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Currently, the world is facing the problem of bacterial resistance, which threatens public health, and bacterial antimicrobial susceptibility testing (AST) plays an important role in biomedicine, dietary safety and aquaculture. Traditional AST methods take a long time, usually 16–24 h, and cannot meet the demand for rapid diagnosis in the clinic, so rapid AST methods are needed to shorten the detection time. In this study, by using an in-house built centrifuge to centrifuge bacteria in a liquid medium onto the inner wall of the bottom surface of a counting plate, and using a phase contrast microscope to track bacterial growth under the effect of different antibiotic concentrations, the results of the minimum inhibitory concentration (MIC) of bacteria under the effect of antibiotics can be obtained in as early as 4 h. We used a combination of E. coli and tigecycline and obtained MIC results that were consistent with those obtained using the gold standard broth micro-dilution method, demonstrating the validity of our method; due to the time advantage, the complete set can be used in the future for point of care and clinical applications, helping physicians to quickly obtain the MIC used to inhibit bacterial growth.

Background: the aim of this study was to evaluate the performance of the Liquid Colony™ (LC) generated directly from positive blood cultures (PBCs) by the FAST System (Qvella, Richmond Hill, ON, Canada) for rapid identification (ID) and antimicrobial susceptibility testing (AST) compared with the standard of care (SOC) workflow. Methods: Anonymized PBCs were processed in parallel by the FAST System and FAST PBC Prep cartridge (35 min runtime) and SOC. ID was performed by MALDI-ToF mass spectrometry (Bruker, Billerica, MA, USA). AST was performed by reference broth microdilution (Merlin Diagnostika, Bornheim, Germany). Carbapenemase detection was carried out with the lateral flow immunochromatographic assay (LFIA) RESIST-5 O.O.K.N.V. (Coris, Gembloux, Belgium). Polymicrobial PBCs and samples containing yeast were excluded. Results: 241 PBCs were evaluated. ID results showed 100% genus-level concordance and 97.8% species-level concordance between LC and SOC. The AST results for Gram-negative bacteria showed a categorical agreement (CA) of 99.1% (1578/1593), with minor error (mE), major error (ME), and very major error (VME) rates of 0.6% (10/1593), 0.3% (3/1122), and 0.4% (2/471), respectively. The results from Gram-positive bacteria showed a CA of 99.6% (1655/1662), with mE, ME, and VME rates of 0.3% (5/1662), 0.2% (2/1279), and 0.0% (0/378), respectively. Bias evaluation revealed acceptable results for both Gram-negatives and Gram-positives (−12.4% and −6.5%, respectively). The LC yielded the detection of 14/18 carbapenemase producers by LFIA. In terms of turnaround time, the ID, AST, and carbapenemase detection results were generally obtained one day earlier with the FAST System compared with the SOC workflow. Conclusions: The ID, AST, and carbapenemase detection results generated with the FAST System LC were highly concordant with the conventional workflow. The LC allowed species ID and carbapenemase detection within around 1 h after blood culture positivity and AST results within approximately 24 h, which is a significant reduction in the turnaround time of the PBC workflow.

International audience; Enterobacterales bloodstream infections are life-threatening and require rapid, targeted antibiotherapy based on antibiotic susceptibility testing (AST). A new method using Muller-Hinton Rapid-SIR (MHR-SIR) agar (i2a, Montpellier, France) allows complete direct AST (dAST) to be read from positive blood culture bottles (BCBs) for all Enterobacterales species after 6-8 h of incubation. We evaluated (i) the performance of dAST from positive BCBs on MHR-SIR agar using two different inoculum protocols; (ii) the categorical agreement between dAST results obtained with MHR-SIR agar vs. those obtained with Muller-Hinton (MH) agar; and (iii) the ability of the MHR-SIR medium to detect beta-lactam resistant Enterobacterales. Finally, we estimated the saved turnaround time (TAT) with MHR-SIR compared with MH agar in our 24/7 laboratory. Our results showed that the most suitable inoculation protocol for dAST on MHR-SIR agar was 1 drop of BCB/5 mL H2O. For monomicrobial Enterobacterales BCBs, dAST performed on MHR-SIR medium showed 99.3% categorical agreement with AST on MH agar. Furthermore, MHR-SIR agar allows early detection of beta-lactam resistance mechanisms, including AmpC hyperproduction, extended-spectrum beta-lactamase, and carbapenemase. Finally, TAT reduction in our 24/7 laboratory was 16 h, enabling a significantly faster provision of antibiotic advice.

Several MALDI-TOF MS-based methods have been proposed for rapid detection of antimicrobial resistance. The most widely studied methods include assessment of β-lactamase activity by visualizing the hydrolysis of the β-lactam ring, detection of biomarkers responsible for or correlated with drug-resistance/non-susceptibility, and the comparison of proteomic profiles of bacteria incubated with or without antimicrobial drugs. Antimicrobial-resistance to a number of antibiotics belonging to different classes has been successfully tested by MALDI-TOF MS in a variety of clinically relevant bacterial species including members of Enterobacteriaceae family, non-fermenting Gram-negative bacteria, Gram-positive cocci, anaerobic bacteria and mycobacteria, opening this field to further clinically important developments. Early detection of drug-resistance by MALDI-TOF MS can be particularly helpful for clinicians to streamline the antibiotic therapy for a better outcome of patients with systemic infection, in all cases where a prompt and effective antibiotic treatment is essential to preserve organ function and/or patient survival.

Staphylococcus aureus remains a leading, virulent pathogen capable of expressing complex drug resistance that requires up to 2-4 days for laboratory analysis. In this study, we evaluate the ability of automated microscopy of immobilized live bacterial cells to differentiate susceptible from non-susceptible responses of S. aureus isolates (MRSA/MSSA, clindamycin resistance/susceptibility and VSSA/hVISA/VISA) to an antibiotic based on the characterization of as few as 10 growing clones after 4 h of growth, compared to overnight growth required for traditional culture based methods. Isolates included 131 characterized CDC isolates, 3 clinical isolates and reference strains. MRSA phenotype testing used 1 h of 1 μg/mL cefoxitin induction followed by 3 h of 6 μg/mL cefoxitin. Clindamycin susceptibility testing used 1h of induction by 0.1 μg/mL erythromycin followed by 3h of 0.5 μg/mL clindamycin. An automated microscopy system acquired time-lapse dark-field images, and then computed growth data for individual immobilized progenitor cells and their progeny clones while exposed to different test conditions. Results were compared to concurrent cefoxitin disk diffusion and D-test references. For CDC organisms, microscopy detected 77/77 MRSA phenotypes and 54/54 MSSA phenotypes, plus 53/56 clindamycin-resistant and 75/75 clindamycin susceptible strains. Automated microscopy was used to characterize heterogeneous and inducible resistance, and perform population analysis profiles. Microscopy-based hVISA population analysis profiles (PAPs) were included as an extended proof of concept, and successfully differentiated VSSA from hVISA and VISA phenotypes compared to plate-based PAP.