Field of Art

The invention relates to a method of identification of proteins of periplasmic space and of the outer membrane of a cell wall of Gram-negative bacteria using mass spectrometry. In particular it relates to a method of identification of beta-lactamases and to the detection of Gram-negative bacteria resistance towards beta-lactam antibiotics.

Background Art

Beta-lactamases are enzymes produced by some bacteria. They are responsible for bacterial resistance towards beta-lactam antibiotics (e.g. penicillins, cephalosporins, carbapenems). A suitable method for the determination of bacterial resistance is detection of beta-lactamases. It is important for an effective antibiotic treatment and for setting of suitable precautions to block the spreading of their producers.

Beta-lactamase identification is carried out using phenotype methods based on the sensitivity towards different inhibitors. A precise identification takes place by the use of PCR amplification of the genes thereof followed by sequencing of the amplicons. There are several laboratories dealing with the development of beta-lactamase identification methods using mass spectrometry (e.g. Schaumann R, Knoop N, Genzel GH, Losensky K, Rosenkranz C, Stingu CS, Schellenberger W, Rodloff AC, Eschrich K. 2012. A step towards the discrimination of beta-lactamase-producing clinical isolates of Enterobacteriaceae and Pseudomonas aeruginosa by MALDI-TOF mass spectrometry. Med. Sci. Monit. 18: MT71-MT77).

The method of protein (beta-lactamase) detection using MALDI-TOF (matrix assisted laser desorption/ionization time-of-flight) mass spectrometry is capable of affording results comparable with molecular genetics assays (PCR, microchips), yet making the whole process considerably faster and cheaper. So far, no effective method of detection of these enzymes using MALDI-TOF mass spectrometry has been published. The only published work, in which beta-lactamase has been identified using MALDI-TOF mass spectrometry, is the work of Camara JE, Hays FA. 2007. Discrimination between wild- type and ampicillin-resistant Escherichia coli by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Anal. Bioanal. Chem. 389: 1633-1638 from 2007. The results published by the above-mentioned authors were, however, ireproducible. At the same time there exist no publications confirming the conclusions of the above-cited work.

It is known from WO 2012/143535 and WO 2012/143534 that carbapenemases and cephalosporinases can be detected by mass spectrometry, however, the proteins are first cleaved into short peptides and the type of carbapenemases and cephalosporinases is then determined according to the spectra typical for those peptides.

Furthermore, the detection of bacterial resistance is known, wherein products of treatment with enzymes causing the resistance are followed by mass spectrometry. It involves covalently modified antibiotics or covalently modified model compounds (US 2012/196309). Likewise, there is known a determination of the presence of beta- lactamases, based on the detection of products of beta-lactamase activity, thus based on the detection of products of hydrolytic cleavage of the beta-lactam ring amide bond of beta-lactam anibiotics (WO 2011/154517).

All methods of detecting the enzymes of bacterial resistance known heretofore require either enzyme degradation into peptides or carrying out of the enzymatic reaction followed by the detection of the products of this reaction. This generates the risk of false positive results by incorporating additional components into the mixture being analyzed.

Disclosure of the Invention The present invention provides a method of detection of gram-negative bacteria periplasmic space and cell wall outer membrane proteins by mass spectrometry, wherein the periplasmic space and cell wall outer membrane proteins are extracted from the bacteria, and the proteins to be detected are stabilized by an inhibitor and/or a substrate of the respective protein to be detected. The stabilized proteins are then dissolved, deposited on a MALDI-TOF plate, covered by matrix solution and measured by a MALDI-TOF (matrix assisted laser desorption/ionization time-of-flight) mass spectrometry method. The resulting spectra are then compared with reference peaks for a given protein. Protein reference peaks are obtained from measurements of protein standards of known - or verified using known methods - identities.

The extraction is preferably carried out as follows: bacteria grown in a liquid cultivation medium appropriate for Gram-negative bacteria, e.g. Mueller-Hinton (MH) bouillon or brain-heart infusion (BHI), are cooled down to a temperature in the range of from 0 °C to 6 °C, followed by a centrifugation in order to sediment the bacteria. The supernatant is discharged, the pellet is re-suspended in an aqueous solution of saccharose having the concentration of at least 20 % (w/w), preferably from 20 % to 40 % (w/w), more preferably in a 40 % (w/w) saccharose aqueous solution. The incubation is carried out for at least 1 hour at the temperature of from 2 °C to 8 °C, followed by an addition of a buffer suitable for a lysozyme activity (for example Tris-HCl buffer) and the lysozyme. The mixture is then incubated at a temperature of from 35 °C to 37 °C for a time necessary to destroy the outer membrane of the cell wall and to liberate the proteins of periplasmic space (generally 1 hour minimum). The quality of the extraction/destruction of the outer membrane of a cell wall can be verified microscopically - the rod-shaped bacteria change into a spherical form (spherocytes).

In a preferred embodiment, the present invention provides a method of detection of beta-lactamases of gram-negative bacteria using mass spectrometry, wherein periplasmic space and cell wall outer membrane proteins are extracted from the bacteria, beta-lactamases in this mixture are stabilized with a beta-lactamase inhibitor and/or substrate. Beta-lactamases are further precipitated, the precipitated beta- lactamases are dissolved and deposited on a MALDI-TOF plate, covered by a matrix solution destined for ionization of proteins with Mr > 10 000 and measured using MALDI-TOF mass spectrometry method. The resulting spectra are compared with reference peaks of beta-lactamases.

In a preferred embodiment, the inhibitor or substrate used for the stabilization is a beta- lactam antibiotic or a beta-lactam inhibitor, e.g. meropenem, ampicillin, clavulanic acid, cefepime, phenyl boric acid. The precipitation is preferably performed using CI to C4 alcohol, more preferably ethanol, or acetone, into which trifluoroacetic acid can preferably be added, more preferably 0.1 vol. % of trifluoroacetic acid. Preferably, the precipitated stabilized beta-lactamase is dissolved in a mixture of acetonitrile and water with an addition of trifluoroacetic acid, more preferably in the mixture containing 50 vol. % of acetonitrile, 2.5 vol. % trifluoroacetic acid and water.

Beta-lactamase reference peaks can be obtained from measurements of beta-lactamase standards, whose identity is known or can be verified using known methods.

The present invention also provides a kit for the detection of beta-lactamases of Gram- negative bacteria using mass spectrometry, containing Tris-HCl buffer (pH 8.0), a lysozyme, a beta-lactamase inhibitor and/or substrate, CI to C4 alcohol or acetone, a mixture of acetonitrile and water with an addition of trifluoroacetic acid, matrix, matrix solvent.

Brief Overview of Figures Fig. 1 represents examples of detection of beta-lactamases of the CMY type. The peaks corresponding to beta-lactamase are marked by an arrow. A - spectrum of the purified beta-lactamase CMY-2, B - spectrum of a sample of Proteus mirabilis producing beta- lactamase CMY- 15, C - spectrum of a sample of Escherichia coli producing beta- lactamase CMY-2.

Fig. 2 represents the spectrum of Escherichia coli, wherein the thin arrow shows the peak of CMY-2 and the bold arrow shows the peak of the protein OmpC.

Fig. 3 represents the spectrum of Klebsiella pneumoniae, wherein the thin arrow shows the peak of CMY-2 and the bold arrow shows the peak of the outer membrane protein II.

Fig. 4 represents the spectrum of Klebsiella pneumoniae, wherein the thin arrow shows the peak of CMY-2 and the bold arrow shows the peak of the OmpK36.

Fig. 5 represents the spectrum of Proteus mirabilis, wherein the thin arrow shows the peak of CMY-2 and the bold arrow shows the peak of the flagellin. Examples Example 1

Cultivation of bacteria and beta-lactamase extraction:

The isolate of bacteria of the family Enterobacteriaceae (the bacteria tested were Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Citrobacter freundii) was inoculated into 50 mL of Mueller-Hinton bouillon (MH bouillon) or brain-heart infusion (BHI) with an addition of 50 mg/L of ampicillin; the culture was cultivated for from 12 to 18 hours at the temperature of from 35 °C to 37 °C.

The media with the cultivated culture was then cooled down to the temperature of ca. 4 °C (incubation on ice for ca. 10 min) and centrifuged for 20 min. The supernatant was discharged. The pellet was re-suspended in 90 xL of aqueous solution of saccharose (40 % w/w) and incubated for 1 hour at 4 °C.

In the next step, 10 μΐ, of 1M Tris-HCl buffer (pH 8.0) was added to the mixture together with 1 uL of Iysozyme (concentration 10 mg/mL); the incubation was carried out for 90 min at the temperature of from 35 °C to 37 °C. The quality of the extraction can be verified microscopically - the rod-shaped bacteria change into a spherical form (spherocytes).

The following centrifugation lasted 5 min at 14 000 g. The supernatant containing extracted beta-lactamases was further used.

Preparation and stabilization of beta-lactamases in a mixture:

100 Ε of the prepared extract was mixed with 25 μΐ, of 100 mM meropenem (phenyl boronic acid was also tested, giving similar results) and incubated for 10 min at room temperature. 1 mL of ice-cold ethanol with 0.1 vol. % of trifiuoroacetic acid was added to the mixture, vortexed for 30 seconds and centrifuged for 20 min at 14 000 g at the temperature of 4 °C. The supernatant was then discharged, the pellet was dried at the temperature of from 35 °C to 37 °C for 10 min, and further dissolved in 50 μΕ of acetonitrile solution (500 μΐ, of acetonitrile, 475 μΐ, of de-ionized water, 25 uL of trifiuoroacetic acid). The mixture was then vortexed for 1 min. Measurement itself:

1 μΐ, of the solution was placed on a MALDI plate used for MALDI-TOF mass spectrometry measurements and let to dry at room temperature. The sample was then covered by 1 of matrix (50 % (vol.) of ethanol in water, saturated with sinapic acid) and allowed to dry. MALDI-TOF measurement followed.

Fig. 1 shows spectra with separated peaks which are easy to integrate and which have a very good signal-to-noise ratio, obtained by using the method according to the present invention.

Figures 2 to 5 show spectra with separated peaks of beta-lactamases and other periplasmic space and cell wall outer membrane proteins, obtained by the procedure described in example 1, using meropenem for stabilization of the proteins.