The recently developed direct-on-target microdroplet growth assay (DOT-MGA) allows rapid universal antimicrobial susceptibility testing (AST) using matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). Here, we investigated a direct application of this method on positive blood cultures (BCs) for the acceleration of sepsis diagnostics.
KEYWORDS: MALDI-TOF, direct-on-target microdroplet growth assay, rapid susceptibility testing, blood culture, carbapenem resistance, AST
ABSTRACT
The recently developed direct-on-target microdroplet growth assay (DOT-MGA) allows rapid universal antimicrobial susceptibility testing (AST) using matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). Here, we investigated a direct application of this method on positive blood cultures (BCs) for the acceleration of sepsis diagnostics. Blood samples spiked with meropenem-nonsusceptible and meropenem-susceptible Enterobacterales isolates were inoculated into Bactec Plus Aerobic/F bottles and incubated in the Bactec automated system. Positive-BC broth was processed using four different methods, filtration/dilution, dilution, lysis/centrifugation, and differential centrifugation. For both dilution-based methods, AST was performed from 1:100, 1:1,000, and 1:10,000 dilutions of positive-BC broth in cation-adjusted Mueller-Hinton broth (CA-MHB). For both centrifugation-based methods, a 0.5 McFarland standard turbidity suspension was prepared from a bacterial pellet and adjusted to a final inoculum of 5 × 105 CFU/ml in CA-MHB. Six-microliter microdroplets with or without meropenem at the breakpoint concentration were spotted in triplicate onto a MALDI-TOF MS target, followed by incubation in a humidity chamber for 3 or 4 h and subsequent broth removal. Spectra were evaluated by MALDI Biotyper software. The test was considered valid if the growth control without antibiotic achieved an identification score of ≥1.7. For samples with meropenem, successful identification (score, ≥1.7) was interpreted as a nonsusceptible result, whereas failed identification (score, <1.7) defined susceptibility. The best test performance was achieved with the lysis/centrifugation method after a 4-h incubation. At this time point, 96.3% validity, 91.7% sensitivity, and 100% specificity were reached. This study demonstrated the feasibility and accuracy of a rapid DOT-MGA from positive BCs. Parallel to susceptibility determination, this method provides simultaneous species identification.
INTRODUCTION
Growing antimicrobial resistance emphasizes the need for swift and accurate detection of resistant pathogens (1–3). Rapid microbiological diagnostics have been shown to have a positive effect on patient management (4, 5) and even on reduction of mortality (6, 7). This is particularly important in the case of sepsis (8, 9). As inadequate antimicrobial treatment is reportedly associated with unfavorable outcomes in sepsis patients (10, 11), a prompt change to the targeted treatment is critical (12). The identification of sepsis pathogens has been considerably accelerated within the last decade due to the introduction of matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) into clinical diagnostics (13, 14). However, rapid antimicrobial susceptibility testing (AST) remains a problem in routine diagnostics (15), and the result is usually provided to clinicians only on the next day. As molecular tests detect resistance caused by selected resistance mechanisms only (16), they are not able to provide reliable information on a microorganism's susceptibility pattern. Rapid phenotypic tests are greatly needed, but there is still a lack of methods that are affordable and capable of being integrated into routine diagnostics (15). The recently developed MALDI-TOF MS-based direct-on-target microdroplet growth assay (DOT-MGA) allows easy-to-perform universal AST (17). This technology is suitable for further applications. In this study, we investigated different procedures for a direct application of this method on positive blood cultures (BCs) to accelerate sepsis diagnostics. Carbapenem resistance in Enterobacterales was chosen in this study as an example of important resistance phenotypes, recently categorized as first priority by the World Health Organization (http://www.who.int/medicines/publications/global-priority-list-antibiotic-resistant-bacteria/en/). (This work was presented in part at the 28th European Congress of Clinical Microbiology and Infectious Diseases (ECCMID), Madrid, Spain, 21 to 24 April 2018 [P1793] [18].)
MATERIALS AND METHODS
Bacterial strains.
Fourteen consecutive Enterobacterales isolates determined as meropenem nonsusceptible by phenotypic AST during routine diagnostics at the Institute of Medical Microbiology, University Hospital Münster, Germany, were included in this study. This group comprised 8 Klebsiella pneumoniae, 2 Enterobacter cloacae, 2 Enterobacter aerogenes, 1 Proteus mirabilis, and 1 Klebsiella oxytoca isolate. The same number of consecutive susceptible isolates for each species was included. Only one isolate per patient was eligible, and isolates from cystic fibrosis patients were excluded.
Determination of MIC.
The meropenem MIC was determined for each isolate using the broth microdilution reference method according to CLSI (19) and ISO (20) guidelines. In brief, bacterial suspensions were prepared in cation-adjusted Mueller-Hinton broth (CA-MHB) to contain approximately 5 × 105 CFU/ml as the final concentration. Real inoculum size was controlled by plating serial dilutions from growth control onto tryptic soy agar (TSA) plates in triplicate and counting colonies after an overnight incubation. Meropenem (TCI Deutschland GmbH, Eschborn, Germany) was tested in doubling concentrations from 0.008 to 256 μg/ml. The results were read after incubation for 18 ± 2 h at 35 ± 1°C. MIC results were interpreted according to the EUCAST breakpoints (21). For the purpose of this study, intermediate and resistant categories were combined into the nonsusceptible category. Reference strain Escherichia coli ATCC 25922 was used for quality control (QC).
Detection of carbapenemase genes.
Meropenem-nonsusceptible isolates were investigated for the presence of carbapenemase genes by isothermal amplification (eazyplex SuperBug CRE; AmplexDiagnostics, Gars Bahnhof, Germany). The panel of this commercial assay detects carbapenemase genes KPC, NDM, OXA-48, OXA-181, and VIM.
Inoculation of blood culture bottles.
Ten microliters of human blood was spiked with 100 μl of bacterial suspension in a 15-ml tube (Greiner Bio-One, Essen, Germany) to simulate a bacterial load of approximately 10 CFU/ml, which is common in bacteremic patients (22). Inoculum was controlled by seeding serial dilutions onto the TSA plates in triplicate and counting colonies after overnight incubation. The spiked blood was introduced into an aerobic glass culture vial (Bactec Plus Aerobic/F; BD Diagnostics, Heidelberg, Germany) after the disinfection of the septum with 70% ethanol and incubated in an automated BC system (Bactec 9240; BD Diagnostics). The time to positivity was automatically documented.
Processing of positive blood cultures.
The positive BCs were removed from the incubator within the personnel's working hours and processed immediately after removal. Four different methods were investigated in comparison for the processing of positive blood cultures upon positivity, namely, filtration/dilution, dilution, lysis/centrifugation, and differential centrifugation (see Fig. S1 in the supplemental material).
Filtration/dilution.
Positive-BC broth was filtered through a sterile 5-μm syringe filter (Sartorius, Göttingen, Germany), followed by preparation of decimal dilutions of the filtrate in CA-MHB. It was assumed that the bacterial concentration in positive-BC broth is approximately 108 CFU/ml. Therefore, filtrate dilutions of 1:100, 1:1,000, and 1:10,000 were used to prepare the inoculum. After the addition of antibiotic solution, this inoculum was assumed to result in final concentrations of approximately 5 × 106, 5 × 105, and 5 × 104 CFU/ml, respectively. The real bacterial concentration in positive-BC broth, and thus in suspensions used for AST, was determined by colony counting after plating serial dilutions onto TSA plates in triplicate and overnight incubation.
Dilution.
The dilution procedure was identical to that described above, but the filtration step was skipped, and positive-BC broth was directly used for preparing serial dilutions.
Lysis/centrifugation.
The inoculum was prepared and standardized using the Sepsityper kit (Bruker Daltonik, Bremen, Germany) as previously described (23). Briefly, 200 μl of lysis buffer was added to 1 ml of positive-BC broth and vortexed for 10 s. This sample was centrifuged for 2 min at 13,000 rpm, and the supernatant was discarded by pipetting. The pellet was resuspended in 1 ml of wash buffer, followed by centrifugation for 2 min at 13,000 rpm. After removal of the supernatant, the pellet was resuspended in 1 ml of 0.85% saline. This suspension was standardized to a 0.5 McFarland standard turbidity using a nephelometer (Densimat, bioMérieux, Marcy-l'Étoile, France) and diluted 1:100 in CA-MHB to achieve the bacterial concentration of approximately 1 × 106 CFU/ml. After this suspension was mixed 1:1 with antibiotic solution, the final inoculum size was expected to be approximately 5 × 105 CFU/ml, as recommended by the CLSI (19) and ISO (20). The real bacterial concentration in the test was determined by vital cell counting as described above.
Differential centrifugation.
Positive-BC broth was processed using low-speed/high-speed differential centrifugation. For this procedure, a 1.5-ml sample of positive-BC broth was centrifuged for 5 min at 2,000 rpm, followed by transferring 1 ml of the supernatant into another tube and discarding the rest of the supernatant and the pellet. The transferred supernatant was centrifuged for 2 min at 13,000 rpm, and the resulting supernatant was discarded. The pellet was resuspended in saline, and the inoculum was standardized and used for AST as described above for the lysis/centrifugation method.
MALDI-TOF MS direct-on-target microdroplet growth assay.
After positive-BC broth was processed using the four different methods described above, the resulting inoculum was used for rapid AST by the DOT-MGA. Fifty microliters of the meropenem solution in CA-MHB was added to 50 μl of bacterial suspension in CA-MHB in a well of a 96-well microtiter plate. Thereby, the final meropenem concentration of 2 μg/ml was established, which is the breakpoint concentration of meropenem differentiating between susceptible and nonsusceptible Enterobacterales isolates according to EUCAST (21). Additionally, growth control was set up for each sample by adding 50 μl of CA-MHB to 50 μl of bacterial suspension in CA-MHB in a well of a 96-well microtiter plate. This was performed in triplicate so that three wells of a microtiter plate contained sample with 2 μg/ml meropenem and three wells contained growth control for that sample. These microtiter plates were incubated for 18 ± 2 h at 35 ± 1°C in air to ensure the broth microdilution control of each experiment throughout the study. The experimental setup for the DOT-MGA was prepared in triplicate. Directly after inoculation of the above-mentioned microtiter plates, 6 μl was transferred from wells onto a spot of the disposable MALDI-TOF MS target (MBT Biotarget 96; Bruker Daltonik), resulting in three spots being inoculated with the sample with meropenem and three spots containing growth control for the corresponding sample. Inoculated target plates were placed in the plastic transport boxes (Bruker Daltonik), which were used as humidity chambers after adding 4 ml of deionized water onto the bottom of the box. Use of such humidity chambers was previously demonstrated as advantageous for avoiding the evaporation of microdroplets (17). Two target plates were inoculated in parallel, one of them being incubated for 3 h and another one for 4 h at 35 ± 1°C in air. After the incubation period, the broth was separated from microbial cells and removed from the MALDI-TOF MS target as previously described (17). In brief, this separation was performed by simple “touching” of a microdroplet sidewise at its bottom with an absorptive material (Fig. 1). In this study, we used filter papers (37 × 100 mm; GE Healthcare GmbH, Freiburg, Germany) for this purpose (Fig. 1). One microliter α-cyano-4-hydroxycinnamic acid matrix containing an internal standard (MBT MASTeR prototype kit; Bruker Daltonik) was added to each spot. After the matrix dried, MALDI-TOF MS was performed using the microflex LT/SH instrument (Bruker Daltonik), with instrument settings optimized for the DOT-MGA assay (acceptance criteria of the intensity were lowered, and the random walk mode of spectrum acquisition was selected). Interpretation of acquired mass spectra and categorization of isolates as susceptible or nonsusceptible were performed as described in previous work (17). Briefly, the spectra were evaluated by the standard MALDI Biotyper 3.1 software (Bruker Daltonik), which is usually used for microbial identification. The test was considered valid if the growth control without meropenem achieved a score of ≥1.7 for species identification. A score of <1.7 for growth control designated an invalid test. For samples with meropenem, successful species identification (score, ≥1.7) was interpreted as a nonsusceptible result for the given isolate, whereas failed species identification (score, <1.7) resulted in categorization as a susceptible isolate. A median result for three spots was calculated and used for analysis. The spectra from processing methods and the incubation time, which resulted in best test performance, were additionally used for the pilot evaluation of a novel dedicated prototype software (MBT MASTeR). This software directly compares the spectra of samples grown in the presence of antibiotics to the growth control spectra considering the number and the intensity of corresponding peaks to quantitatively calculate a resistance score value. The threshold for the score values was empirically set to 1.5, indicating resistance for strains with a score higher than 1.5.
FIG 1.
MALDI-TOF MS direct-on-target microdroplet growth assay (DOT-MGA).
RESULTS
Characterization of bacterial isolates using standard methods.
The meropenem MIC50, MIC90, and MIC range of meropenem-nonsusceptible isolates were 16 μg/ml, 128 μg/ml, and 4 to 128 μg/ml, respectively. In one isolate from the meropenem-nonsusceptible group, the nonsusceptibility to meropenem was not reproducible during the broth microdilution controls carried out in parallel to each experiment throughout the study. For this reason, this isolate was excluded from the analysis. For meropenem-susceptible isolates, the MIC50, MIC90, and MIC range of meropenem were 0.016 μg/ml, 0.06 μg/ml, and 0.008 to 0.06 μg/ml, respectively. The MIC of QC strain E. coli ATCC 25922 was within the recommended range throughout the study. In 6/14 (42.9%) nonsusceptible isolates, the carbapenemase gene was detected. Of these, three isolates were OXA-48 positive, one isolate was VIM positive, one isolate was NDM positive, and one isolate possessed both NDM and OXA-181 genes. In 8/14 (57.1%) isolates, no carbapenemase gene was detected.
Microbial cell counts.
The real bacterial concentration in inoculated blood samples was 11.4 CFU/ml on average (range, 6 CFU/ml to 22 CFU/ml). The mean time to positivity was 10.7 h (range, 7.1 h to 20.6 h). The mean bacterial concentration in positive BCs was 3.0 × 108 CFU/ml (range, 8.4 × 107 CFU/ml to 7.8 × 108 CFU/ml). The BC samples were processed 3.0 ± 2.9 (SD) hours, on average, after positivity signal.
A significant correlation (r = −0.43, P = 0.02) was demonstrated between real bacterial concentration in inoculated blood and the time to positivity of BCs; higher bacterial counts in simulated blood were associated with shorter times to positivity. There was no statistically significant correlation between bacterial concentration in positive BCs and time to positivity, between bacterial concentration in positive BCs and bacterial concentration in inoculated blood, or between bacterial concentration in positive BCs and time from positivity signal to processing of positive BCs (i.e., duration of additional incubation after positivity).
The real final bacterial concentrations achieved with different processing methods of positive BCs are presented in Table 1. The lysis/centrifugation and differential centrifugation methods provided the mean inoculum size closest to that recommended by the CLSI and ISO (19, 20). There was no considerable loss of microbial cells through the filtration of positive BCs during the filtration/dilution processing method (Table 1).
TABLE 1.
Final bacterial concentrations achieved with different processing methods for positive blood culturesa
| Processing method | Dilution | Final concentrations in test (CFU/ml) |
||
|---|---|---|---|---|
| Mean | Minimum | Maximum | ||
| Filtration/dilution | 1:100 | 1.2 × 106 | 1.8 × 105 | 3.5 × 106 |
| 1:1.000 | 1.2 × 105 | 1.8 × 104 | 3.5 × 105 | |
| 1:10,000 | 1.2 × 104 | 1.8 × 103 | 3.5 × 104 | |
| Dilution | 1:100 | 1.5 × 106 | 4.2 × 105 | 3.9 × 106 |
| 1:1,000 | 1.5 × 105 | 4.2 × 104 | 3.9 × 105 | |
| 1:10,000 | 1.5 × 104 | 4.2 × 103 | 3.9 × 104 | |
| Lysis/centrifugation | 4.4 × 105 | 8.3 × 104 | 1.1 × 106 | |
| Differential centrifugation | 4.6 × 105 | 1.0 × 105 | 8.9 × 105 | |
n = 28.
Performance of the MALDI-TOF MS DOT-MGA.
The performance of the DOT-MGA for direct detection of carbapenem nonsusceptibility in Enterobacterales from positive blood cultures using the different processing methods is shown in Table 2. The lysis/centrifugation method provided the most accurate results, particularly after a 4-h incubation. A direct DOT-MGA method using differential centrifugation demonstrated a similar performance. Table 3 shows the results of the DOT-MGA for these two processing methods after a 4-h incubation when the same spectra were reanalyzed by the novel prototype software.
TABLE 2.
Performance of MALDI-TOF MS DOT-MGA for direct detection of carbapenem nonsusceptibility in Enterobacterales from positive blood cultures using evaluation with MALDI Biotyper scoresa
| Processing method | Dilution | Values for 3-h incubation (%) |
Values for 4-h incubation (%)b |
||||
|---|---|---|---|---|---|---|---|
| Validityc | Sensitivityd | Specificityd | Validity | Sensitivity | Specificity | ||
| Filtration/dilution | 1:100 | 96.3 | 100 | 42.9 | 100 | 92.3 | 14.3 |
| 1:1,000 | 96.3 | 91.7 | 71.4 | 100 | 84.6 | 64.3 | |
| 1:10,000 | 85.2 | 80.0 | 100 | 88.9 | 81.8 | 100 | |
| Dilution | 1:100 | 100 | 92.3 | 71.4 | 96.3 | 100 | 46.2 |
| 1:1,000 | 96.3 | 83.3 | 85.7 | 100 | 84.6 | 92.9 | |
| 1:10,000 | 92.6 | 63.6 | 100 | 92.6 | 90.9 | 100 | |
| Lysis/centrifugation | 92.6 | 90.9 | 100 | 96.3 | 91.7 | 100 | |
| Differential centrifugation | 92.6 | 90.9 | 92.9 | 96.3 | 83.3 | 100 | |
n = 27.
The values in bold indicate the processing methods and incubation time which resulted in the best test performance.
Validity, percentage of tests with the growth control successfully detected.
Calculated for valid tests.
TABLE 3.
Performance of MALDI-TOF MS DOT-MGA for direct detection of carbapenem nonsusceptibility in Enterobacterales from positive blood culturesa
| Processing method | Validity (%)b | Sensitivity (%)c | Specificity (%)c |
|---|---|---|---|
| Lysis/centrifugation | 96.4 | 100 | 100% |
| Differential centrifugation | 96.4 | 92.3 | 100% |
Evaluated with MBT MASTeR prototype software. Incubation time, 4 h; n = 27.
Percentage of tests with growth control successfully detected.
Calculated for valid tests.
DISCUSSION
Recent study has demonstrated the potential of a MALDI-TOF MS-based DOT-MGA as a rapid AST method that provides phenotypic results within a few hours independently from the resistance mechanism (17). In septic patients, direct AST from positive BCs would further reduce the time to result in comparison with the standard testing from subcultivated colonies. In this study, we compared four processing methods for positive-BC broth to develop a direct AST procedure. Both the accuracy of resistance detection and the good suitability for laboratory workflows are essential criteria for comparing the methods because time- and labor-consuming processing methods have poor chances for being well accepted for routine diagnostics (15, 24).
Dilution methods can be performed with minimum hands-on time and are also suitable for robotized automation, whereas centrifugation methods require a technician's attention and are difficult to automatize. On the other hand, centrifugation procedures may result in a clean microbial pellet that can be used for the preparation of standardized inoculum for AST (23).
The principal feasibility of direct susceptibility testing from positive blood cultures using a MALDI-TOF MS-based DOT-MGA was demonstrated for all processing methods implemented in this study. However, the experiments revealed accuracy issues with the simple dilution methods. At least for the group of microorganisms investigated in this study, it appears important for direct testing from positive BCs to prepare a clean sample and standardized inoculum prior to initiating the MALDI-TOF MS-based DOT-MGA itself. Both methods applying standardization of inoculum by preparing 0.5 McFarland standard suspension, lysis/centrifugation, and differential centrifugation resulted in mean bacterial concentration within the range (2 × 105 to 8 × 105 CFU/ml) recommended by the CLSI and ISO (19, 20). The lysis/centrifugation method provided the most accurate results, particularly after a 4-h incubation. The lysis buffer did not affect the results for the microorganisms included in this study. A recent study also demonstrated high accuracy of results and no negative impact of the lysis reagent after using the lysis/centrifugation method for direct AST of Gram-negative rods from positive BCs with another technology (23). However, the impact of lysis buffer on the viability of bacteria and their growth characteristics can be organism dependent and should be considered whenever a new group of microorganisms is studied. Differential centrifugation methods showed comparable accuracy of results (Tables 2 and 3).
One of the dilution processing methods used in our study included filtration of positive-BC broth prior to the preparation of decimal dilutions from the resulting filtrate. The second dilution processing method matched this method, but the filtration step was skipped. The comparison of these two dilution methods was intended to elucidate whether filtration provides any improvement in the results accuracy by retention of blood cells and, hence, higher quality of MALDI-TOF MS spectra. Interestingly, the results showed no considerable difference between filtration/dilution and dilution methods. Although there was no remarkable bacteria loss due to filtration (Table 1), it also did not contribute to sample purification and test accuracy (Table 2). Similarly, filtration of positive BCs did not have the purification effect in a recent study which implemented optical measurements for AST (23). One possible explanation might be the ability of erythrocytes to pass through filter pores even smaller than 5 μm due to their deformability (25). Additionally, other blood components passing into filtrate may also disturb measurements. The filtration step provided no advantage in our study and, hence, can be skipped. However, it does not eliminate a possibility that an alternative filtration procedure would provide some benefit.
A comparison of the lysis/centrifugation method to differential centrifugation was performed to investigate whether the use of lysis and wash reagents can be avoided to enable more cost- and time-efficient processing. Both centrifugation methods allowed a precise standardization of inoculum size. However, bacterial pellets resulting from lysis/centrifugation were visually cleaner than pellets after differential centrifugation (see Fig. S2 in the supplemental material), which appeared red due to the remaining blood components. Furthermore, slightly higher sensitivity and specificity were achieved after lysis/centrifugation than after differential centrifugation (Table 2).
Interestingly, the bacterial concentration in positive BCs was relatively similar among the samples. Even BCs which were left in the incubator for several hours after positivity signal did not achieve significantly higher concentrations, presumably due to the saturation of bacterial growth in the BC vials. In this study, we did not remove the positive BCs from the incubator outside of working hours. This study design was chosen intentionally to simulate the situation in the laboratories not staffed overnight. This allowed us to investigate the impact of further incubation on the variability of bacterial concentrations in positive-BC bottles and the respective AST results. A recent study showed similar mean bacterial concentration in positive BCs (26). However, the range of achieved concentrations was broader, with a 2.75-log span (26), whereas the concentration range in our study did not exceed 1 log. This may be explained by the additional inclusion of nonfermentative Gram-negative rods, which may have growth characteristics different from Enterobacteriaceae and other members of the Enterobacterales order, and by the use of three different automated BC incubators in the study of Chandrasekaran et al. (26).
Due to similar bacterial concentrations in positive BCs, comparable inoculum size was achieved in different samples for correspondent dilutions (Table 1). The final inoculum size closest to that recommended by CLSI and ISO (19, 20) was achieved with the 1:1,000 dilution of positive-BC broth. The higher sensitivity was achieved with lower dilution factors, whereas the higher specificity was reached with higher dilution factors (Table 2). That is, the growth of nonsusceptible isolates was better detected when the inoculum size was large, whereas this large inoculum size more frequently led to the false-resistant results with susceptible isolates. Low inoculum size facilitated correct categorization of susceptible isolates but led to more false-susceptible results with nonsusceptible isolates. Notably, 1:1,000 dilution showed suboptimal sensitivity and specificity results for the filtration/dilution and dilution methods (Table 2) in spite of almost optimal average inoculum size (Table 1). One possible cause might be the disturbance of MALDI-TOF MS measurement by remaining blood components. Centrifugation methods, particularly lysis/centrifugation, provide much cleaner samples and result in higher sensitivity and specificity.
The scope of current and future applications of MALDI-TOF MS in clinical microbiology was recently reviewed (27). The direct MALDI-TOF MS-based DOT-MGA adds to this array of methods and has the potential to further accelerate sepsis diagnostics. Notably, the MALDI-TOF MS-based DOT-MGA not only provides susceptibility determination directly from positive BC but also offers simultaneous species identification. Depending on the workflows of individual laboratories, a combination of rapid AST and rapid identification can be organized in different ways. Those different options for diagnostic workflow are presented in Fig. 2.
FIG 2.
Suggested workflow options for rapid identification and antimicrobial susceptibility testing using MALDI-TOF MS DOT-MGA.
In conclusion, this study demonstrated the feasibility and accuracy of rapid susceptibility testing by a DOT-MGA directly from positive BCs. Future studies should focus on assay standardization and the determination of further major resistance phenotypes.
Supplementary Material
ACKNOWLEDGMENTS
This study was funded by grants from the German Federal Ministry of Education and Research (BMBF) to E.A.I. and K.B. (grant number 16GW0150) and M.K. (grant number 16GW0149K).
We are thankful to Damayanti Kaiser and Barbara Grünastel for expert technical assistance.
E.A.I. and K.B. are inventors of a pending patent owned by the University of Münster and licensed to Bruker. K.S., O.D., and M.K. are employees of Bruker Daltonik GmbH. L.M.S. declares no conflict of interest.
Footnotes
Supplemental material for this article may be found at https://doi.org/10.1128/JCM.00913-18.
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