Rapid Phenotypic Detection of Microbial Resistance in Gram-Positive Bacteria by a Real-Time Laser Scattering Method.

We developed a methodology for antimicrobial susceptibility testing (AST) based on the BacterioScanTM216R laser scattering technology, using methicillin resistance in Staphylococcus aureus and vancomycin resistance in enterococci as exemplar for important resistance phenotypes. Fifty methicillin-resistant (MRSA) and 50 methicillin-susceptible (MSSA) S. aureus, as well as 50 vancomycin-resistant enterococci (VRE) and 50 vancomycin-susceptible enterococci (VSE) isolates were used for the study. Optimal test conditions were derived by investigating the effects of inoculum size, medium, incubation temperature and broth filtration. We proposed four different statistical approaches for rapid discrimination between resistant and susceptible bacteria. The statistical approach based on raw measurements of bacterial concentrations delivered sensitivity of 100% and specificity of 94% for discrimination between MRSA and MSSA already after 3 hours of incubation. Categorical agreement of ≥90% was achieved after 140 min with this approach. Differentiation between VRE and VSE was possible with 98% sensitivity and 92% specificity after 3 hours, using a sophisticated statistical approach based on concentration slopes derived from the raw concentration measurements. This approach provided categorical agreement of ≥90% after 165 min. The sensitivity and specificity estimates were confirmed by leave-one-out cross validation. In conclusion, the phenotypic AST methods developed in this study are promising for rapid detection of MRSA and VRE. The development and application of this technology would allow early detection of the resistant pathogens, thus facilitating swift change to the targeted antimicrobial treatment as well as timely initiation of appropriate infection control measures. Further studies are warranted to validate this approach for the detection of other resistance phenotypes, including direct testing from clinical specimens.

Rapid Detection and Identification of Candidemia by Direct Blood Culturing on Solid Medium by Use of Lysis-Centrifugation Method Combined with Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS).

Candida sepsis is a life-threatening condition with increasing prevalence. In this study, direct blood culturing on solid medium using a lysis-centrifugation procedure enabled successful Candida species identification by matrix-assisted laser desorption-ionization time of flight mass spectrometry on average 3.8 h (Sabouraud agar) or 7.4 h (chocolate agar) before the positivity signal for control samples in Bactec mycosis-IC/F or Bactec Plus aerobic/F bottles, respectively. Direct culturing on solid medium accelerated candidemia diagnostics compared to that with automated broth-based systems.

Acceleration of antimicrobial susceptibility testing of positive blood cultures by inoculation of Vitek 2 cards with briefly incubated solid medium cultures.

Briefly incubated agar cultures from positive blood cultures were used for antimicrobial susceptibility testing (AST) by Vitek 2. The cultivation time until inoculation was 3.8 h for Gram-positive cocci and 2.4 h for Gram-negative rods. The error rates were low, providing early and reliable AST without additional time or cost expenditure.