MALDI TOF

A concise but explanatory presentation on the use, advantage and limitations of MALDI TOF in molecular diagnosis

1. MALDI-TOF as a diagnostic tool in Microbiology (Advantages and Limitations) Dr. Suprakash Das Senior Resident Dept. of Microbiology

2. Introduction Rapid and accurate species identification of bacteria, fungi, and viruses is a fundamental requirement in clinical and food microbiology and other fields of microbiology diagnostics. Whereas virus recognition is usually achieved within hours by either serological tests or genotyping approaches using various nucleic acid detection systems, the conventional identification of bacteria and fungi still mainly relies on methods that include laborious and time-consuming initial cultivation and ensuing isolation of the microorganism. Though species identification of a pure culture is achievable within 24–48 h with various (semi-)automated systems, additional isolation steps are frequently necessary, which can extend the time until diagnosis by days, e.g., if the potential pathogen must be separated from the physiological background flora. Realistically species assignment of a putative pathogen from a non-sterile specimen takes at least 2–3 days.

3. Introduction In many areas of patient care, elapsed time until diagnosis may considerably reduce the therapeutic quality of care due to a lack of information about the infecting pathogen. Therefore, a rapid species diagnosis is of high priority as a focused therapy might be lifesaving for the patient. Molecular techniques which targets discriminatory genes offer excellent species identification often missed by phenotypic methods but they are limited by their high price per test, need of highly expert persons and most of them lack vast database. Here, the power of matrix-assisted laser desorption/ionization time-of- flight mass spectrometry (MALDI-TOF MS) is demonstrated as a redevelopment that has evolved to revolutionize the identification of prokaryotic and eukaryotic pathogens in microbial laboratories during recent years.

4. History of MALDI-TOF Mass spectrometry (MS) technology has been used for several decades in chemistry. In 1975 , Anhalt and Fenselau suggested the use of this tool for bacterial characterization, as they observed that different and unique mass spectra were produced from bacterial extracts of different genera and species. In the 1980s , desorption/ionization techniques (plasma desorption, laser desorption, and fast atom bombardment) that allow the generation of molecular biomarker ions from microorganisms were developed, opening the road for bacterial profiling.

5. History of MALDI-TOF The first experiments were based on a bio-molecule ionization processes that allowed the generation of biomarker molecules of low molecular masses, mostly bacterial lipids. Only in the late 1980s , Tanaka and Fenn ,[ Noble-2002 ] thanks to the development of soft ionization techniques (matrix-assisted laser desorption/ionization [MALDI] and electrospray ionization), made possible the analysis of large biomolecules such as intact proteins.

6. History of MALDI-TOF For the first time, in 1996 , MALDI-time of flight (TOF) spectral fingerprints could be obtained from whole bacterial cells by Holland et al ., avoiding pretreatment before the MS analysis. Nowadays , the matrix-assisted laser desorption ionization time-of flight mass spectrometry (MALDI-TOF MS) has emerged as a rapid, accurate, and sensitive tool for microbial characterization and identification of bacteria, fungi, and viruses, as demonstrated by the exponential number of publications about the issue.

7. Basic Terminology Adduct- Ion formed by the interaction of an ion with one or more atoms or molecules to form an ion containing all the constituent atoms of the precursor ion as well as the additional atoms from the associated atoms or molecules. Analyte - Biomolecule or sample that is being analyzed. Chromophore - Functional group in a molecule that is known to absorb light; this is necessary for the MALDI matrix in order to absorb the energy of the laser beam. Desorption- The opposite of absorption; here a substance is released from or through the surface rather than going into it. TOF- Time taken by the ions to travel through the flight tube when an electrostatic potential is applied at its ends

8. Basic Terminology Detector- The ions generated in a mass spectrometer after traveling through the flight tube ultimately hit the analyzer, where they are detected and converted into a digital output signal. Mass analyzer- Chamber having an electrostatic field; its purpose is to separate the ions coming from the source depending on their mass-to-charge ratio so that they can be detected by the detector. Matrix- Compound that is mixed with the sample that is being analyzed; the matrix protects the sample molecules from being destroyed by direct focus of the laser beams and facilitates the sample’s vaporization and ionization. Sublimation- Passing from solid to gas without going through a liquid phase.

9. Basic Principle of MALDI-TOF The genomic information within a microbial cell translates into more than 2000 proteins, a substantial number of which can be studied using proteomics. It is estimated that for genomes which contain less than 1000 genes, more than 50% of predicted proteome may be identified from the genome. Similarly 30 and 10% predicted proteome may be identified from genomes which carry 2500 and 4000 genes respectively ( Jungblut and Hecker , 2004) . Thus, a microbial genome containing 600–7000 predicted genes represents a medium-sized complex system where application of proteomics may provide knowledge of a substantial part of the microbe’s proteome.

10. Basic Principle of MALDI-TOF Mass spectrometry is an analytical technique in which chemical compounds are ionized into charged molecules and ratio of their mass to charge (m/z) is measured. Though MS was discovered in the early 1900s, its scope was limited to the chemical sciences. However, the development of electron spray ionization (ESI) and matrix assisted laser desorption ionization (MALDI) in 1980s increased the applicability of MS to large biological molecules like proteins. In both ESI and MALDI, peptides are converted into ions by either addition or loss of one or more than one protons. Both are based on “soft ionization” methods where ion formation does not lead to a significant loss of sample integrity.

11. Basic Principle of MALDI-TOF MALDI- TOF MS has certain advantages over ESI-MS viz. MALDI- TOF MS produces singly charged ions, thus interpretation of data is easy comparative to ESI-MS, for analysis by ESI- MS, prior separation by chromatography is required which is not needed for MALDI-TOF MS analysis. Consequently, the high throughput and speed associated with complete automation has made MALDI-TOF mass spectrometer an obvious choice for proteomics work on large-scale

12. MALDI – Principle and Methodology The sample for analysis by MALDI MS is prepared by mixing or coating with solution of an energy-absorbent, organic compound called matrix. When the matrix crystallizes on drying, the sample entrapped within the matrix also co-crystallizes. The sample within the matrix is ionized in an automated mode with a laser beam . Desorption and ionization with the laser beam generates singly protonated ions from analytes in the sample. The protonated ions are then accelerated at a fixed potential , where these separate from each other on the basis of their mass-to- charge ratio (m/z).

13. MALDI – Principle and Methodology The charged analytes are then detected and measured using different types of mass analyzers like quadrupole mass analyzers, ion trap analyzers, time of flight (TOF) analyzers etc. For microbiological applications mainly TOF mass analyzers are used. During MALDI-TOF analysis, the m/z ratio of an ion is measured by determining the time required for it to travel the length of the flight tube. A few TOF analyzers incorporate an ion mirror at the rear end of the flight tube, which serves to reflect back ions through the flight tube to a detector. Thus, the ion mirror not only increases the length of the flight tube , it also corrects small differences in energy among ions (Yates, 1998). Based on the TOF information, a characteristic spectrum called peptide mass fingerprint (PMF) is generated for analytes in the sample.

14. MALDI – Principle and Methodology Identification of microbes by MALDI-TOF MS is done by either comparing the PMF of unknown organism with the PMFs contained in the database, or by matching the masses of biomarkers of unknown organism with the proteome database. In PMF matching, the MS spectrum of unknown microbial isolates is compared with the MS spectra of known microbial isolates contained in the database. For species level identification of microbes, a typical mass range m/z of 2–20 kDa is used, which represents mainly ribosomal proteins along with a few housekeeping proteins. The characteristic pattern of highly abundant ribosomal proteins, which represent about 60–70% of the dry weight of a microbial cell, in the mass range of 2–20 kDa is used to identify a particular microorganism by matching its PMF pattern with the PMFs of the ribosomal proteins contained in an extensive open database .

15. MALDI – Principle and Methodology Although, the culture conditions might profoundly affect the microbial physiology and protein expression profile (Welker, 2011) they do not influence microbial identification by MALDI- TOF MS. Valentine et al. (2005) cultured three bacterial species on four different culture media and found that microbial MALDI-TOF MS identification was independent of culture conditions. A number of organic compounds have been used as matrices for MALDI-TOF MS but for microbiological applications, α- cyano-4-hydroxycinnamicacid(CHCA ), 2,5-dihydroxybenzoicacid(DHB ) , and 3,5-dimethoxy-4-hydroxycinnamic acid ( sinapinic acid ) have been found to be the most useful.

16. MALDI – Principle and Methodology The matrix solution consists of water and a mixture of organic solvents containing ethanol/methanol or acetonitrile and a strong acid like trifluoro acetic acid (TFA), which dissolves the matrix. The solvents penetrate the cell wall of microorganisms and extract out the intracellular proteins. When the solvent evaporates, ‘co-crystallization’ of protein molecules and other cellular compounds entrapped within the matrix solution takes place. Investigators have evaluated different sample preparation methods for different groups of microorganisms. Some microbes might be identified directly by MS, called direct cell profiling , while for some others whole cell lysates or crude cell extracts are prepared. In direct cell profiling , a single colony of microorganism is picked and spotted directly on to the sample plate and immediately overlaid with the matrix solution.

17. Role of the Matrix The energy pooling theory results in the following mechanism, whereby the absorbed photon from the excited-state matrix molecule ( M*) is transferred to the second excited matrix molecule, resulting in the formation of a cationic matrix radical ( M + ), a nonradical matrix molecule ( M), and a free electron (e): MM→2hv M*M*→ M + + M + e - The second step of this two-part reaction involves a proton transfer event from the excited matrix molecule to the clinical sample ( A), resulting in ionization of a molecule of the clinical sample:

18. Role of the Matrix M* + A→ (M - H ) + AH + Additional ions of the clinical specimen are formed by secondary ion-molecule reactions between matrix-matrix and matrix specimen interactions. These reactions are thermodynamically favorable because the proton affinity of MALDI matrices is typically lower than that of peptides and proteins to be analyzed in clinical material. This is modeled by the following equations: M + + M→ MH + + (M - H) and MH + + A→ M + AH +

19. List of commonly used matrices in UV-MALDI PA- Picolinic Acid DHAP-2,6 dihydroxyacetophenone . HPA- 3- hydroxy picolinic acid THAP- 2,4,6, tri hydroxyacetophenone SA- 3,5 dimethoxy-4-hydroxy cinnamic Acid HABA- 2,4-Hydroxy phenylazo benzoic Acid MBT- 2- mercaptobenzothiazole

20. Time of Flight Mass Analyzer The time of flight (TOF) analyzer is dependent upon the principle that applying an electrostatic field ( eV ) to the ionized clinical material causes a generated ion with a charge ( z) to accelerate, imparting to it some amount of kinetic energy (KE). The ions then move into a field-free drift region, where the only force affecting ionic movement is the kinetic energy from the acceleration step. The velocity ( v) of the ionized molecule from a clinical specimen can therefore be calculated by using the following equation, where KE is kinetic energy, m is mass, v is velocity, z is the charge of the ion (1 for MALDI), eV is the voltage applied, D is the distance to the detector, and t is time:

21. Time of Flight Mass Analyzer In this context, D and eV are constant and t is measured, allowing the m/z ratio to be determined. A simple mathematical rearrangement results in the following equation. KE= ½ mv 2 = zeV , v= D/t, t= D.(Square root of m/2zeV) . This equation demonstrates that drift time is directly proportional to the m/z ratio. Larger ions will have a longer drift time and smaller molecules will have a shorter drift time, demonstrating separation of molecules based on mass. This allows for separation of ions originating from clinical material based on the m/z ratio.

22. Sample Preparation Gram negative bacteria like Neisseria spp. , Yersinia spp. and Vibrio spp. were identified by MALDI-TOF MS using direct cell profiling. A ‘preparatory extraction’ of microbes with formic acid (FA) reportedly increased the ability of MALDI-TOF MS in identifying Gram-positive species. Due to the complex nature of their cell walls aerobic actinomycetes , Nocardia and mycobacteria require specialized processing procedures prior to MALDI-TOF analysis.

23. Sample Preparation Verroken et al. (2010) described a modified procedure for identification of Nocardia spp . by MALDI-TOF MS. Bacteria were lysed in boiling water, followed by ethanol precipitation of proteins. The precipitated proteins were dried, re-suspended in 70% formic acid and acetonitrile and analyzed by MALDI-TOF MS. EI Khéchine et al. (2011) described a procedure which combined inactivation and processing methods.

24. Sample Preparation Mycobacterial colonies collected in screw-cap tubes containing water and 0.5% Tween-20 were inactivated by heating at 95◦C for 1h. Inactivated samples were centrifuged and vortexed with glass beads to disrupt the mycobacterial cell wall, the resultant pellet was resuspended in formic acid, acetonitrile,and centrifuged again. Finally, the supernatant was deposited on to the MALDI target plate and over laid with matrix.

25. Sample Preparation As in bacteria, various sample processing methods have been investigated for identification of yeasts by MALDI-TOF MS, out of which ‘preparatory extraction’ with formic acid was reported to be most suitable . Investigators finally devised a protocol wherein fungi were cultivated on Sabouraud gentamicin-chloramphenicol agar for 72h at 27◦C , extracted with formic acid following incubation in ethanol . Acetonitrile was added, the mixture was centrifuged and supernatant was used for MALDI-TOF MS analysis .

26. Sample Preparation Lau et al. (2013) reported a method based on mechanical lysis for sample preparation of fungal hyphae and spores . A small specimen from the mold isolate was suspended in ethanol and zirconia -silica beads , vortexed and centrifuged . The pellet was re suspended in 70% FA , vortexed again, and centrifuged . The supernatant was used for subsequent analysis by MALDI-TOF MS. e.g Penicillium spp.

27. Sample Preparation Suggestions for MALDI-TOF MS sample preparations for use with different classes of microbes. Different groups of microorganisms vary fundamentally in their cellular composition and architecture. These differences have been demonstrated to affect the quality of spectra generated during MS experiments and, thus, the accuracy of MALDI-TOF MS-derived identifications. As such, investigators from a number of independent studies have evaluated different methods for sample preparation of different groups of microorganisms, ranging directly from intact-cell to full-protein extraction- based methodologies.

28. Present Scenario The discovery of suitable matrices, usage of whole/intact cells for recording PMF of microbes in the typical mass range (m/z) of 2–20 kDa , followed by availability of dedicated databases for microbial identification has made MALDI-TOF MS a lucrative alternative for microbial identification. The first MALDI-TOF MS system capable of microbial identification, termed “ MALDI Biotyper ” was developed by Bruker Daltonics . The MALDI Biotyper consisted of a basic MALDI-TOF platform, operating and analysis softwares , an onsite database and a simple method for extraction/preparation of sample. This system also allowed for up gradation by providing option of adding internal libraries of organisms.

29. Present Scenario Another MALDI platform for microbial identification was introduced by Shimadzu in collaboration with bioMérieux . Later, Andromas introduced a different kind of database and software for routine bacteriological identification which was compatible with both Bruker and Shimadzu instruments. In the last 2 years, MALDI Biotyper CA System ( Bruker Daltonics Inc.) has been approved by the US Food and Drug Administration (FDA) for identification of cultured bacteria from human specimens ( in vitro diagnosis).

30. Present Scenario Similarly, VITEK R MS ( bioMerieux Inc.) was approved by the USFDA for identification of cultured bacteria and yeasts. The list of microorganisms approved for identification is unique to the individual system. The organism databases are the key components of commercial MALDI platforms. They are continually increasing in size and are regularly updated by the manufacturers with discovery of new microbial species and annotations.

31. General schematic for the identification of bacteria and yeast by MALDI-TOF MS using the intact-cell method . Bacterial or fungal growth is isolated from plated culture media (or can be concentrated from broth culture by centrifugation in specific cases) and applied directly onto the MALDI test plate. Samples are then overlaid with matrix and dried. The plate is subsequently loaded into the MALDI-TOF MS instrument and analyzed by software associated with the respective system, allowing rapid identification of the organism.

32. General schematic for MS analysis of ionized microbiological isolates and clinical material. Once appropriately processed samples are added to the MALDI plate, overlaid with matrix, and dried, the sample is bombarded by the laser. This bombardment results in the sublimation and ionization of both the sample and matrix. These generated ions are separated based on their mass-to-charge ratio via a TOF tube, and a spectral representation of these ions is generated and analyzed by the MS software, generating an MS profile. This profile is subsequently compared to a database of reference MS spectra and matched to either identical or the most related spectra contained in the database, generating an identification for bacteria or yeast contained within the sample.

33. Comparison of 2 FDA approved systems Specification VITEK MS ( Biomeriux ) MALDI- Biotyper ( Bruker ) Calibrator- Escherichia coli ATCC 8739 US IVD Bacterial Test Standard (BTS) Matrix- -cyano-4-hydroxycinnamic acid (VITEK MS-CHCA) -cyano-4-hydroxycinnamic acid (US IVD HCCA portioned); must be reconstituted in accordance with instructions provided by using recommended solvent (standard solvent: 50 vol % ll acetonitrile , 47.5 vol % ll water, 2.5 vol % ll trifluoroacetic acid) Extraction- formic acid (VITEK MS-FA) (1) Deionized water (2) Absolute ethanol ( EtOH ) (3) Acetonitrile (ACN) (4) Formic acid (70%) (5) Microtube ( Eppendorf ), PCR clean 1.5 ml

34. Comparison of 2 FDA approved systems Specification VITEK MS ( Biomeriux ) MALDI- Biotyper ( Bruker ) Supplies- Target slides: single-use, disposable slides consisting of two acquisition groups, each with 16 sample spots (VITEK MS-DS target slides); each group includes one calibration spot. reusable, steel plates consisting of spots for 48 test organisms (US IVD 48 Spot Target); there are also five cross-joint positions that should be used for the BTS control. Sterile inoculating loops (1 µl) Sterile inoculating loops (1 µL) Precision micropipette (0.5 to 2.0 ll) Precision micropipette (0.5 to 2.0 ll) Sterile pipette tips without filter to reduce any protein contamination Sterile pipette tips without filter.

35. Comparison of 2 FDA approved systems Specification VITEK MS ( Biomeriux ) MALDI- Biotyper ( Bruker ) Equipment- Instrument : VITEK MS, floor model with a class 1, 337-nm fixed focus, nitrogen laser Microflex LT/SH mass spectrometer; desktop model with a class 1 337-nm fixed focus, nitrogen laser. Preparation station: VITEK MS Prep Station; location for preparing target slides, including a computer workstation with barcode reader. not applicable to MBT-CA system. Software: 1 . VITEK MS Acquisition Station ; operates the MS to acquire spectral data from each sample. The signal is recorded as a spectrum of intensity versus mass (in daltons [Da]). 1. MBT-CA System Software Package , including the MBT-CA System client software, the MBT-CA System Server, and the MBT-CA System, DB Server 2. VITEK MS Analysis Server ; manages the VITEK MS workflow and computes identification results 2. flexControl Software Package ,

36. Comparison of 2 FDA approved systems Specification VITEK MS ( Biomeriux ) MALDI- Biotyper ( Bruker ) Database VITEK MS Knowledge Base; current version (V2.0) of the reference database contains 755 approved taxa (645 bacteria and 100 fungi). MALDI Biotyper for Clinical Applications (MBT-CA); reference database containing the identity of 210 species or species groups, covering 280 clinically relevant bacteria and yeast species. The reference library was established using type strains combined with 5 to 38 additional clinical or culture collection strains per species.

37. Comparison of 2 FDA approved systems VITEK MS ( Biomeriux ) MALDI- Biotyper ( Bruker ) Media types: a. Columbia blood agar with 5% sheep blood b. TSA with 5% sheep blood c. TSA d. Chocolate polyvitex agar e. Campylosel agar f. MAC (use of this medium from some suppliers may show less optimal performance) g. Modified Sabouraud dextrose agar (glucose: 20 g/liter) h. chromID CPS a. Columbia blood agar with 5% sheep blood (Gram-negative aerobic bacteria) b. TSA with 5% sheep blood (Gram-negative aerobic bacteria, Gram-positive aerobic bacteria, yeast) c. Chocolate agar (Gram-negative aerobic bacteria and Gram-positive aerobic bacteria) d. MAC (Gram-negative aerobic bacteria) e. Columbia CNA agar with 5% sheep blood (Gram-positive aerobic bacteria) f. Brucella agar with 5% horse blood (Gram-negative and Gram-positive anaerobic bacteria) g. CDC anaerobe agar with 5% sheep blood (Gram-negative and Gram-positive anaerobic bacteria)

38. Comparison of 2 FDA approved systems VITEK MS ( Biomeriux ) MALDI- Biotyper ( Bruker ) Age of culture: For both bacteria and yeast protocols, isolates should be obtained from cultures after 24 to 72 h of incubation under appropriate growth conditions. In general, bacteria and yeast isolates should be obtained from cultures after 18 to 48 h of incubation, with continued stability up to 12 additional hours at room temperature (maximum 60 h). The following organisms, however, require specific considerations: a. Bordetella : incubation on BG agar should not be longer than 24 h (+12 h storage at RT) b. Campylobacter: incubation can be prolonged to 72 h (+12h storage at RT) c. Streptococcus pneumoniae : incubation should not be longer than 24 hours (+12 h storage at RT) due to possible autolysis of organism.

39. Comparison of 2 FDA approved systems Specification VITEK MS ( Biomeriux ) MALDI- Biotyper ( Bruker ) Identification Results. (1) Single identification : When only one significant organism or organism group is identified, a single identification is displayed with a confidence value of 60 to 99.9. (2) Low discrimination identification : When more than one significant organism or organism group is retained, but not more than four, low discrimination identifications for each organism are displayed. The sum of the confidence values equals 100. (3 ) Nonidentified : When more than four organisms or groups are identified, the list of possible organisms is displayed but the sum of confidence values is less than 100. (4 ) Nonidentified , U (unclaimed identification): No match is found. Using the FDA-approved algorithm, an organism identification is reported with high confidence if the log(score) is greater than or equal to 2.000. An organism identification is reported with low confidence if the log(score) is between 1.700 and 2.000. No identification is given if the log(score) is less than 1.700. (1) In general practice, a score of greater than or equal to 2.000 allows for species-level identification , whereas a score of 1.700 to 1.999 allows for genus-level identification . Again, a log(score) of less than 1.700 indicates no identification.

40. MALDI-TOF in Clinical Bacteriology A number of researchers have shown that MALDI-TOF MS can be used for early identification of bacteria in blood cultures, urinary tract infections (UTIs), cerebrospinal fluids, respiratory tract infections, stool samples etc. Many studies have shown that MALDI-TOF MS equal or even surpassed the conventional diagnostic methods in speed and accuracy in detecting blood stream infections. A few studies suggested that additional pre-treatment of body fluids by ammonium chloride, formic acid or short-term incubation on solid medium further improved the diagnostic potential of MALDI-TOF MS.

41. MALDI-TOF in Clinical Bacteriology When conventional methods for identification of urinary tract pathogens for diagnosis of UTIs were compared with MALDI-TOF MS based identification systems, it was found that MALDI-TOF MS required minimal processing time and identified bacteria from urine samples in the presence of even more than two uro -pathogens . Diagnosis of infectious diarrhea in laboratory is usually done by culture and identification of bacteria in the stool samples. This is a costly and time consuming process requiring 3–5 days for detection and identification of enteric bacterial pathogens. He et al. (2010) found that the entire procedure for identification by MALDI- TOF MS, from smear preparation to reporting of the final result was completed within 30 min, thus shortening the turnaround time of the test by 2–3 days.

42. MALDI-TOF in Clinical Bacteriology Bacterial meningitis is a neurological emergency. MALDI-TOF MS has been used for direct detection of bacteria causing meningitis in cerebrospinal fluids ( Segawa et al., 2014). It has also been used for rapid identification of atypical, Gram-negative environmental organisms and respiratory tract pathogens which chronically infect patients with cystic fibrosis . Guembe et al. (2014) reported that MALDI-TOF MS can perform better than conventional culture methods in diagnosis of catheter- related bloodstream infections .

43. MS Identification of Bacteria Directly from Patient Specimens While MALDI-TOF MS has been extensively evaluated as a universal platform for the proteomic analysis and identification of bacteria and yeasts from culture media, The technology is also being exploited to analyze patient specimens directly, completely bypassing the need for culture by detecting the presence or absence of pathogens in the clinical specimen proper. This type of direct analysis proved impossible before the advent of molecular analysis. The ability of both molecular and proteomic approaches to identify targets in these types of samples can also be enhanced by preliminary processing of these samples, removing some of the elements (proteins, nucleic acids, and cellular debris, etc.) that can inhibit analysis.

44. MS Identification of Bacteria Directly from Patient Specimens Current position of MALDI-TOF MS in the workflow of the clinical microbiology laboratory, including the current options for analysis of bacteria directly from patient specimens. The MALDI-TOF MS instrument fits easily into the clinical microbiology workflow, occupying the position once held by instruments for automated phenotypic-based identifications (blue arrows) . Evaluated mechanisms for the processing of samples directly from patient specimens are included (hatched red arrows) , as are options for the use of traditional (green arrows) and MALDI-TOF MS (hatched green arrows) mechanisms. Finally, results are imported into the laboratory information system from the MADLI-TOF MS instrument or other instruments and reported to physicians and pharmacists as indicated.

45. Food- and Water-Borne Bacteria The genus Aeromonas which is indigenous to surface waters is currently composed of 17 species, of which seven can cause severe water-borne outbreaks. Donohue et al. (2007) used the m/z signature of known strains of Aeromonas to assign species to unknown environmental isolates. Their results showed that MALDI-TOF MS rapidly and accurately classified unknown species of the genus Aeromonas , which was suitable for environmental monitoring.

46. Food- and Water-Borne Bacteria MALDI-TOF MS has also been applied successfully in food microbiology for various purposes like, Identification and classification of lactic acid bacteria in fermented food Detection of bacteria involved in spoilage of milk and pork Identification of bacteria isolated from milk of dairy cows Identification of bacteria present in probiotics and yogurt Identification of pathogenic bacteria contaminating powdered infant formula-food Characterization of biogenic amine-producing bacteria responsible for food poisoning and Identification of causative agents of seafood-borne bacterial gastroenteritis.

47. Detection and Identification of Agents of Biological Warfare Fast and reliable identification of microbes which pose threats as agents of bioterrorism is required, not only to combat biological-warfare attacks , but also to prevent natural outbreaks caused by these organisms. Conventionally, organisms which pose severe threats as agents of bioterrorism have been identified by phenotypic, genotypic, and immunological identification systems which are slow, cumbersome and pose significant risk to the laboratory personnel. Recently various researchers reported MALDI-TOF MS as a simple, rapid and reliable approach to identify highly pathogenic organisms like Brucella spp., Coxiella burnetti , Bacillus anthracis , Francisella tularensis , and Y. pestis .

48. Detection and Identification of Agents of Biological Warfare Further work is being carried out to develop safe and MS- compatible protocols for inactivation of vegetative cells and spores of highly pathogenic organisms (TFA & Ethanol) , which can be integrated into a routine microbiological laboratory. MALDI-TOF MS has also been shown to be useful for detection of protein toxins , such as staphylococcal enterotoxin B , botulinum neurotoxins, Clostridium perfringens epsilontoxin , shiga toxin etc. which can be used as potential agents for bioterrorism when delivered via an aerosol route.

49. Detection and Identification of Agents of Biological Warfare Alam et al. (2012) developed a simple method of sample processing for identification of protein toxins by MALDI-TOF/TOF MS method. Nebulizer was used to generate aerosols which were collected using a cyclone collector. Tandem MS data with information from peptide sequences was used for detecting toxins that originated from organisms of any geographical location.

50. Bacteria Identified in various studies using MALDI-MS Bacteria Genus Species or Group Evaluated Gram-positive organisms Staphylococcus Coagulase -negative staphylococci S. aureus Coagulase positive, non- S. aureus Micrococcus Micrococcus spp. Streptococcus Beta-hemolytic species Group A streptococci Group B streptococci Streptococcus pneumoniae Viridans group streptococci Nutritionally variant streptococci Enterococcus Enterococcus spp. Lactococcus Lactococcus spp. Bacillus Bacillus spp.

51. Bacteria Identified in various studies using MALDI-MS Bacteria Genus Species or Group Evaluated Gram-positive organisms Listeria Listeria spp. Corynebacterium Corynebacterium spp. Arcanobacterium / Trueperella Trueperella spp./A. haemolyticum Nocardia / myc-obacteria Nocardia Nocardia spp. Mycobacterium Mycobacterium spp. Gram-negative bacteria Enterobacteriaceae Salmonella Salmonella spp. Escherichia/ Shigella E. coli/ Shigella spp. Cronobacter Cronobacter spp.

52. Bacteria Identified in various studies using MALDI-MS Bacteria Genus Species or Group Evaluated Enterobacteriaceae Enterobacter Enterobacter cloacae complex Pantoea Pantoea spp. Plesiomonas P. shigelloides Klebsiella / Raoultella K. oxytoca / Raoultella spp. Yersinia Yersinia spp. Y. enterocolitica Y. pestis /Y. pseudotuberculosis Nonfermenting rods Acinetobacter Acinetobacter Burkholderia B. cepacia complex B. mallei /B. pseudomallei Pseudomonas Pseudomonas spp.

53. Bacteria Identified in various studies using MALDI-MS Bacteria Genus Species or Group Evaluated Nonfermenting rods Stenotrophomonas Stenotrophomonas maltophilia Fastidious organisms Brucella Brucella spp. Bartonella Bartonella spp. Francisella Francisella spp. Haemophilus Haemophilus spp. Vibrio Vibrio spp. Aeromonas Aeromonas spp. Campylobacter Campylobacter spp. Helicobacter Helicobacter spp. Neisseria Neisseria gonorrhoeae /N. meningitidis Moraxella Moraxella catarrhalis Legionella Legionella spp.

54. Bacteria Identified in various studies using MALDI-MS Bacteria Genus Species or Group Evaluated Anaerobic bacteria Propionibacterium P. acnes Bacteroides Bacteroides spp. Clostridium Clostridium spp. Clostridium difficile

55. Bacteria in which MALDI-TOF MS was used for identification and strain typing.

56. Detection of Antibiotic Resistance in Bacteria MALDI-TOF MS has been shown to generate PMFs capable of discriminating lineages of methicillin -resistant S. aureus strains MALDI-TOF MS has been shown to be of great use in identifying vancomycin -resistant enterococci . The production of β- lactamases is detected by MALDI-TOF MS employing a‘mass spectrometric β- lactamase (MSBL) assay . The MSBL assay has been applied for detection of resistance to β- lactam antibiotics like penicillin, ampicillin , piperacillin , cetazidime , cefotaxime , ertapenem , meropenem , and imipenem .

57. Detection of Antibiotic Resistance in Bacteria Using MSBL assay researchers have successfully detected β- lactamase producing organisms like Escherichia coli , Klebsiella pneumoniae , Pseudomonas aeruginosa , Acinetobacter baumanni , Citrobacter freundii , Enterobacter cloaceae , Salmonalla spp . etc .

58. Detection of Antibiotic Resistance in Bacteria Recently, Johansson et al. (2014) developed a MALDI-TOF MS method for detection and verification of carbapenemase production in anaerobic bacterium, Bacteroides fragilis as early as2.5h . Hart et al. (2015) suggested that instead of using intact bacterial cells for MS, the periplasmic compartment should be extracted (since β- lactamases are located in the periplasm ); in-solution digested with trypsin , separated by nano -LC before MALDI-TOF MS analysis. Using this approach they reported the peptide sequence of biomarkers for several classes of β- lactamases like CTX-M-1group extended spectrum β- lactamase , TEM β- lactamase , VIM a metallo - β- lactamase and CMY-2, an ampC β- lactamase .

59. Bacterial Strain Typing and Taxonomy Proteomics represents the functional aspect of genomics and can be used as a taxonomic tool. Gel-based whole cell protein profiling may be as cumbersome and time consuming as any other genomic technique. On the other hand MALDI-TOF MS intact cell or whole cell PMF based typing is a rapid and sensitive method for bacterial identification. In many cases it has shown resolution and reproducibility which is better than gel- based protein or DNA fingerprinting techniques.

60. MALDI-TOF MS in Clinical Virology The use of MALDI-TOF MS in virology has advanced less as it has in bacteriology or mycology. This might be a consequence of the relatively low protein content of viruses ( Kliem andSauer,2012), higher molecular weight of viral proteins(>20,000 Da )and a probable carry over of debris of the cell substrate in which viruses are cultured in vitro. Many researchers have proved the utility of MALDI-TOF MS for diagnosis of various infectious viruses in clinical samples like influenza viruses enteroviruses human papilloma viruses (HPVs) herpes virus hepatitis virus etc. Interestingly in most of the studies, the viral genetic material was amplified by PCR and the amplicons were analyzed/identified by MALDI.

61. MALDI-TOF MS in Clinical Virology Yi et al. (2011) reported the use of a PCR-based MS method for detection of high-risk HPVs , a prime cause of human cervical cancer. Piao et al. (2012) combined the multiplex PCR with MALDI-TOF MS and developed a PCR-Mass assay which simultaneously detected eight distinct viruses associated with enteric infections in humans . Calderaro et al. (2014) reported that MALDI- TOF MS was an effective, rapid and inexpensive tool which identified various poliovirus serotypes from different clinical samples.

62. Viral Genotyping, Sub-typing, and Epidemiological Studies Apart from viral identification, MALDI-TOF MS has also been used in virology for genotyping of JC polyomaviruses , hepatitis B and hepatitis C viruses and for detection of mutations in hepatitis B viruses. Many researchers have demonstrated the application of MALDI-TOF MS for screening of influenza virus subtypes and for tracking epidemiology of influenza viruses. Downard (2013) described a method for detection of strains of influenza viruses using whole virus protein digests. This ‘ proteotyping approach’ was successful in typing, subtyping , and tracing the lineage of human influenza viruses. MALDI-TOF MS has proved efficacy in detecting drug resistance to ganciclovir in cytomegaloviruses which frequently infect transplant recipients

63. MALDI-TOF MS in Clinical Mycology Conventional methods for identification of fungi are based on morphological, biochemical, and/or immunological properties which might span 2–5 days, or more, and often require combining several phenotypic methods for conclusive interpretations. The molecular methods based on analysis of genes encoding 18S rRNA and the internal transcribed spacer regions 1 and/or 2 (ITS 1/2) are tedious and time consuming. Fungal identification by MALDI-TOF MS in the medical mycology laboratory has moved at a slower pace than bacterial identification, owing to their inherent biological complexity which makes their study as a whole difficult.

64. MALDI-TOF MS in Clinical Mycology In order to obtain reproducible PMF results, parameters like culture media, quantity/type of colony material and incubation time, need to be carefully standardized. Also, the fungal cells might require additional treatment with trifluoroacetic acid, formic acid, or acetonitrile along with beating with beads to disrupt strong cell walls. Among fungi, highly reproducible PMF spectra have been reported for the ascomycetous and basidiomycetous yeasts including organisms like Candida, Cryptococcus, and Pichia .

65. Fungus Identified using MALDI-TOF in various studies Fungi Genus Species Yeasts Candida Candida spp. Cryptococcus Cryptococcus spp. Filamentous fungi/molds Aspergillus Aspergillus spp. Fusarium Fusarium spp. Dermatophytes Pseudallescheria - Scedosporium Pseudallescheria - Scedosporium Penicillium Penicillium spp.

66. Limitations of MALDI-TOF identification. The sensitivity of MALDI-TOF MS analysis for identification of microorganisms largely depends on the quality of the reference database used Difficult to identify microorganisms using MALDI-TOF MS- Enterobacteriaceae - E. coli/ Shigella : As with many genotypic and phenotypic identification systems, E. coli and Shigella species cannot be differentiated by using MALDI-TOF MS because of their high degree of similarity at the genomic level. Salmonella: Per FDA approval of the VITEK MS system, confirmatory testing is recommended before a final identification of Salmonella is made using MALDI-TOF MS. Enterobacter cloacae complex: Resolution to the species-level cannot always be accomplished by using MALDI-TOF MS analysis for strains within the E. cloacae complex. Combination of MALDI-TOF MS analysis and other molecular approaches, such as use of polymerase chain reaction (PCR) detection of specific gene targets, may be required for reliable identification of species within this complex.

67. Limitations of MALDI-TOF identification. Nonfermenting Gram-negative bacteria- Neisseria : Per FDA approval of the VITEK MS system, confirmatory testing is recommended before a final identification of Neisseria gonorrhoeae is made using MALDI-TOF MS. Burkholderia cepacia complex: MALDI-TOF MS analysis can reliably identify isolates within the B. cepacia complex to the genus-level but may misidentify or fail to identify isolates at the species-level. Gram-Positive Bacteria- Streptococcus mitis group: MALDI-TOF MS does not reliably distinguish between members of the S. mitis group. Therefore, additional testing, such as Optochin sensitivity and bile solubility, may be required to differentiate Streptococcus pneumoniae from other members of the group.

68. References Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry: a Fundamental Shift in the Routine Practice of Clinical Microbiology. Clinical Microbiology Reviews p. 547–603July 2013 Volume 26 Number 3. Singhal N, Kumar M, Kanaujia PK and Virdi JS (2015) MALDI-TOF mass spectrometry: an emerging technology for microbial identification and diagnosis. Front. Microbiol . 6:791. doi : 10.3389/fmicb.2015.00791 . Clinical Microbiology Procedures Handbook, 4 th Ed. Advanced Techniques in Diagnostic Microbiology. 2 nd Ed.

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