. baumannii, and 6 P. aeruginosa). Overall performance varied markedly by species and antibiotic. E. coli showed frequent unreadable or ATU zones at early timepoints and wide CA variability (50–100%), with high very major error (VME) rates for AMP, TZP, and CAZ, particularly at 6–8 h. K. pneumoniae displayed consistently high CA (mostly 100%) for carbapenems, CAZ, and TZP. A. baumannii demonstrated excellent agreement (100% for most agents), except for GEN at 6–8 h. P. aeruginosa could be evaluated only at 16–20 h, showing high CA for AMK, CAZ, and CIP; lower CA for MEM (83%); non-calculable CA for IMI due to universal ATU readings; and a CA value of 0% for TZP due to the predominance of the ATU results. VMEs ranged from 0% to 26.1% across species and reading times, but carbapenems did not generate VMEs. Molecular analysis revealed blaKPC in 66.7%, blaNDM in 46.7%, and blaOXA-48 in 33.3% of isolates, with co-occurrence in several strains. Sequencing identified blaKPC-2 and blaNDM-1 as the predominant variants, with one isolate harboring blaNDM-5. Conclusions: EUCAST RAST markedly accelerates susceptibility reporting from positive blood cultures, but its accuracy is species- and time-dependent. Performance was excellent for K. pneumoniae (including CRKP) and A. baumannii and acceptable for P. aeruginosa at 16–20 h. In contrast, E. coli showed frequent ATU results at early timepoints and high ME/VME rates, making readings before 8 h unreliable for clinical decisions. Overall, RAST can effectively support rapid antimicrobial stewardship when species-specific limitations are recognized, and early-timepoint results are interpreted with caution.

Background: Bloodstream infections (BSIs) are medical emergencies associated with high morbidity and mortality, where delays in initiating appropriate antimicrobial therapy adversely affect outcomes. Although automated blood culture systems reduce time to pathogen detection, conventional antimicrobial susceptibility testing (AST) requires additional incubation, leading to delayed targeted therapy. The EUCAST Rapid Antimicrobial Susceptibility Testing (RAST) method was developed to provide early phenotypic susceptibility results directly from positive blood culture bottles. Aim: To evaluate the diagnostic accuracy of the EUCAST RAST method, compare its performance with conventional disc diffusion AST using FDA Class II criteria, and determine the prevalent pathogens and resistance patterns in bloodstream infections. Methods: A prospective laboratory-based observational study was conducted over six months on monomicrobial positive blood cultures. RAST was performed directly from flagged blood culture bottles, with inhibition zones read at 8 hours and interpreted using EUCAST RAST breakpoints. Conventional Kirby–Bauer disc diffusion testing, interpreted as per CLSI guidelines, served as the reference standard. Categorical agreement and error rates were analyzed. Results: EUCAST RAST showed high categorical agreement (&gt;90%) across all tested organisms. Perfect agreement was observed for Enterococcus faecium, Acinetobacter baumannii, and Salmonella enterica. Minor discrepancies were mainly associated with fluoroquinolones among Enterobacterales. Conclusion: EUCAST RAST is a rapid, reliable AST method that significantly reduces turnaround time and supports early targeted therapy and antimicrobial stewardship in BSIs.

&amp;lt;i&amp;gt;Introduction&amp;lt;/i&amp;gt;: The escalating prevalence of multidrug resistance is a global threat to human health particularly in critically ill patients with bloodstream infections (BSIs). Delay in the administration of the appropriate antimicrobial treatment is associated with higher mortality rates and adverse consequences. This study attempted to estimate the rapid antimicrobial susceptibility testing (RAST) breakpoints directly from flagged BacT/Alert blood culture bottles in clinical practice. &amp;lt;i&amp;gt;Material &amp; Methods&amp;lt;/i&amp;gt;: A descriptive, cross-sectional study conducted at a tertiary care hospital in Delhi over a period of two months. The RAST was performed directly from the clinical samples for blood cultures received in our laboratory in parallel with the routine antimicrobial testing as per standard CLSI guidelines. Blood cultures were routinely incubated in BacT/Alert 3D. The inhibition zones were read at 4, 6, 8 and 16-20 hour of incubation as per European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. The identification of the isolates was confirmed by Vitek-2 compact system. &amp;lt;i&amp;gt;Results&amp;lt;/i&amp;gt;: In our study, the area of technical uncertainty (ATU) percentage was initially high at 4 hours but decreased significantly in later incubation periods. At 4 hours, none of the &amp;lt;i&amp;gt;S. aureus&amp;lt;/i&amp;gt; isolates showed &amp;gt;90% categorical agreement (CA) for any antimicrobial tested. However, clindamycin achieved the highest CA (100%) at 6 hours and 90% thereafter, with no very major errors (VME) or major error (ME). Cefoxitin required 8 hours to reach &amp;gt;90% CA, with no VME observed at any time point, but up to 75% ME at 8 hours. At 4 hours, most antimicrobials had high (&amp;gt;1.5%) rates of VME among &amp;lt;i&amp;gt;Enterobacteriales&amp;lt;/i&amp;gt;. By 6 hours, only Meropenem and Gentamicin had &amp;gt;90% CA, with no VME observed for other antibiotics. &amp;lt;i&amp;gt;Conclusion&amp;lt;/i&amp;gt;: The RAST method is relatively easy to implement in clinical microbiology labs, offering cost-effectiveness, simplicity, and rapid results, especially in resource-limited settings. However, reporting RAST results can be complex due to potential challenges with CA, VME, and ME, particularly in the initial hours of incubation and within the ATU.

Combining diagnostic methods for antimicrobial susceptibility testing – A comparative approach

“Barcode” cell sensor microfluidic system: Rapid and sample-to-answer antimicrobial susceptibility testing applicable in resource-limited conditions

ABSTRACTHelicobacter pylori is an important human pathogen associated with peptic ulcer disease, dyspepsia, and gastric malignancy. Antimicrobial susceptibility testing (AST) is often requested for patients who fail eradication therapy. The Clinical and Laboratory Standards Institute (CLSI) reference method, agar dilution (AD), is not performed in most laboratories and maintaining organism viability during transit to a reference laboratory is difficult. We assessed the performance of the Etest (bioMérieux) as a method for H. pylori AST in comparison to AD. Etest MICs were determined for 83 H. pylori isolates at ARUP and Cleveland Clinic (CC). Categorical agreement (CA), very major, major, and minor errors (VME, ME, and mE) were determined for Etest using AD performed at Mayo Clinic Laboratories as the reference method. Testing on isolates with errors was repeated to determine final results summarized below. For clarithromycin, 66.3% of isolates were resistant (R) by AD; Etest results at each laboratory showed 1mE (1.2%) and 1 ME (3.8%). For tetracycline, only 2 isolates were R by AD; a single VME occurred at both sites (98.8% CA, 50% VME) with the same isolate. Applying EUCAST levofloxacin breakpoints to interpret ciprofloxacin results, 60.2% of isolates were R by AD; ARUP CA was 97.6% (1 ME (3%), 1 VME (2%)) and CC CA was 96.3% (1 ME (3%), 2 VMEs (4%)). Despite high error rates, the categorical agreement was acceptable (>90%) for all three antibiotics between AD and Etest. In-house susceptibility testing by gradient diffusion can allow for testing of fastidious organisms that may not survive transport to specialized laboratories; however, the method is not without technical challenges. Characterization of resistance mechanisms, increased AD dilutions, and testing from the same inoculum may determine if the observed errors reflect technical issues or breakpoints that need optimization.IMPORTANCE Routine antimicrobial susceptibility testing (AST) of Helicobacter pylori by agar dilution is difficult to perform and not practical in most clinical microbiology laboratories. The Etest gradient diffusion method can be a reliable alternative for H. pylori AST with the advantage of being a less laborious quantitative method. This work reveals that an optimized Etest method can provide acceptable performance for H. pylori AST and describes the challenges associated with this methodology.

Assessment of bacterial viability by laser desorption ionization mass spectrometry for antimicrobial susceptibility testing

Rapid antimicrobial susceptibility testing by stimulated Raman scattering metabolic imaging and morphological deformation of bacteria

Gone are the days when the antimicrobial susceptibility pattern of a bacterial isolate could be predicted simply on the basis of its species identifi cation. Although Streptococcus pyogenes isolates remain susceptible to penicillin, one has to continually ask – for how long? With the discovery of strains of Staphylococcus aureus that are highly resistant to vancomycin (1) and strains of Acinetobacter species that are pan resistant (2, 3), the role of antimicrobial susceptibility testing in guiding therapy for infectious diseases is becoming more and more important (4). Yet, ironically, many of these novel resistance phenotypes are not easily detected using the automated susceptibility testing methods so prevalent in today's clinical laboratories (5–7). The ability of the clinical laboratory to detect emerging resistance profi les is often directly related to the extra efforts expanded to catch novel resistance mechanisms. Although resistant bacteria were common previously only in intensive care units of hospitals, multidrug resistance has become an issue among strains of community-acquired pathogens such as Salmonella, Shigella, and even Neisseria gonorrhoeae (8, 9). To complicate matters even further, resistant organisms that arise in the community are now also spreading into healthcare settings (10, 11). Therefore, it is imperative that changes in resistance patterns of a wide range of bacterial pathogens be monitored continually to ensure optimal treatment both of the individual patients and for maintaining the effi cacy of empiric therapy regimens. This chapter will explore the methods used for antimicrobial susceptibility testing of bacterial pathogens.

The development of new antimicrobial agents is essential to combat antimicrobial resistance. Reliable antimicrobial susceptibility testing (AST) methods should be established early and thoughtfully to ensure timely patient access to these agents. The standard reference method for AST is broth microdilution (BMD) in cation-adjusted Mueller-Hinton broth (CAMHB), as defined by CLSI M07 (CLSI. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically. 12th ed. CLSI Standard M07. Clinical and Laboratory Standards Institute, 2024) and ISO 20776-1 (ISO. Susceptibility Testing of Infectious Agents and Evaluation of Performance of Antimicrobial Susceptibility Test Devices-Part 1: Broth Micro-dilution Reference Method for Testing the In Vitro Activity of Antimicrobial Agents Against Rapidly Growing Aerobic Bacteria Involved in Infectious Diseases. ISO 20776-1. International Organization for Standardization, 2019). While some agents may require modifications to this method to better reflect clinical activity, such changes must be scientifically justified. Modifications aimed solely at producing lower minimal inhibitory concentration (MIC) values-or to make one antimicrobial agent appear superior to others-are not scientifically valid and are strongly discouraged. Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) caution developers that unnecessary deviations from reference AST methods can lead to increased costs, regulatory hurdles, delays in test availability and reduced clinical adoption. Until joint guidance under development by CLSI and EUCAST is finalized, early and rigorous evaluation using the reference method is essential.

To assess the expected clinical and financial benefits of rapid reporting of microbiology results, we compared patients whose cultured samples were processed in the normal manner to patients whose samples were processed more rapidly due to a minor change in work flow. For the samples tested in the rapid-reporting time period, the vast majority of bacterial identification and antimicrobial susceptibility testing (AST) results were verified with the Vitek system on the same day that they were available. This time period was called rapid AST (RAST). For RAST, a technologist on the evening shift verified the data that became available during that shift. For the control time period, cultures were processed in the normal manner (normal AST [NAST]), which did not include evening-shift verification. For NAST, the results for approximately half of the cultures were verified on the first day that the result was available. The average turnaround time for the reporting of AST results was 39.2 h for RAST and 44.4 h for NAST (5.2 h faster for RAST [P = 0.001]). Subsequently, physicians were able to initiate appropriate antimicrobial therapy sooner for patients whose samples were tested as part of RAST (P = 0.006). The mortality rates were 7. 9 and 9.6% for patients whose samples were tested as part of RAST and NAST, respectively (P = 0.45). The average length of stay was 10. 7 days per patient for RAST and 12.6 days for NAST, a difference of 2.0 days less for RAST (P = 0.006). The average variable cost was $4, 927 per patient for RAST and $6,677 for NAST, a difference of $1,750 less per patient for RAST (P = 0.001). This results in over $4 million in savings in variable costs per year in our hospital.

BackgroundLife-threatening syndromic hospital infections including sepsis, ventilator acquired pneumonia, catheter-associated urinary tract infection (CAUTI), and surgical site infections are often caused by multidrug-resistant pathogens. Implementing the targeted narrow-spectrum antimicrobial therapy as rapidly as possible at the onset of infection is critical for lowering morbidity and mortality for these infections. We present the new MultiPath technology for rapid syndromic infection detection, pathogen identification, and phenotypic antimicrobial susceptibility testing (AST). Our feasibility data demonstrate the technology’s potential application as a rapid CAUTI diagnostic.MethodsThe MultiPath technology detects and counts cells in a 30-minute assay using nonmagnified digital imaging. For identification, target pathogen cells are labeled using fluorescent in situ hybridization (FISH) with rRNA-specific probes, tagged with magnetic nanoparticles, deposited on a surface, imaged, and quantified. For AST, samples are mixed with growth medium, incubated for 4 hours in the presence of serial dilutions of antibiotics, FISH-labeled, magnetically selected, and quantified by digital imaging. The MultiPath assays use a dye-cushion layer to optically sequester the sample and unbound fluorescent probes from the imaging surface, eliminating the need for sample preparation and wash steps.ResultsThe MultiPath ID method specifically detected a range of common CAUTI pathogens including E. coli, K. pneumonia, E. faecium, E. faecalis, and P. aeruginosa. The limit of detection for E. coli was 27 CFU in a 100 µL assay in 10% urine. We present data demonstrating target inclusivity, specificity, and dynamic range. Our AST feasibility study results show excellent correlation with the broth micro dilution reference test for 5 antibiotics. Variable inoculum levels had little impact on MICs in the study.ConclusionThe data presented demonstrate the potential of the rapid ID/AST technology to achieve excellent analytical and clinical performance. This, combined with the method’s simplicity, robustness to sample matrix, and ease-of-use may make the method valuable for rapid syndromic infection diagnostics.Disclosures All authors: No reported disclosures.

Urinary tract infection (UTI) is the most common infection for people with neurogenic lower urinary tract dysfunction (NLUTD) due to spinal cord injury/disease (SCI/D). While the current diagnostic pillars include symptoms, inflammation, and bacterial load, each is uniquely problematic when applied to NLUTD-related UTI. Authoritative guidelines have not taken into account the impact of bladder management on symptoms; baseline inflammation in the absence of symptoms limits usefulness of white blood count; and bacteriuria in the absence of symptoms and limitations of standard urine culture (SUC) limits utility of SUC-based determination of bacterial load. Newer approaches are being developed to better guide diagnosis and antibiotic use. These include the Urinary Symptom Questionnaires for Neurogenic Bladder for symptom measurement and decision-making around symptoms, novel inflammatory markers (urine NGAL, IL-1, and IL-8), and culture-independent microbial detection methods. Rapid antimicrobial susceptibility testing (AST) methods have been developed utilizing a number of different approaches, increasing the likelihood of point-of-care application in the future. Advancements in UTI diagnosis will include reconceptualization of "urine is sterile" to "eubiosis and dysbiosis," symptom complexes to aid decision-making, use of novel inflammatory markers, and less reliance on SUC with increased emphasis on culture-independent methods for bacterial identification, AST, and decision-making.