Characterization of β-Lactamase Enzyme Activity in Bacterial Lysates

ARTICLE pubs.acs.org/jpr

Characterization of β-Lactamase Enzyme Activity in Bacterial Lysates using MALDI-Mass Spectrometry Gero P. Hooff,*,† Jeroen J. A. van Kampen,‡ Roland J. W. Meesters,† Alex van Belkum,‡,§ Wil H. F. Goessens,‡ and Theo M. Luider† †

Department of Neurology, Laboratory of Neuro-Oncology and Clinical and Cancer Proteomics, Erasmus University Medical Center (Erasmus MC), Rotterdam, The Netherlands ‡ Department of Microbiology and Infectious Diseases, Erasmus University Medical Center (Erasmus MC), Rotterdam, The Netherlands ABSTRACT: Plasmid-encoded β-lactamases are a major reason for antibiotic resistance in Gram negative bacteria. These enzymes hydrolyze the β-lactam ring structure of certain β-lactam antibiotics, consequently leading to their inactivation. The clinical situation demands for specific first-line antibiotic therapy combined with a quick identification of bacterial strains and their antimicrobial susceptibility. Strategies for the identification of β-lactamase activity are often cumbersome and usually lack sensitivity and specificity. The current work demonstrates that matrix assisted laser desorption/ ionization mass spectrometry (MALDI-MS) is an ideal tool for these analytical investigations. Herein, we describe a fast and specific assay to determine β-lactamase activity in bacterial lysates. The feasibility of the analytical read-out was demonstrated on a MALDI-triple quadrupole (QqQ) and a MALDI timeof-flight (TOF) instrument, and the results allow the comparison of both approaches. The assay specifically measures enzyme-mediated, time-dependent hydrolysis of the β-lactam ring structure of penicillin G and ampicillin and inhibition of hydrolysis by clavulanic acid for clavulanic acid susceptible β-lactamases. The assay is reproducible and builds the basis for future in-depth investigations of β-lactamase activity in various bacterial strains by mass spectrometry. KEYWORDS: mass spectrometry (MS), MALDI-MS, E. coli, bacterial resistance, β-lactamase, antibiotics

’ INTRODUCTION With a rapidly increasing resistance to available antibiotics (ABs), physicians are facing tremendous problems in the treatment of bacterial infections as potent ABs for multiresistant strains become scarce. A relatively unreserved usage of antimicrobial agents in combination with diminished pipelines of the pharmaceutical industry over the last decades led to a critical number of resistant and multiresistant strains against drugs from various classes of antibiotics.1 In Gram-negative pathogens, β-lactamase production remains the most important contributing factor to β-lactam resistance. β-Lactamase proteins are bacterial enzymes that inactivate β-lactam antibiotics by hydrolysis, which results in the inactivation of the drugs. Until now more than 700 different β-lactamases have been described with different characteristics with respect to their hydrolyzing capacities. Plasmid-encoded β-lactamases are among the most important acquired resistance determinants emerging worldwide in members of the Enterobacteriaceae.2 4 During the 1990s, the TEM and SHV ESBL derivatives were the most common extended spectrum lactamases (ESBLs) observed worldwide. This trend was followed by the alarming appearance of CTX-M type enzymes with a substrate preference for the third generation cephalosporin, cefotaxime.5 Nowadays we are facing yet another challenge—the occurrence of carbapenemases, which are plasmid-encoded enzymes r 2011 American Chemical Society

capable of hydrolyzing carbapenem ABs, for instance, Klebsiella pneumoniae carbapenemases (KPC) and New Delhi metallo-β-lactamase (NDM) in K. pneumoniae and E. coli, aggravates a targeted treatment of infections caused by the aforementioned bacterial strains.6 For epidemiological purposes and infection prevention purposes, it is necessary to determine the nature and prevalence of these different enzymes. However, due to the huge variety and complexity of bacterial enzyme expression and regulation, leading to their resistance against many common ABs, phenotypic methods are no longer fully reliable for an adequate fast characterization and confirmation. An alternative quick-and-easy method determining substrate specificity of these β-lactamases is needed and will be beneficial for patient care in terms of speed of diagnosis, optimal treatment with specific antibiotics (first-line treatment) and infection prevention. Technical advancements, especially in the field of mass spectrometry, allow an easy implementation into common workflows, as instrumentation and software became increasingly user-friendly. Matrix-assisted laser desorption/ionization MS Special Issue: Microbial and Plant Proteomics Received: August 31, 2011 Published: October 21, 2011 79

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The reaction was stopped using 180 μL of acetonitrile (ACN) and, where applicable, 2 μL of cefoxitin (used as internal standard (IS)) solution in water (5 μmol/mL final conc.) were added. The solution was transferred to Eppendorf vials and centrifuged for 10 min (20000 g, RT). The supernatant was mixed 1:1 with αcyano-4-hydroxycinnamic acid (CHCA) and spotted onto a MALDI target plate (Applied Biosystems/MDS Sciex; Foster City, CA) for MALDI-QqQ analysis and onto a Bruker MTP 384 target plate (Bruker Daltonic GmbH, Bremen, Germany) prior to mass spectrometry analysis.

(MALDI-MS) combines beneficial characteristics for routine and high-throughput measurements. Analysis speed, sensitivity and selectivity are key attributes provided by this technique.7 9 The current work describes a novel, fast and highly specific approach for the determination and characterization of bacterial derived β-lactamase activity. The technique presented herein demonstrates the development and implementation of an assay for the determination and characterization of β-lactamase activity in bacterial lysates against selected β-lactam ABs. The analytical read-out of this assay was demonstrated on a common MALDI time-of-flight (TOF) MS and on a more sophisticated MALDI triple quadrupole (QqQ) MS instrument. Consequently, the present work elucidates the advantages and disadvantages of both approaches and demonstrates the convenience of the implementation of the assay into the medical microbiology laboratory.

MALDI-MS/MS Analysis

MALDI-MS/MS analysis was conducted on a MALDI-QqQ system, FlashQuant workstation AB Sciex (Applied Biosystems/ MDS Sciex; Foster City, CA). The mass spectrometer was run in positive multiple reaction monitoring (MRM) mode with nitrogen as collision gas. Following parameters were used: Laser frequency, 1000 Hz; laser power, 60%; plate voltage, 60 V; skimmer voltage, 0 V; source gas, 15 au (arbitrary units); CAD gas, 12 au; collision energy and collision cell energy exit potential, CE/CXP were: 35 eV/20 V for penicillinG (PenG) and its hydrolysate, benzyl penicilloic acid (BPA), 25 eV/10 V for ampicillin-Na+ (AMP), 18 eV/12 V; for cefoxitin-Na+ (CEF); and for imipenem (IMI) 22 eV/12 V. The multiple reaction monitoring (MRM) transitions (quantifier/qualifier) were for PenG m/z 335.0 f 289.0/335.0 f 128.2; for BPA m/z 353.2 f 128.2/353.2 f 174.0; for AMP m/z 372.0 f 182.0/372.0 f 196.0; for CEF m/z 450.2 f 388.8; and for IMI m/z 300.0 f 227.0/300.0 f 272.0, respectively. Laser speed was 1 mm/s for all measurements and samples were measured with 6 12 spots per sample. Mean peak areas were used to calculate the results. Data in all figures are shown as the signal intensity ratios of hydrolysis product and substrate (AB), which simultaneously take the degradation and the product formation into account.

’ MATERIALS AND METHODS Materials

All chemicals and the penicillinase (β-lactamase I from Bacillus cereus (B.cereus)) were from Sigma-Aldrich (SigmaAldrich, Munich; Germany) unless stated otherwise. Solvents were of ULC MS grade and were purchased from Biosolve (Valkenswaard, The Netherlands). E. coli strains

Three different bacterial strains were used for the experiments described in the present work: an Escherichia coli (expressing chromosomally encoded AmpC β-lactamase) and two different E. coli isolates harboring a plasmid-encoding TEM-1 β-lactamase. Susceptibility results for each strain were determined by the Vitek 2 system (bioMerieux, Marcy l’Etoile, France). For each β-lactamase preparation, frozen isolates ( 80 °C) were subcultured on blood agar (Becton Dickinson GmbH, Heidelberg, Germany). After overnight incubation at 37 °C, colonies were picked and suspended in 20 mL of 0.9% NaCl. Turbidity was adjusted to 3 McF using a densitometric analysis (densiCHEK; bioMerieux, Marcy toile, France). Subsequently, the suspension was centrifuged L’E for 10 min (1700 g; 4 °C). Pellets were resuspended in 200 μL of 50 mM Tris-HCl buffer (pH 7.4) and 10 μL of freshly prepared lysozyme (Sigma-Aldrich, St. Louis, MO) solution (40 mg/mL in 50 mM Tris-HCl buffer, pH 7.4) was added. Following incubation at 37 °C for 1 h under constant shaking, 10 μL of 0.5 mM EDTA solution was added and incubation continued for 10 min at 20 °C. The resulting lysates were centrifuged for 5 min (20000 g; room temperature (RT)) and supernatants were stored at 80 °C before being subjected to the β-lactamase activity assay.

MALDI-TOF analysis

MALDI-TOF measurements were conducted on an Ultraflex 3 mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with a 200 Hz nitrogen smart beam laser (337 nm). The instrument was run in linear mode to detect the following mass peaks (m/z values): PenG 335.106; BPA 353.116; AMP 372.349; CEF 450.384. FlexControl version 2.4 software was used to operate the instrument and peak intensities were used to calculate results. Data in all figures are shown as ratios of signal intensity of product and substrate. Inter- and Intraday Reproducibility (Quality Control Assay)

For each freshly prepared lysate, a quality control assay (QCa) was performed with PenG to assess the activity of the lysate prior to further incubation tests. Furthermore, this assay was used to show the reproducibility of the β-lactamase activity assay within one day (intraday reproducibility) and within three consecutive days (interday reproducibility). Samples were incubated for 0 and 30 min and the results were compared to the mean values of an intraday evaluation.

β-Lactamase Activity Assay

The β-lactamase activity assay was performed in 0.5 mL reaction vials or in 96-well plates. After the addition of 2 μL of water or clavulanic acid in water (5 μmol/mL final conc.) and 2 μL of a freshly prepared antibiotic stock solution (5 μmol/mL final conc.), 16 μL of thawed bacteria lysate were added and incubated for the respective time period in a Eppendorf thermomixer (Eppendorf, Wesseling-Berzdorf, Germany) at 37 °C. Samples used for calibration curves (see linearity measurements) were spiked in pooled E. coli lysates, which were previously subjected to freeze/thaw cycles and kept at RT overnight to ensure loss of enzymatic activity. Incubation with penicillin G (PenG) in these samples did not show any hydrolysis (data not shown).

Linearity

To demonstrate the linear response of PenG and AMP of both instruments, the MALDI-TOF and -QqQ, calibration curves were constructed using CEF as IS. To take matrix effects (ion suppression or enhancement) during the MS measurements into account, samples were prepared in bacteria lysates, as described earlier. Linearity was tested in a range of 10 1000 nmol/mL 80

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Figure 2. Enzyme kinetics of pure β-lactamase from B. cereus with penicillin G (50 nmol/mL) measured with MALDI-TOF and MALDIQqQ. Nonlinear regression is shown by a dotted line, excluding values below the quantification limit (0 10 min). Results are shown as signal intensity of the product/substrate ratio. (a) Time dependent degradation with 1  10 2 units enzyme measured by MALDI-TOF (mean ( SEM, 5 spots). (b) Time-dependent degradation with 1  10 2 units enzyme measured by MALDI-QqQ (mean ( SEM, 6 spots); * excluded outlier.

Figure 1. (a) Chemical structure of PenG (m/z 335) and the β-lactam ring-opening through the β-lactamase. The hydrolysis product (BPA) has a m/z of 353. MALDI-TOF spectrum shows the mass spectrum of PenG at t = 0 min (blue trace) and t = 15 min (red trace) of the βlactamase activity assay. (b) MALDI-QqQ MRM trace of PenG (substrate) and BPA (product) at t = 0 min and t = 15 min. Measured with n = 4 for each time point of the incubation. Time points below the peaks show the actual measuring time, which is roughly 5 s/spot.

with the following concentrations: 10, 50, 100, 250, 500, and 1000 nmol/mL. Identical solutions were measured on both instruments.

’ RESULTS Experiments with Purified β-Lactamase Enzyme

Initial experiments in the developmental phase of the project aimed to identify the selected antibiotics and the metabolite of PenG (BPA). BPA derives from bacterial β-lactamase activity, namely hydrolysis of PenG and has a net mass gain of 18 Da. Both compounds could be positively identified by high resolution MALDI-TOF MS analysis (see Figure 1a). Additionally, MS1 scans on the MALDI-QqQ instrument showed an emerging peak at the respective m/z value of 353 after incubation of PenG with isolated β-lactamase (commercially available β-lactamase I from B. cereus). The subsequent MS2 scans (data not shown) of the drugs and the metabolite were optimized to give the MRM mass transitions listed in the Materials and Methods section. All spectra/MRM traces on the MALDI-QqQ were compared to those from identical samples to which no antibiotic was added and did not show any interfering signals (data not shown). Similar experiments were carried out for AMP, CEF and IMI, and all three ABs were identified on both instruments in the relevant concentration range. Conditions of the sample preparation and the measurements on both instruments were optimized to result in optimal peak intensities and measuring times of ∼5 or ∼10 s/ spot on the MALDI-QqQ and MALDI-TOF system, respectively. Figure 1b shows the MRM trace of the decreased peak intensities after 15 min incubation (upper trace) and the simultaneous

Figure 3. Influence of increasing concentrations of clavulanic acid on the time dependent degradation of PenG (5 μmol/mL), expressed as the product/substrate ratio (PenG/BPA ratio) of the mean peak areas measured on the MALDI-QqQ instrument. Experiments were carried out with β-lactamase from B. cereus.

increase of the product peaks (lower trace) showing that the substrate PenG was converted to BPA. The feasibility of the β-lactamase assay was shown by time dependent incubations of PenG with isolated β-lactamase (B. cereus). Figure 2a and b show the MALDI-TOF and -QqQ measurement of identical samples from the time dependent PenG degradation. In addition to a better spot-to-spot reproducibility (average RSD (relative standard deviation) of 9% compared to 44%), an improved correlation (r2 = 0.8849 compared to r2 = 0.4203; nonlinear regression) was observed for the MALDI-QqQ compared to the MALDI-TOF application. The semiquantitative evaluation with the TOF instrument showed a comparable increase of the product/substrate ratios. Both instruments were able to detect (limit of detection) the metabolite (BPA) after 2.5 min incubation (data not shown). However, for 81

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Figure 4. β-Lactamase activity assay results with PenG (5 μmol/mL) over an incubation time of 5 and 20 min. Degradation was measured on the MALDI-QqQ for a lysate of a TEM-1 E. coli isolate. Values are expressed as the product/substrate ratio of the respective mean peak areas. Means ( SEM, n = 5 technical replicates.

measurements of a time-dependent degradation, the BPA peak intensities first had to overcome a threshold limit (limit of quantification) on both instruments, which was roughly reached at t = 20 min. As a control, CEF and IMI were incubated under the same conditions and did not show any degradation over 60 min (data not shown). To further ensure a specific degradation of PenG by β-lactamase activity, PenG was coincubated with clavulanic acid (CA). CA is a suicide inhibitor for certain β-lactamases, especially plasmid encoded β-lactamases, that is, penicillinases, while it does not inhibit most AmpC β-lactamases. For this set of experiments, 5 μmol/mL PenG was incubated with various concentrations (0, 1, 5, and 10 μmol/mL) of CA and the incubation was terminated at the following time points: 0, 15, 30, 45, and 60 min. The bar graph in Figure 3 shows product/substrate ratios over time and visualizes the concentration dependent effect of CA. Compared to the product/ substrate ratio (measured via MALDI-QqQ) after 60 min without inhibitor (100%), 1 μmol/mL CA showed 8.6% turnover while 5 and 10 μmol/mL CA showed an insignificant 3.0 and 2.4% turnover, respectively.

Figure 5. (a) Intra- and (b) interday reproducibility with partially derepressed E. coli isolates and PenG (5 μmol/mL) measured on the MALDI-QqQ. Values are expressed as the product/substrate ratio of the respective mean peak areas. Means ( SEM, n = 3.

and on three consecutive days. Results were obtained via the MALDI-QqQ instrument. The graphs (Figure 5a and b) shows matching product/substrate ratios of the peak areas for 0 and 15 min in both experimental setups. Measurements of Clinical Isolate Extracts and Inhibition by Clavulanic Acid

Finally, the applicability of the developed assay was tested with clinical isolates. An E. coli isolate (TEM-1) was freshly cultivated and after lysozyme treatment submitted to the newly developed β-lactamase activity assay. Identical assay conditions were applied, but instead of PenG the assay was conducted with AMP, another β-lactam antibiotic. The lysate was incubated with and without the addition of CA for 0, 15, 30, and 60 min. In the samples without CA a significant degradation of AMP was observed within the first 15 min, and we were no longer able to detect AMP after 15 min. Addition of CA showed a successful inhibition of β-lactamase activity and no degradation was observed: The respective relative peak areas at 0, 15, 30, and 60 min were 100.0, 100.1, 105.6 and 119.6% under the influence of CA (data not shown).

Proof of Concept with a Clinical Isolate of E. coli

To determine the applicability of the MALDI technique in determining β-lactamase activity in clinical isolates, the extract of a TEM-1 positive E. coli was incubated with PenG (Figure 4). A significant hydrolysis of PenG within the first 5 min of incubation was shown. Assay Linearity

Following the previously described experiment, the linearity of extracted β-lactamase activity was determined. The antibiotics of interest, PenG and AMP were measured over a linear range of 10 1000 nmol/mL (exception MALDI-TOF for AMP: 100 1000 nmol/L, due to a lower sensitivity) in inactivated bacterial lysates. To increase accuracy, CEF was introduced as an IS. For both instruments a linear response for the analyte/IS ratio was observed. PenG was measured with r2 = 0.9984 (y = 0.0009x 0.0244) and r2 = 0.9355 (y = 0.0007x + 0.1012) on the MALDI-QqQ and -TOF, respectively. AMP was measured with r2 = 0.9996 (y = 0.0071x 0.0293) and r2 = 0.9768 (y = 0.0064x 0.6423) on both instruments, respectively.

’ DISCUSSION The current work describes the development and successful application of a quick-and-easy assay for the characterization of β-lactamase activity in bacterial lysates. This assay will be beneficial to characterize β-lactamases causing reduced susceptibility or resistance of various Gram negative bacteria such as E. coli strains against selected β-lactam ABs by a fast and very specific semiquantitative determination of drug hydrolysis. Common susceptibility tests mainly rely on cumbersome and timeconsuming quantitative (broth dilution) and qualitative (disk diffusion) assays or on faster and automated (e.g., Vitek, AutoSCAN, etc.) tests.10 Furthermore, susceptibility results do not give information with regard to the exact mechanisms of resistance and only performing additional experiments gives information in respect to the characteristics of certain β-lactamase

Assay Reproducibility

To further assess the reproducibility of the β-lactamase activity assay, three independent assays with PenG as substrate for the AmpC expressing E. coli was monitored within one day (n = 3) 82

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enzymes. In particular, it is difficult with existing techniques to discriminate between β-lactamase activity of bacteria and decreased influx or increased efflux of β-lactam AB in bacteria. Just recently two publications described for the first time the use of MALDI-TOF for the characterization of carbapenemase activity.11,12 Both contributions demonstrated the validity of this novel approach in a substantial number of different strains. However, the current work demonstrates the possibility to determine hydrolysis rates and substrate preferences of the β-lactamase enzyme, resulting in information with regard to the specificity of the enzyme and the technical reproducibility of the assay. The addition of different ABs (IMI, CEF, AMP) belonging to different classes and that of inhibitors, as demonstrated with CA, gives crucial information about the specificity of the observed enzymatic hydrolysis. CA is an inhibitor useful for characterization of different classes of β-lactamases.13 Inter- and intraday measurements in combination with the determination of the linearity of the detection strengthen the analytical side of this approach. The relevant information is obtained in a timely fashion. Including the lysate preparation from a bacterial colony on an agar plate, the assay presented herein delivers information on β-lactamase activity of a selected strain including information of the inhibiting influence of CA within 1.5 h. An unequivocal identification of the applied antibiotic standards and BPA allowed the reliable measurements of the samples from the newly developed assay via both instruments. As for the MALDI-QqQ this information was mainly obtained via product ion scans (MS2) for structural elucidation. The MALDI-TOF delivered a higher mass accuracy leading to the identification of the drugs and the metabolite by measuring the exact mass of the analytes, thus confirming their identity. Initial investigations with a commercial β-lactamase build the basis for possible enzyme kinetic studies (Michaelis Menten) in the future. These investigations will help to understand the influence of mutations in β-lactamases,14 will allow a detailed assessment of the efficiency of enzyme inhibitors and therefore will allow the research for new lead compounds in drug discovery. For these purposes, the possibility of MRM of the MALDI-QqQ provides an ideal tool for several reasons. Regarding mass spectrometry, MRM measurements on a QqQ system are the most common and reliable way for quantification,15 the software for MRM is more user-friendly than software for profiling and, most importantly, both instruments are designed for automation for high throughput analysis.16 18 Taken together, the present work demonstrates a simple, highly accurate and reproducible assay for the determination and characterization of β-lactamase activity in clinical isolates of bacterial strains. The analytical readout is easy to handle and can be achieved in a very sensitive and ultra fast way, which was demonstrated on two different MS platforms, MALDI-TOF and MALDI-QqQ. The latter provides certain advantages especially in terms of accuracy and precision, which makes it favorable for quantitative assessments. Under the described settings, both instruments were shown to be suitable for qualitative measurements of enzyme-mediated drug degradation. Future studies will include various ABs and strains to expand on this newly applied technique.

Medical Center Rotterdam (Erasmus MC), Dr. Molewaterplein 50, Room Ae-307, 3015 GE Rotterdam, The Netherlands. Phone: +31107044521. Fax: +31107044365. E-mail: g.hooff@erasmusmc.nl. Present Addresses §

BioMerieux, 3 route de Port Michaud, La Balme-Les-Grottes, France

’ ACKNOWLEDGMENT We thank Nicole Lemmens for the technical support for the preparation of bacterial lysates. This work was supported by BioMerieux, Marcy l’Etoile, France. ’ REFERENCES (1) French, G. L. The continuing crisis in antibiotic resistance. Int. J. Antimicrob. Agents 2010, 36 (Suppl 3), S3–7. (2) Bradford, P. A. Extended-spectrum beta-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat. Clin. Microbiol. Rev. 2001, 14 (4), 933–51. (3) Jacoby, G. A. AmpC beta-lactamases. Clin. Microbiol. Rev. 2009, 22 (1), 161–82. (4) Paterson, D. L.; Bonomo, R. A. Extended-spectrum beta-lactamases: a clinical update. Clin. Microbiol. Rev. 2005, 18 (4), 657–86. (5) Canton, R.; Coque, T. M. The CTX-M beta-lactamase pandemic. Curr. Opin. Microbiol. 2006, 9 (5), 466–75. (6) Yong, D.; Toleman, M. A.; Giske, C. G.; Cho, H. S.; Sundman, K.; Lee, K.; Walsh, T. R. Characterization of a new metallo-betalactamase gene, bla(NDM-1), and a novel erythromycin esterase gene carried on a unique genetic structure in Klebsiella pneumoniae sequence type 14 from India. Antimicrob. Agents Chemother. 2009, 53 (12), 5046–54. (7) Meesters, R. J.; Cornelissen, R.; van Klaveren, R. J.; de Jonge, R.; den Boer, E.; Lindemans, J.; Luider, T. M. A new ultrafast and highthroughput mass spectrometric approach for the therapeutic drug monitoring of the multi-targeted anti-folate pemetrexed in plasma from lung cancer patients. Anal. Bioanal. Chem. 2010, 398 (7 8), 2943–8. (8) van Kampen, J. J.; Burgers, P. C.; de Groot, R.; Luider, T. M. Qualitative and quantitative analysis of pharmaceutical compounds by MALDI-TOF mass spectrometry. Anal. Chem. 2006, 78 (15), 5403–11. (9) Wagner, M.; Varesio, E.; Hopfgartner, G. Ultra-fast quantitation of saquinavir in human plasma by matrix-assisted laser desorption/ ionization and selected reaction monitoring mode detection. J. Chromatogr., B: Anal.t Technol. Biomed. Life Sci. 2008, 872 (1 2), 68–76. (10) Smaill, F. Antibiotic susceptibility and resistance testing: an overview. Can. J. Gastroenterol. 2000, 14 (10), 871–5. (11) Burckhardt, I.; Zimmermann, S. Using Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry To Detect Carbapenem Resistance within 1 to 2.5 h. J. Clin. Microbiol. 2011, 49 (9), 3321–4. (12) Hrabak, J.; Walkova, R.; Studentova, V.; Chudackova, E.; Bergerova, T. Carbapenemase Activity Detection by Matrix-Assisted Laser Desorption/Ionisation Time-of-Flight (MALDI-TOF) Mass Spectrometry J. Clin. Microbiol. 2011, 49 (9), 3222-7. Epub 2011 Jul 20. (13) Page, M. G.; Dantier, C.; Desarbre, E.; Gaucher, B.; Gebhardt, K.; Schmitt-Hoffmann, A. In vitro and in vivo properties of BAL30376, a beta-lactam and dual beta-lactamase inhibitor combination with enhanced activity against Gram-negative Bacilli that express multiple betalactamases. Antimicrob. Agents Chemother. 2011, 55 (4), 1510–9. (14) Pfeifer, Y.; Cullik, A.; Witte, W. Resistance to cephalosporins and carbapenems in Gram-negative bacterial pathogens. Int. J. Med. Microbiol. 2010, 300 (6), 371–9. (15) Hopfgartner, G.; Bourgogne, E. Quantitative high-throughput analysis of drugs in biological matrices by mass spectrometry. Mass Spectrom. Rev. 2003, 22 (3), 195–214.

’ AUTHOR INFORMATION Corresponding Author

*G. P. Hooff, Laboratory of Neuro-Oncology and Clinical and Cancer Proteomics, Department of Neurology, University 83

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(16) Pusch, W.; Kostrzewa, M. Application of MALDI-TOF mass spectrometry in screening and diagnostic research. Curr. Pharm Des. 2005, 11 (20), 2577–91. (17) Rathore, R.; Corr, J. J.; Lebre, D. T.; Seibel, W. L.; Greis, K. D. Extending matrix-assisted laser desorption/ionization triple quadrupole mass spectrometry enzyme screening assays to targets with small molecule substrates. Rapid Commun. Mass Spectrom. 2009, 23 (20), 3293–300. (18) van Kampen, J. J.; Burgers, P. C.; de Groot, R.; Gruters, R. A.; Luider, T. M. Biomedical application of MALDI mass spectrometry for small-molecule analysis. Mass Spectrom. Rev. 2011, 30 (1), 101–20.

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