Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Dec;52(12):4155-62.
doi: 10.1128/JCM.01872-14. Epub 2014 Sep 17.

Quantitative matrix-assisted laser desorption ionization-time of flight mass spectrometry for rapid resistance detection

Christoph Lange  1 , Sören Schubert  2 , Jette Jung  2 , Markus Kostrzewa  3 , Katrin Sparbier  1
Affiliations

Quantitative matrix-assisted laser desorption ionization-time of flight mass spectrometry for rapid resistance detection

Christoph Lange et al. J Clin Microbiol. 2014 Dec.

Abstract

Antibiotic resistance in Gram-negative microorganisms is an increasing health care problem. The rapid detection of such resistance is crucial for starting an early specific therapy and to enable initiation of the required hygiene measures. With continued emphasis on reducing the cost of laboratory testing, only economical/low-cost approaches have a chance of being implemented. During recent years, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been developed to be a standard method in microbiology laboratories for the rapid and cost-efficient identification of microorganisms. Extending the usage of MALDI-TOF MS in the clinical microbiology laboratory to the area of resistance testing is an attractive option. Quantitative MALDI-TOF MS using an internal standard facilitates the measurement of the quantity of peptides and small proteins within a spectrum. These quantities correlate to the number of microorganisms and therefore to the growth of a microorganism. The comparison of growth in the presence or absence of an antibiotic allows for analysis of the susceptibility behavior of a strain. Here, we describe a novel method and its application in the analysis of 108 Klebsiella sp. isolates. After 1 h of incubation at a meropenem concentration of 8 μg/ml, a sensitivity of 97.3% and a specificity of 93.5% were achieved (compared to Etest results).

PubMed Disclaimer

Figures

FIG 1
FIG 1
Pseudogel views of the mass range between 3 and 10 kDa of a susceptible (A, B) and a resistant (C, D) K. pneumoniae strain after incubation in the absence (lower panels) or presence (upper panels) of meropenem (64 μg/ml) for 1 h. For each incubation, four spectra acquired from two different spots are shown. Internal standard peaks are marked by arrows.
FIG 2
FIG 2
Box plots of the results of the automated evaluation. (A) AUC values obtained for four selected K. pneumoniae strains in the absence or presence of meropenem (64 μg/ml) for 1 h. Results were obtained from four spectra from two separate spots. Susceptible strains show a clear reduction of AUC in the presence of antibiotic. (B) The ratio of the AUC in the presence of antibiotic to that in the absence of antibiotic provides the relative growth. The horizontal line indicates the susceptibility/resistance threshold, the medians are indicated by bold lines, the minima and maxima are indicated by whiskers, and the 25th and 75th percentiles are indicated by boxes.
FIG 3
FIG 3
Determination of breakpoint concentrations by meropenem titration (0.025, 0.075, 1, 2, 4, 8, 16, and 32 μg/ml) for five different K. pneumoniae strains. Etest-derived MICs are given in the legend for each strain. The gray bar indicates the concentration of 8 μg/ml used for the analysis of the clinical isolates. The susceptibility/resistance threshold used for the analysis of the clinical isolates is indicated by the dashed line.
FIG 4
FIG 4
Reproducibility of the MBT-ASTRA. RG values were obtained for five different strains at the indicated meropenem concentration on four different days. Although absolute values varied, classification results were consistent.
FIG 5
FIG 5
Analysis of resistance status of 108 Klebsiella strains. For each strain, the MIC obtained from Etest results (x axis) was plotted against the RG value from the MS-based resistance test (y axis). The susceptibility/resistance threshold for the MS-based assay was set at a relative growth value of 0.4 (dashed line). The MIC-based definition of resistance was determined according to EUCAST guidelines (gray vertical line). Except for five susceptible strains and one resistant strain, classification of the strains was in agreement for the two tests.
FIG 6
FIG 6
Analysis of 18 positive blood cultures using the MS-based resistance test. Samples were incubated for 1 h at 37°C with 8 μg/ml meropenem. All strains except one (highlighted in gray) were correctly classified. A prolonged incubation time enabled correct classification for this incorrectly classified strain. In the box plots, medians are indicated by bold lines, the minima and maxima are indicated by whiskers, and the 25th and 75th percentiles are indicated by boxes.
See this image and copyright information in PMC

References

    1. WHO. 2014. Antimicrobial resistance: global report on surveillance 2014. WHO, Geneva, Switzerland.
    1. Woodford N, Eastaway AT, Ford M, Leanord A, Keane C, Quayle RM, Steer JA, Zhang J, Livermore DM. 2010. Comparison of BD Phoenix, Vitek 2, and MicroScan automated systems for detection and inference of mechanisms responsible for carbapenem resistance in Enterobacteriaceae. J. Clin. Microbiol. 48:2999–3002. 10.1128/JCM.00341-10. - DOI - PMC - PubMed
    1. Van der Zee A, Steer N, Thijssen E, Nelson J, van't Veen A, Buiting A. 2003. Use of multienzyme multiplex PCR amplified fragment length polymorphism typing in analysis of outbreaks of multiresistant Klebsiella pneumoniae in an intensive care unit. J. Clin. Microbiol. 41:798–802. 10.1128/JCM.41.2.798-802.2003. - DOI - PMC - PubMed
    1. Mellmann A, Bimet F, Bizet C, Borovskaya AD, Drake RR, Eigner U, Fahr AM, He Y, Ilina EN, Kostrzewa M, Maier T, Mancinelli L, Moussaoui W, Prévost G, Putignani L, Seachord CL, Tang YW, Harmsen D. 2009. High interlaboratory reproducibility of matrix-assisted laser desorption ionization–time of flight mass spectrometry-based species identification of nonfermenting bacteria. J. Clin. Microbiol. 47:3732–3734. 10.1128/JCM.00921-09. - DOI - PMC - PubMed
    1. Seng P, Drancourt M, Gouriet F, La Scola B, Fournier P-E, Rolain JM, Raoult D. 2009. Ongoing revolution in bacteriology: routine identification of bacteria by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Clin. Infect. Dis. 49:543–551. 10.1086/600885. - DOI - PubMed

Publication types

MeSH terms

Substances