Research Article: Rapid antibiotic susceptibility testing on blood cultures using MALDI-TOF MS

Date Published: October 11, 2018

Publisher: Public Library of Science

Author(s): Marlène Sauget, Xavier Bertrand, Didier Hocquet, Karsten Becker.


Antibiotic resistance is a major public health problem requiring the early optimization of antibiotic prescriptions. Matrix-Assisted Laser Desorption Ionization-Time Of Flight Mass Spectrometry (MALDI-TOF MS) has been shown to accurately identify bacteria from positive blood culture. Here, we developed a rapid detection of Escherichia coli resistance to amoxicillin (AMX) and cefotaxime (CTX) from positive blood culture based on MALDI-TOF MS. Potential sparing of broad-spectrum antibiotics was also evaluated. We tested 103 E. coli-positive blood cultures. Blood cultures were subculture 1-hour in antibiotic-free rich liquid media before further incubation with and without AMX for 2.5 h or CTX for 2 h. Protein extracts associated with an internal control were spotted on a MALDI-TOF target and spectra were analyzed with the MBT-ASTRA prototype software (Bruker Daltonik GmbH, Bremen, Germany). Bacterial growth ratio was calculated from the AUC spectra obtained in the presence and absence of the antibiotic and compared to a threshold which classified E. coli as susceptible or resistant. Results were interpreted with MICs determined using agar dilution method as reference technique. MBT-ASTRA recognized 95% and 84% of the AMX- and CTX-susceptible isolates, respectively. Overall, quantitative analysis of mass spectra allows susceptibility testing within 4 hours after the positivity of blood culture with E. coli. At the first report of positive blood culture, MALDI-TOF MS would then provide the prescribers with the bacterial identification and the susceptibility to AMX and CTX, thus limiting the use of broad-spectrum compounds.

Partial Text

In recent decades, antibiotic resistance has become a major public health problem requiring the optimization of antibiotic prescriptions. However, the current methods for the determination of bacterial resistance require time. Indeed, after bacterial identification, antibiotic testing needs from 7 h for automated systems with fast-growing species [1] to 24 h for agar dilution method with a return to the clinician the next day [2]. Simultaneously, the increasing proportion of blood infections with antibiotic-resistant Enterobacteriaceae limits treatment options [3]. The antibiotic susceptibility testing should be reported as soon as possible to avoid the overuse of broad-spectrum antibiotics (i.e. third-generation cephalosporins, carbapenems) and to ensure the timely appropriate antibiotherapy. Hence, rapid antibiotic testing is an area of intense research.

MBT ASTRA method indirectly estimates the growth of bacterial strains. The peak normalization combined with an internal quantitative control permits the comparison of the total intensity of the MALDI-TOF MS spectra. The peak intensity reflects the amount of bacterial proteins, which directly depends on the bacterial growth [9]. Recently MBT-ASTRA assay was implemented in several studies for the detection of bacterial resistance either from clinical isolates or directly from positive blood cultures [9, 13–15]. Gram-negative bacterial species (Enterobacteriaceae, Pseudomonas sp., and Acinetobacter sp.) were tested in combination with different antibiotic classes (penicillins, cephalosporins, carbapenems, fluoroquinolones, or aminoglycosides) [13, 14]. Mycobacteria tuberculosis was tested with rifampicin and izoniazid, respectively [15]. The protocol optimization (i.e. antibiotic concentration and incubation time) is necessary for each species-antibiotic combination. Unfortunately, the antibiotic (ATB) concentrations to be tested (ATBMS-ASTRA) cannot be predicted from the susceptible clinical breakpoints (ATBS breakpoint). For instance, the ratio CTXMS-ASTRA/CTXS breakpoint varies from 2 to 20 depending on the bacterial species and studies [13, 14]. Moreover, the optimal incubation time depends on the nature and the concentration of the antibiotic and varies from 1 to 4 h.




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