Antimicrobial Susceptibility Testing Methods Research Articles

Selection and Evaluation of Suitable Quality Control Strains for Meropenem Antimicrobial Susceptibility Testing through Preliminary External Quality Assessment

BackgroundQuality control (QC) of carbapenem susceptibility testing for Gram-negative bacteria faces challenges due to limited measuring ranges and the lack of suitable QC strains. This study aimed to select and evaluate QC strains for meropenem antimicrobial susceptibility testing (AST) through a pilot external quality assessment (EQA). MethodsCandidate QC strains for meropenem AST were selected based on primary AST data and genomic information from the Japan Antimicrobial Resistant Bacterial Bank. Phenotype stability was verified through serial passaging and MIC comparison with original strains. The validated broth microdilution method was used to determine the target MIC value in a pilot EQA involving 47 clinical laboratories in Japan using ten different AST methods. ResultsTwo strains, Citrobacter freundii JBBDAJB-19-0032 (Strain-A) and Enterobacter hormaechei subsp. steigerwaltii JBEBAAB-19-0102 (Strain-B), both carrying blaIMP-1, were selected as candidate QC strains. The meropenem MICs for Strain-A and Strain-B were 4 mg/L and 2 mg/L, respectively. In the pilot EQA, 43 laboratories reported appropriate results for Strain-A and 40 laboratories reported appropriate results for Strain-B. The MIC range was 2-8 mg/L for Strain-A and 1-4 mg/L for Strain-B. However, 19 and 12 laboratories, respectively, reported out-of-range MICs using AST plates on the MicroScan WalkAway (Beckman Coulter). Inappropriate results were reported by four and seven laboratories, respectively, using common methods for Strain-A and Strain-B, respectively. ConclusionsThe candidate QC strains selected for this study are suitable for meropenem AST EQA, except when the measuring range of certain methods does not match their QC range.

CRISPR/Cas9-Mediated Genome Editing of T4 Bacteriophage for High-Throughput Antimicrobial Susceptibility Testing.

The accurate and effective determination of antimicrobial resistance is essential to limiting the spread of infectious diseases and ensuring human health. Herein, a simple, accurate, and high-throughput phage-based colorimetric sensing strategy was developed for antimicrobial susceptibility testing (AST). Taking advantage of the CRISPR/Cas9 system, the genome of the T4 phage was modularly engineered to carry lacZ-α (lacZa), a marker gene encoding the α-fragment of β-galactosidase (β-gal). T4lacZa phages were identified by blue-white selection and then used for a biosensing application. In this strategy, the bacterial solution is exposed to the T4lacZa phage, causing target bacteria to overexpress β-gal. Upon the addition of a colorimetric substrate, the β-gal initiates an enzymatic reaction, resulting in a solution color change from yellow to red. This sensing strategy offers a visual way to monitor bacterial growth in the presence of antibiotics, enabling the determination of bacterial antimicrobial susceptibility. As a proof of concept, our developed sensing strategy was successfully applied to identify 9 different multidrug-resistant Escherichia coli (E. coli) in urine samples with 100% specificity. Compared with conventional disk diffusion susceptibility tests, the engineered phage-based sensing strategy can shorten the detection time by at least half without losing detection sensitivity, providing an alternative high-throughput method for AST in clinical diagnosis.

Ten Years Later: Still Unchanged Susceptibility to Antibiotics of Listeria monocytogenes from Poultry Processing Environments.

Listeria monocytogenes is still recognized as being commonly susceptible to antibiotics; however, there have been reports of reduced susceptibility in recent years. The significance of this resistance is not clear, in part due to the disparity in the antimicrobial susceptibility testing methods used. EUCAST (European Committee on Antimicrobial Susceptibility Testing) has recently proposed a standardized method for antibiotic susceptibility testing of L. monocytogenes. The aim of this work was to evaluate the susceptibility to 11 antibiotics in clinical use of 50 pulsed-field gel electrophoresis types of L. monocytogenes representing 347 isolates from a poultry industry setting using the EUCAST method and to compare the results with those obtained 10 years before. All poultry strains were sensitive to all the antibiotics tested but one strain was resistant to benzylpenicillin according to the EUCAST criteria. The current findings supported the previous study and confirmed that in certain food-associated L. monocytogenes populations, antibiotic sensitivity has remained stable.

Clinical pilot of bacterial transcriptional profiling as a combined genotypic and phenotypic antimicrobial susceptibility test.

Antimicrobial resistance is a growing health threat, but standard methods for determining antibiotic susceptibility are slow and can delay optimal treatment, which is especially consequential in severe infections such as bacteremia. Novel approaches for rapid susceptibility profiling have emerged that characterize either bacterial response to antibiotics (phenotype) or detect specific resistance genes (genotype). Genotypic and Phenotypic AST through RNA detection (GoPhAST-R) is a novel assay, performed directly on positive blood cultures, that integrates rapid transcriptional response profiling with the detection of key resistance gene transcripts, thereby providing simultaneous data on both phenotype and genotype. Here, we performed the first clinical pilot of GoPhAST-R on 42 positive blood cultures: 26 growing Escherichia coli, 15 growing Klebsiella pneumoniae, and 1 with both. An aliquot of each positive blood culture was exposed to nine different antibiotics, lysed, and underwent rapid transcriptional profiling on the NanoString platform; results were analyzed using an in-house susceptibility classification algorithm. GoPhAST-R achieved 95% overall agreement with standard antimicrobial susceptibility testing methods, with the highest agreement for beta-lactams (98%) and the lowest for fluoroquinolones (88%). Epidemic resistance genes including the extended spectrum beta-lactamase blaCTX-M-15 and the carbapenemase blaKPC were also detected within the population. This study demonstrates the clinical feasibility of using transcriptional response profiling for rapid resistance determination, although further validation with larger and more diverse bacterial populations will be essential in future work. GoPhAST-R represents a promising new approach for rapid and comprehensive antibiotic susceptibility testing in clinical settings.IMPORTANCEExposure to antibiotics causes differential transcriptional signatures in susceptible vs resistant bacteria. These differences can be leveraged to rapidly predict resistance profiles of Escherichia coli and Klebsiella pneumoniae in clinically positive blood cultures.

Repurposing MALDI-TOF MS for effective antibiotic resistance screening in Staphylococcus epidermidis using machine learning

The emergence of Staphylococcus epidermidis as a significant nosocomial pathogen necessitates advancements in more efficient antimicrobial resistance profiling. However, existing culture-based and PCR-based antimicrobial susceptibility testing methods are far too slow or costly. This study combines machine learning with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to develop predictive models for various antibiotics using a comprehensive dataset containing thousands of S. epidermidis isolates. Optimized machine learning models utilized feature selection and achieved high AUROC scores ranging from 0.80 to 0.95 while maintaining AUPRC scores up to 0.97. Shapley Additive exPlanations were employed to analyze relevant features and assess the significance of corresponding protein biomarkers while also verifying that predictive power was derived from the detection of proteins rather than noise. Antimicrobial resistance models were validated externally to evaluate model performance outside the original data collection site. The approaches and findings in this study demonstrate a significant advancement in rapid, cost-effective antimicrobial resistance profiling, offering a promising solution for improving treatments for nosocomial infections and being potentially applicable to other microbial pathogens in the future.

A-273 Rapid, Portable Invasive Fungal Infection Diagnostic and Candida Antimicrobial Susceptibility Testing

Abstract Background Traumatic wounds are associated with a higher risk of invasive fungal infections (IFI), which lead to a significant increase in amputation or death. Actionable treatment decisions are hampered because true infections can be difficult to differentiate from external wound contaminates, and tissue biopsy and quantification of fungal load is required to determine if treatment is necessary, which currently takes several days. GeneCapture is developing the CAPTURE 60-minute, portable infection identification platform to detect infectious levels of common fungi including the genera Aspergillus, Fusarium, and Scedosporium, the family Herpotrichiellaceae, and the order Mucorales. The company is also pursuing rapid identification and antimicrobial susceptibility testing for Candida species from multiple infection types. Methods This study seeks to determine the Limit of Detection (LoD), sensitivity and specificity of the CAPTURE ID assay for fungal pathogens. Since tissue biopsy samples must be used to assess the presence of IFI in wounds, GeneCapture has developed a manual process for the crude homogenization of muscle tissue to allow this matrix to be used as the starting sample for the ID assay. Mock samples were tested for inclusivity and exclusivity on a series of IFI ID panels against multiple strains of the included fungi, against fungi not yet specifically detected on the panel, and against bacteria that commonly infect wounds. While the LoD for yeasts may be determined in CFU/mL, there is no similar, easy-to-define metric for molds. GeneCapture has developed a quantitative PCR method to detect the single copy beta-tubulin gene to roughly correlate fungal dry mass and nuclei number to assay signal for molds cultured in Potato Dextrose Broth. Determining beta-tubulin copy numbers for extracts from mock samples then allowed detection of the progression of IFI over the course of infection. Lastly, antimicrobial susceptibility testing (AST) was performed on two species of Candida to validate utilizing GeneCapture’s rapid phenotypic AST method for fungal infections. Results To date, the CAPTURE ID panel has been tested against 27 included or excluded strains of fungi or bacteria with 97% sensitivity and 100% specificity. The current LoD for mold organisms varies from approximately 1,000 nuclei for Aspergillus species, to 100,000 nuclei for Fusarium species. Additional work to understand this range is underway. Lastly, we have correctly determined the susceptibility of Candida species using our novel approach within 4-6 hours. Conclusions GeneCapture’s ID and AST technology offer a unique solution to address the increasing prevalence of fungal infections. Our platform can identify the most common fungal components in a wound within 60 minutes of receiving a tissue biopsy sample. Candida infections occur across multiple matrices and rapidly identifying the species of Candida and then determining its phenotypic susceptibility profile will have a large impact on treating these difficult pathogens.

A One Health exploration of antimicrobial resistance in Escherichia coli originated from urban and rural lakes ecosystem.

Antimicrobial resistance (AMR) has become one of the most serious threats to One Health. Aquatic environments are an ideal non-clinical AMR reservoir and can act as a key battlefront for tackling the AMR. However, AMR data using the One Health approach remain scarce in aquatic environments worldwide. Here, we extensively assessed AMR in Escherichia coli isolated from urban and rural lake ecosystems using the One Health perspective. A total of 162 E. coli isolates obtained from lakes were tested against 25 antimicrobials using an in-vitro antimicrobial susceptibility testing method. A low (2%) to moderate (45%) drug resistance rate was found for all antimicrobials used in human/veterinary medicine or animal/plant agriculture. However, <80% E. coli isolates exhibited multidrug resistance (MDR) phenotype to highly important (amikacin, gentamicin, trimethoprim) or critically important (amoxicillin, ampicillin, colistin) drugs of both human and veterinary medicine. Of concern, >50% of E. coli isolates exhibited MDR to drugs used as last-resorts (chloramphenicol, colistin) or as frontline (nitrofurantoin, sulfamethoxazole, ampicillin, gentamicin) against E. coli infections. In conclusion, the presence of MDR E. coli strains in urban or rural lake ecosystems highlights their possible role as AMR reservoirs with potential One Health risks.

Verification of the Mycoplasma IST3 for urogenital mycoplasma culture in comparison to the Mycoplasma IST2

Background: Unlike the Mycoplasma IST2 kit (bioMérieux), the Mycoplasma IST3 kit has been updated to comply with the standardized antimicrobial susceptibility test (AST) method for Ureaplasma spp. (Up) and Mycoplasma hominis (Mh). We aimed to verify the use of the Mycoplasma IST3 kit for genital mycoplasma cultures. Methods: From September 2023 to January 2024, the R1 medium remaining after inoculation with IST2 was refrigerated until the next day. For IST2-positive samples, 300 μL of residual R1 medium was inoculated into the IST3. Species identification, enumeration, and AST results obtained using IST3 were compared with those obtained using IST2. Results: A total of 48 IST2-positive samples were inoculated into IST3, including 35, 1, and 12 Up-only, Mh-only, and both Up- and Mh-positive samples, respectively. Among Up-only samples, 2.8%, 91.4%, and 100.0% were susceptible to ciprofloxacin, tetracycline, and erythromycin, respectively. With IST3, 45 (93.8%) samples grew genital mycoplasmas; 42 (89.4%) of the 47 Up-positive samples and 6 (46.2%) of the 13 Mh-positive samples showed growth of the same organisms. All seven samples that failed to grow Mh were from mixed cultures, of which four Mh concentrations of &lt; 104/mL. Up was susceptible to levofloxacin, tetracycline, and erythromycin at the rates of 64.3 %, 88.1 %, and 95.2 %, respectively. Conclusion: IST3 showed good performance in detecting genital Mycoplasma except for its tendency to not detect Mh of low concentrations in mixed cultures. IST3 is preferable to IST2 because it can accurately screen for erythromycin resistance in Up and reduce falseresistances for fluoroquinolone.

Activity of antibiotics against Burkholderia cepacia complex in artificial sputum medium.

Burkholderia cepacia complex (Bcc) is a collection of intrinsically drug-resistant Gram-negative bacteria that cause life-threatening disease in people with cystic fibrosis (CF). Standard antimicrobial susceptibility testing methods have poor predictive value for clinical outcomes in Bcc infections, probably due in part to differences between in vitro testing conditions and the environment in which Bcc grow in the lungs of people with CF. To compare the activity of commonly used antibiotics under standard in vitro testing conditions with activity in conditions mimicking those found in vivo. Two Bcc strains were grown alone and with six different antibiotics (minocycline, ceftazidime, meropenem, tobramycin, levofloxacin, trimethoprim-sulfamethoxazole) in two different media: standard cation-adjusted Mueller-Hinton broth and an artificial sputum medium designed to simulate the environment in the lungs of people with CF through addition of components including mucin, free DNA and amino acids. Two different starting conditions were used for time-kill assays: a standard ∼5 × 106 cfu/mL inoculum, and a high-density inoculum in which bacteria were grown for 72 hours before addition of antibiotics. Growth detection was performed by colony enumeration and by detection of resazurin reduction. There were major discrepancies between standard susceptibility results and activity in our models. Some antibiotics, including ceftazidime, showed minimal activity in all time-kill assays despite low minimal inhibitory concentrations, while others, notably tobramycin, were more active in high-density growth conditions than in standard time-kill assays. This work underscores the urgent need to develop more clinically relevant susceptibility testing approaches for Bcc.

Rapid Phenotypic and Genotypic Antimicrobial Susceptibility Testing Approaches for Use in the Clinical Laboratory.

The rapid rise in increasingly resistant bacteria has become a major threat to public health. Antimicrobial susceptibility testing (AST) is crucial in guiding appropriate therapeutic decisions and infection prevention practices for patient care. However, conventional culture-based AST methods are time-consuming and labor-intensive. Therefore, rapid AST approaches exist to address the delayed gap in time to actionable results. There are two main types of rapid AST technologies- phenotypic and genotypic approaches. In this review, we provide a summary of all commercially available rapid AST platforms for use in clinical microbiology laboratories. We describe the technologies utilized, performance characteristics, acceptable specimen types, types of resistance detected, turnaround times, limitations, and clinical outcomes driven by these rapid tests. We also discuss crucial factors to consider for the implementation of rapid AST technologies in a clinical laboratory and what the future of rapid AST holds.

Real-time flow cytometry to assess qualitative and quantitative responses of oral pathobionts during exposure to antiseptics.

With increasing evidence that microorganisms are becoming more tolerant to standard antimicrobials, faster and more accessible antimicrobial susceptibility testing methods are needed. However, traditional susceptibility assays are laborious and time-consuming. To overcome the abovementioned limitations, we introduce a novel approach to define antimicrobial susceptibility in a much shorter time frame with the use of real-time flow cytometry. Furthermore, phenotypic fingerprinting analysis can be applied on the data to study the way antiseptics affect the bacterial cell morphology over time and, thus, gain information on the mode of action of a certain compound.

Rapid detection of antimicrobial susceptibility of the Bacteroides fragilis group by flow cytometry: A preliminary study

A total of nine Bacteroides fragilis group strains and B. fragilis ATCC 25285 were studied. Six antibiotics were used in the study. Broth dilution method was used for flow cytometry (FCM) analysis. Cell suspensions with antibiotics and antibiotic-free were stained with thiazole orange and propidium iodide (PI) to differentiate dead/live cells. The percentage of dead and live cells was calculated using FCM device. Cut-off values for antibiotics (26,7 %, 35,5 % and 30,2 % for meropenem, AMC and clindamycin, respectively) were calculated for dead/live cell differentiation. A common cut-off value was calculated for bactericidal and bacteriostatic (31,8 % and 25,7 % respectively). The PI staining ratios of the B. fragilis ATCC 25285 calculated in the MIC ranges for each antibiotic were under the cut-off values calculated with clinical isolates. The cut-off values we calculated are compatible with MBC rather than MIC values. The FCM method is one of the candidate methods for antimicrobial susceptibility testing.

Blood culture-free ultra-rapid antimicrobial susceptibility testing.

Treatment assessment and patient outcome for sepsis depend predominantly on the timely administration of appropriate antibiotics1-3. However, the clinical protocols used to stratify and select patient-specific optimal therapy are extremely slow4. In particular, the major hurdle in performing rapid antimicrobial susceptibility testing (AST) remains in the lengthy blood culture procedure, which has long been considered unavoidable due to the limited number of pathogens present in the patient's blood. Here we describe an ultra-rapid AST method that bypasses the need for traditional blood culture, thereby demonstrating potential to reduce the turnaround time of reporting drug susceptibility profiles by more than 40-60 h compared with hospital AST workflows. Introducing a synthetic beta-2-glycoproteinI peptide, a broad range of microbial pathogens are selectively recovered from whole blood, subjected to species identification or instantly proliferated and phenotypically evaluated for various drug conditions using a low-inoculum AST chip. The platform was clinically evaluated by the enrolment of 190 hospitalized patients suspected of having infection, achieving 100% match in species identification. Among the eight positive cases, six clinical isolates were retrospectively tested for AST showing an overall categorical agreement of 94.90% with an average theoretical turnaround time of 13 ± 2.53 h starting from initial blood processing.

What's old is new: leveraging existing antimicrobial susceptibility test methods for rapid results in patients with bloodstream infections.

The use of rapid disk diffusion or modified automated antimicrobial susceptibility testing (AST) system approaches demonstrates excellent performance for gram-negative organisms directly from blood cultures. In a recent study, S. Khan, A. Das, A. Mishra, A. Vidyarthi, et al. (Microbiol Spectr 12:e03081-23, 2024, https://doi.org/10.1128/spectrum.03081-23) compared the performance of three direct-from-blood AST methods against standard of care disk diffusion and automated AST. The results demonstrated high categorical agreements and low error rates across three protocols. The study suggests that locally validated direct-from-blood AST protocols offer reliable and fast results, particularly for resource-limited settings. However, local context and workflows should be considered prior to implementing rapid AST protocols, and more research is needed on the performance of rapid AST protocols for gram-positive organisms.

Development of Rapid Disk Diffusion Device Using Laser Speckle Formation Technology for Rapid Antimicrobial Susceptibility Testing

The escalation of antimicrobial resistance (AMR) due to the excessive and inappropriate use of antimicrobials has prompted the urgent need for more rapid and effective antimicrobial susceptibility testing (AST) methods. Conventional AST techniques often take 16–24 h, leading to empirical prescription practices and the potential emergence of AMR. The study aimed to develop a rapid disk diffusion (RDD) method utilizing laser speckle formation (LSF) technology to expedite AST results. The study aimed to evaluate the performance of LSF technology in determining antimicrobial susceptibility. In this study, preclinical and clinical settings were established to compare the LSF technology with conventional disk diffusion (DD) methods to measure the inhibition zones. Preclinical experiments with different bacterial strains demonstrated more than 70% categorical agreement (CA) against most antimicrobials. Further, clinical experiments with multiple strains and antibiotics revealed CA ranging from 40 to 79%, while major and minor discrepancies were observed around 30% and 11%, respectively. These observations revealed high concordance between RDD and DD for multiple antimicrobials in multiple species. The results underscore the potential of RDD-based LSF technology for hastening AST procedures. The current study is marked by a unique equipment setup and analysis approach. Collectively, the suggested laser-based RDD showed greater potential than previously developed comparable methods. The proposed method and design have a higher application potential than formerly developed similar technologies. Together, the study contributes to the ongoing development of rapid AST methods.

Clinical Pilot of Bacterial Transcriptional Profiling as a Combined Genotypic and Phenotypic Antimicrobial Susceptibility Test.

Antimicrobial resistance is a growing health threat, but standard methods for determining antibiotic susceptibility are slow and can delay optimal treatment, which is especially consequential in severe infections such as bacteremia. Novel approaches for rapid susceptibility profiling have emerged that characterize either bacterial response to antibiotics (phenotype) or detect specific resistance genes (genotype). GoPhAST-R is a novel assay, performed directly on positive blood cultures, that integrates rapid transcriptional response profiling with detection of key resistance gene transcripts, thereby providing simultaneous data on both phenotype and genotype. Here, we performed the first clinical pilot of GoPhAST-R on 42 positive blood cultures: 26 growing Escherichia coli, 15 growing Klebsiella pneumoniae, and 1 with both. An aliquot of each positive blood culture was exposed to 9 different antibiotics, lysed, then underwent rapid transcriptional profiling on the NanoString® platform; results were analyzed using an in-house susceptibility classification algorithm. GoPhAST-R achieved 95% overall agreement with standard antimicrobial susceptibility testing methods, with the highest agreement for beta-lactams (98%) and the lowest for fluoroquinolones (88%). Epidemic resistance genes including the extended spectrum beta-lactamase bla CTX-M-15 and the carbapenemase bla KPC were also detected within the population. This study demonstrates the clinical feasibility of using transcriptional response profiling for rapid resistance determination, although further validation with larger and more diverse bacterial populations will be essential in future work. GoPhAST-R represents a promising new approach for rapid and comprehensive antibiotic susceptibility testing in clinical settings.

Antimicrobial resistance surveillance of Bacteroides fragilis isolated from blood cultures, Europe, 2022 (ReSuBacfrag)

ObjectivesBacteroides fragilis is the most frequent cause of anaerobic bacteraemia. Although recent data suggest a rise in antimicrobial resistance (AMR) of this and other anaerobic bacteria, surveillance remains limited due to a lack of both data availability and comparability. However, a newly introduced standardised method for antimicrobial susceptibility testing (AST) of anaerobic bacteria has made larger scale surveillance possible for the first time. The aim of this study was to investigate phenotypic AMR of Bacteroides fragilis isolates from bacteraemia across Europe in 2022. MethodsIn a multicentre approach, clinical microbiology laboratories in Europe were invited to contribute results of AST for Bacteroides fragilis blood culture isolates (including only the first isolate per patient and year). AST of a selection of four antibiotics was performed locally by participating laboratories in a prospective or retrospective manner, using the new EUCAST disc diffusion method on fastidious anaerobe agar (FAA-HB). ResultsA total of 16 European countries reported antimicrobial susceptibilities in 449 unique isolates of Bacteroides fragilis from blood cultures in 2022. Clindamycin demonstrated the highest resistance rates (20.9%, range 0 - 63.6%), followed by piperacillin-tazobactam (11.1%, 0–54.5%), meropenem (13.4%, 0–45.5%), and metronidazole (1.8%, 0–20.0%), all with wide variation between countries. ConclusionConsidering that the mean resistance rates across Europe were higher than expected for three of the four anti-anaerobic antibiotics under surveillance, both local AST of clinically relevant isolates of Bacteroides fragilis and continued surveillance on an international level is warranted.

Sedimentation field-flow fractionation for rapid phenotypic antimicrobial susceptibility testing: a pilot study.

The increase in antibiotic resistance is a major public health issue. The development of rapid antimicrobial susceptibility testing (AST) methods is becoming a priority to ensure early and appropriate antibiotic therapy. To evaluate sedimentation field-flow fractionation (SdFFF) as a method for performing AST in less than 3 h. SdFFF is based on the detection of early biophysical changes in bacteria, using a chromatographic-type technology. One hundred clinical Escherichia coli strains were studied. A calibrated bacterial suspension was incubated for 2 h at 37°C in the absence (untreated) or presence (treated) of five antibiotics used at EUCAST breakpoint concentrations. Bacterial suspensions were then injected into the SdFFF machine. For each E. coli isolate, retention times and elution profiles of antibiotic-treated bacteria were compared with retention times and elution profiles of untreated bacteria. Algorithms comparing retention times and elution profiles were used to determine if the strain was susceptible or resistant. Performance evaluation was done according to CLSI and the ISO standard 20776-2:2021 with broth microdilution used as the reference method. AST results from SdFFF were obtained in less than 3 h. SdFFF showed high categorical agreement (99.8%), sensitivity (99.5%) and specificity (100.0%) with broth microdilution. Results for each antimicrobial were also in agreement with the ISO 20776-2 recommendations, with sensitivity and specificity of ≥95.0%. This study showed that SdFFF can be used as a rapid, accurate and reliable phenotypic AST method with a turnaround time of less than 3 h.

A multisite validation of a two hours antibiotic susceptibility flow cytometry assay directly from positive blood cultures

BackgroundRapid antimicrobial susceptibility testing (AST) is urgently needed to provide safer treatment to counteract antimicrobial resistance. This is critical in septic patients, because resistance increases empiric therapy uncertainty and the risk of a poor outcome. We validate a novel 2h flow cytometry AST assay directly from positive blood cultures (PBC) by using a room temperature stable FASTgramneg and FASTgrampos kits (FASTinov® Porto, Portugal) in three sites: FASTinov (site-1), Hospital Ramon y Cajal, Madrid, Spain (site-2) and Centro Hospitalar S. João, Porto, Portugal (site-3). A total of 670 PBC were included: 333 spiked (site-1) and 337 clinical PBC (151 site-2 and 186 site-3): 367 gram-negative and 303 gram-positive. Manufacturer instructions were followed for sample preparation, panel inoculation, incubation (1h/37ºC) and flow cytometry analysis using CytoFlex (Site-1 and -2) or DxFlex (site-3) both instruments from Beckman-Coulter, USA.ResultsA proprietary software (bioFAST) was used to immediately generate a susceptibility report in less than 2 h. In parallel, samples were processed according to reference AST methods (disk diffusion and/or microdilution) and interpreted with EUCAST and CLSI criteria. Additionally, ten samples were spiked in all sites for inter-laboratory reproducibility. Sensitivity and specificity were >95% for all antimicrobials. Reproducibility was 96.8%/95.0% for FASTgramneg and 95.1%/95.1% for FASTgrampos regarding EUCAST/CLSI criteria, respectively.ConclusionFASTinov® kits consistently provide ultra-rapid AST in 2h with high accuracy and reproducibility on both Gram-negative and Gram-positive bacteria. This technology creates a new paradigm in bacterial infection management and holds the potential to significantly impact septic patient outcomes and antimicrobial stewardship.

MultiRapid ATB NP test for detecting concomitant susceptibility and resistance of last-resort novel antibiotics available to treat multidrug-resistant Enterobacterales infections

BackgroundRecently developed therapeutics against Gram-negative bacteria include the β-lactam-β-lactamase inhibitor combinations ceftazidime–avibactam (CZA), meropenem–vaborbactam (MEV), and imipenem–relebatam (IPR), and the siderophore cephalosporin cefiderocol (FDC). The aim of this study was to develop a test for rapid identification of susceptibility/resistance to CZA, MEV, IPR, and FDC for Enterobacterales in a single test for rapid clinical decision making. MethodsThe MultiRapid ATB NP test is based on the detection of glucose metabolism occurring after bacterial growth in the presence of defined concentrations of CZA, MEV, IPR, and FDC, followed by visual detection of colour change of the pH indicator red phenol (red to yellow) generated by the acidification of the medium upon bacterial growth. This test is performed in 96-well microplates. The MultiRapid ATB NP test was evaluated using 78 Enterobacterales isolates and compared to the reference method broth microdilution. ResultsThe MultiRapid ATB NP test displayed 97.0% (confidence interval [CI] 92.6–98.8) sensitivity, 97.7% (CI 94.3–99.1) specificity, and 97.4% (CI 95.0–98.7) accuracy. The results were obtained after 3 h of incubation at 35 °C ± 2 °C, representing at least a 15-h gain-of-time compared with currently used antimicrobial susceptibility testing methods. ConclusionThe MultiRapid ATB NP test provided accurate results for the concomitant detection of susceptibility/resistance to CZA, MEV, IPR, and FDC in Enterobacterales, independent of the resistance mechanism. This test may be suitable for implementation in any microbiology routine laboratory.