Rapid identification of the causative microorganism is important for appropriate antimicrobial therapy of bloodstream infections. Bacteria from positive blood culture (BC) bottles are not readily available for identification by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Lysis and centrifugation procedures suggested for direct MALDI-TOF MS from positive BCs without previous culture are associated with additional hands-on processing time and costs. Here, we describe an alternative approach applying MALDI-TOF MS from bacterial cultures incubated very briefly on solid medium. After plating of positive BC broth on Columbia blood agar (n = 165), MALDI-TOF MS was performed after 1.5, 2, 3, 4, 5, 6, 7, 8, 12 and (for control) 24 h of incubation until reliable identification to the species level was achieved (score ≥2.0). Mean incubation time needed to achieve species-level identification was 5.9 and 2.0 h for Gram-positive aerobic cocci (GPC, n = 86) and Gram-negative aerobic rods (GNR, n = 42), respectively. Short agar cultures with incubation times ≤2, ≤4, ≤6, ≤8 and ≤12 h yielded species identification in 1.2%, 18.6%, 64.0%, 96.5%, 98.8% of GPC, and in 76.2%, 95.2%, 97.6%, 97.6%, 97.6% of GNR, respectively. Control species identification at 24 h was achieved in 100% of GPC and 97.6% of GNR. Ethanol/formic acid protein extraction performed for an additional 34 GPC isolates cultivated from positive BCs showed further reduction in time to species identification (3.1 h). MALDI-TOF MS using biomass subsequent to very short-term incubation on solid medium allows very early and reliable bacterial identification from positive BCs without additional time and cost expenditure.

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.

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.KEYWORDS Candida, candidemia, MALDI-TOF, blood culture, direct blood culturing, lysis-centrifugation, mass spectrometry, rapid tests, sepsis C andidemia is associated with a particularly high mortality rate that is increasing, especially among immunocompromised and critically ill patients (1-3). While early and appropriate treatment is vital (3), the slow growth of yeasts delays a timely diagnosis (4). The current diagnostic standard includes inoculation of the patient's blood into special bottles with liquid medium and incubation in an automated blood culture (BC) instrument (5). In cases where growth is detected, Gram staining is performed and the broth is subcultivated onto a solid medium to grow colonies for identification and antimicrobial susceptibility testing (AST) (4). The lysis-centrifugation blood culture (LC BC) method is an alternative approach, which has been available for decades but whose use has declined with the advent of blood culture automation (6-9). LC BC involves the selective lysis of blood cells with subsequent centrifugation and culturing of the sediment directly on agar plates (6). A recent study found that this approach provides a dramatic reduction in the time required for BC diagnostics of bacteria when combined with the modern methods of identification and susceptibility testing (10).In this study, we investigated whether the direct cultivation on solid medium using the LC BC method combined with identification by matrix-assisted laser desorptionionization time of flight mass spectrometry (MALDI-TOF MS) would result in a more rapid detection and differentiation of yeasts from blood compared to that with the currently used liquid medium-based automated BC instruments. We also investigated the impact of specific fungal or general media used with each 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.

Pathogen identification and antimicrobial susceptibility testing (AST) should be available as soon as possible for patients with bloodstream infections. We investigated whether a lysis-centrifugation (LC) blood culture (BC) method, combined with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) identification and Vitek 2 AST, provides a time advantage in comparison with the currently used automated broth-based BC system. Seven bacterial reference strains were added each to 10 mL human blood in final concentrations of 100, 10 and 1 CFU/mL. Inoculated blood was added to the Isolator 10 tube and centrifuged at 3000 g for 30 min, then 1.5 mL sediment was distributed onto five 150-mm agar plates. Growth was observed hourly and microcolonies were subjected to MALDI-TOF MS and Vitek 2 as soon as possible. For comparison, seeded blood was introduced into an aerobic BC bottle and incubated in the BACTEC 9240 automated BC system. For all species/concentration combinations except one, successful identification and Vitek 2 inoculation were achieved even before growth detection by BACTEC. The fastest identification and inoculation for AST were achieved with Escherichia coli in concentrations of 100 CFU/mL and 10 CFU/mL (after 7 h each, while BACTEC flagged respective samples positive after 9.5 h and 10 h). Use of the LC-BC method allows skipping of incubation in automated BC systems and, used in combination with rapid diagnostics from microcolonies, provides a considerable advantage in time to result. This suggests that the usefulness of direct BC on solid medium should be re-evaluated in the era of rapid microbiology.