Mass spectrometry detection of bacterial toxins and antibiotic resistance

Mass spectrometry (MS) and proteomics are gaining popularity in bacterial research and applications; notably, bacterial toxin detection and antibiotic resistance. Currently, several MS platforms exist (Fig. 1) and are described below.

by Angela Sloan, Dr Keding Cheng

Mass pattern and spectra comparison using MALDI-TOF-MS
The United States Food and Drug Administration (US FDA)-approved MALDI Biotyper or VITEK MS are both matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry (MALDI-TOF-MS)-based instruments which perform by ionizing molecules extracted from whole cell culture without specific protease treatment. The bacterial culture is normally treated with a strong solvent such as 1% trifluoroacetic acid (TFA) in 50% acetonitrile (ACN) [1] or 70% formic acid (FA) followed by 50% ACN [2] and centrifuged. Extracted molecules are mixed with chemicals (matrices) such as cyano-4-hydroxy-cinnamic acid (CHCA) and loaded onto a MALDI plate for MS detection. Sample spots are shot by laser energy and mass spectra, represented by mass to change ratios (m/z), are then obtained [1–3]. MS spectra are compared against different MS patterns/fingerprints harboured in the spectra library, and the microorganism is identified as the best match. Alternately, the bacterial culture can be smeared directly onto a MALDI plate and covered by the matrix of choice [3]. Each matching spectra result is a potential identification (ID) and given a confidence score. Generally, an ID showing a score equal to or greater than 2 is considered a correct identification [3]. A score below 1.7 is considered unreliable, from 1.7 to 1.9 indicates probable genus identification, from 2.0 to 2.29 indicates confident genus identification, and from 2.3 to 3.0 indicates highly confident species identification [4]. Culture conditions may affect spectra quality and ID [5], with pure cultures producing the most stable and consistent results [6–7]. MALDI-TOF-MS has been used in to test antibiotic resistance by growing cells in media containing normal and isotope-labelled lysine residues with and without antibiotics. The mass shift of many MS peaks, observed using bioinformatics software, demonstrated cell growth in the presence of the antibiotics, hence signifying antibiotic resistance [8].

Mass fingerprinting for molecules of interest using MALDI-TOF-MS or LC-MS
Peptide mass fingerprinting (PMF) is another form of mass fingerprinting of peptides, often obtained after protease treatment. Experimental mass spectra are compared to theoretical mass spectra using protease digestion patterns, which subsequently allows the protein/molecule of interest to be identified [9]. Proteins are typically prepared using an enrichment process such as gel electrophoresis or chromatography, and enzymatically digested either within the gel or in solution. During in-gel digestion, trypsin penetrates dried gel pieces and digests the protein within. The resulting tryptic peptides are then easily extracted from the gel, vacuum-dried, and analysed by MS.  When loaded onto a MALDI plate, samples will be covered with a matrix such as CHCA, for downstream MS analysis [10]. Bacterial extracts for routine MALDI-TOF-MS testing can also be digested, fractionated through chromatography, and loaded onto a MALDI plate to reduce sample complexity on each MALDI spot. Further fragmentation of the selected masses can be performed to obtain peptide sequences and subspecies level identification [11].

MALDI-TOF-MS is now able to detect carbapenemase or β-lactamase activities, indicators of antibiotic hydrolysis, by incubating bacteria from positive blood culture with the antibiotic. If the substrate is hydrolysed and shows a mass shift in MS detection, and antibiotic resistance can be inferred [12–13]. Liquid chromatography-mass spectrometry (LC-MS) has also been used for antibiotic susceptibility testing and metabolite profiling. For example, ampicillin (m/z 350 Da) can be hydrolysed into ampicillin-penicilloic acid (m/z 368 Da) and penilloic acid (m/z 324 Da) in ampicillin-resistant cells, resulting in mass changes easily detected by LC-MS [14].

Kalb et al. and Wang et al. used an Endopep-MS (a MALDI-TOF-MS-based method), to analyse the botulinum toxin [15–16], a highly substrate specific endopeptidase. The Endopep-MS method is based on the masses of cleavage products, which will be unique according to which toxin is present. The authors focused on the light chain of the toxin, providing it with a substrate in vitro that mimics the substrate in vivo. The presence/absence of the toxins was detected with 100% accuracy, and the analysis was not disturbed by the complex sample matrices analysed, such as meat and milk.

Targeted LC-MS/MS for protein identification and quantitation
Targeted protein identification and quantitation can be performed by multiple reaction monitoring (MRM) through a quadrupole MS system [17]. Ions (charged peptides) of interest can be selected from one quadrupole field and fragmented in the next. Peptide sequences are obtained and fragmented ions used for quantitation using stable isotope-labelled standard peptides as identification and quantitation references. Ion selection and fragmentation (MRM transition) is very fast and hundreds of targeted proteins can be identified and quantified in a 1-hour LC-MS/MS run. Either protein standards or peptide standards can be synthesized and used [18–19]. The beauty of using protein standards is that they can be spiked into the test sample, and quantitation of the protein performed accurately since the standards go through the sample preparation process [18]. MRM is highly specific, sensitive, accurate and reproducible.  Rees et al. used the unique affinity of organisms for their host, such as bacteriophages for certain bacteria, and analysed phage amplification products through sequence-based LC-MS/MRM to deduce antibiotic resistance properties of host cell [20]. MRM-based quantitation can also be multiplexed to quantitate many molecules of interest per sample run, rendering the process desirable for clinical applications [19]. A recent publication by Charretier et al. showed that with Staphylococcus aureus as a model, bacteria identification, antibiotic resistance, virulence and type profiling could all be obtained using one MRM platform [21].

Moura et al. used two methods of liquid chromatography-tandem mass spectrometry (LC-MS/MS) to detect the presence of Clostridium difficile toxins in cell culture filtrate: ultra-performance liquid chromatography-tandem mass spec (UPLC-MS/MS) and data independent UPLC-MS/MS [22]. Notably, the label-free data independent method could perform protein identification and quantitation in one MS experiment using alternating high and low energy. UPLC-MS/MS confirmed that digestion with trypsin was efficient and robust with sufficient amino acid coverage to identify the two main C. difficile toxins, TcdA and TcdB. They also showed that the most efficient combination of enzymes for differentiation of the two toxins was trypsin and GluC, providing amino acid coverage of 91% for TcdA and 95% for TcdB.  The data independent method quantified toxins at low levels and identified them separately. This is a novel development, as conventional methods typically quantify the total amount of toxin present. The method detected TcdA at 5 ng (1.6 μg/ml) and TcdB at 1.25 ng (0.43 μg/ml). Cell culture filtrate appears to be a novel approach for detection of C. difficile toxins, creating potential for future study.
Staphylococci are Gram-positive bacteria that cause pus-forming infections, food poisoning, and are the major cause of wound infections, nosocomial acquired pneumonia and septicemia [23–26].  Currently different results were obtained for Staphylococci species identification by MS approaches [23–25, 27]. Even so, evidence showed that the emergence of multiple Staphylococcus strains resistant to prescribed antibiotics could be detected by targeted LC-MS/MS by incubating bacteriophages with the bacteria [20]. In addition, Hennekinne et al. used isotope-labelled protein standards spiked into food extracts to identify and quantitate enterotoxins during a S. aureus food-poisoning outbreak using the LC-MS/MS platform. Bacterial toxins were enriched by the immune-affinity method and run on SDS-PAGE. In-gel digestion was performed and the toxin peptides were identified and quantitated based on quantitation standards [26].

Advantages of using MS platforms for bacterial toxin and antibiotic resistance tests
One of the most widely noted benefits of MALDI-TOF-MS is the ability of this platform to produce incredibly fast results, in some cases just a few minutes after loading the sample. MALDI-TOF-MS regularly uses fewer reagents and fewer steps, and requires less information on the organism than methods such as PCR or biochemical testing. Overall analysis is also cheaper since it occupies fewer working hours than traditional methods (the cost per MALDI-TOF-MS sample is reported to be approximately $0.50 to $1.00 [28]. Such rapid analyses would be of great benefit to epidemiological studies and clinical diagnoses where time is of the essence and faster identification will ultimately benefit patients [29].
Conclusion and perspective

An appealing aspect of MS is that any molecule that can be ionized should be detectable by MS with high sensitivity and resolving power. Current studies have confirmed that MS can analyse any cultivatable organism and its related metabolites without much prior knowledge, and it has been shown to be useful in a variety of applications, from fast hospital diagnosis and identification of typical bacteria, to organisms that are difficult to culture. With the gaining popularity of MS instrumentation, growing detectability, and user-friendly hardware and software, MS will certainly play a more substantial role in solving critical problems such as antibiotic resistance and the identification of toxins.

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The authors
Angela Sloan*1 MSc, Keding Cheng1,2 MD
1National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, MB R3E 3R2, Canada
2Department of Human Anatomy and Cell Sciences, Faculty of Medicine, University of Manitoba, Winnipeg, MB R3E 0J9, Canada

*Corresponding author