ESCMID Online Lecture Library. by author

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1 Future of MALDI-ToF Jordi Vila Departament of Clinical Microbiology Hospital Clinic, Barcelona, Spain

2 MALDI-TOF MS Identification of bacteria, yeast and filamentous fungi

3 MALDI-TOF MS - Mycobacteria Centrifuge 1ml. eliminate supernatant and add 300µl H 2 O HPLC to the pellet Inactivate at 100º 30 min Add 900 µl of absolute ethanol, shake for 15 sec Centrifuge at 13,000 g for 2 min Eliminate supernatant Leave to dry a room temperature Add silica beads Add from 20 to 50 µl of acetonitrile Shake for 1 min with homogenizer at max. speed Add the same quantity of formic acid. Shake 5 sec. Centrifuge at 13,000 g for 2 min. Suspend two loops of the culture in 300 µl of H 2 O HPLC Place 1 µl of sample in the MALDI plate Leave to dry Add 1 µl of matrix and leave to dry Tudó G (2015) EJCM 34: 1527

4 MALDI-TOF MS - Mycobacteria Recommendations Substitute the vortex shaking by tissue homogeneizer shaking Calibrate the A MALDI-TOF equipment every run Test samples by duplicate If possible use B manual mode of the equipment, shooting the ionization C laser several time Interpret all profiles classified as A as good identification Categories B should be re-read or re-extracted. Category C should be re-extracted

5 Identification of species of the genus Candida. 200 strains MALDI-TOF MS C. albicans (102) C. glabrata (43) C. parapsilosis (22) C. tropicalis (20) C. krusei (7) C. lusitaniae (2) C. dubliniensis (2) C. norvegiensis (1) C. kefyr (1) - Excellent correlation (100%) between identification by API 20C and MALDI-TOF MS Unpublished data

6 MALDI-TOF MS Protocol to prepare yeast to be identified by MALDI-TOF MS. Inoculate 1mL tubes of Sabouraud Liquid Broth (SLB) 24h. Centrifuge for 2 min at full speed (13,000 rpm). Suspend the pellet in 300μl water, add 900μl ethanol, and vortex. Centrifuge for 2 min at full speed (13,000 rpm). Remove supernatant carefully by pipetting, centrifuge shortly, and remove the residual ethanol completely. Incubate at 37ºC until the pellet is completely dry.

7 MALDI-TOF MS Protocol to prepare yeast to be identified by MALDI-TOF MS. Add 30 μl of formic acid (70%) and approximatelly silica beads. Shake the mix with an automatic homogenizer (Minilys) for 4 cycles of 15 seconds each. Centrifuge shortely. Add 30 μl of acetonitrile, mix carefully and centrifuge for 2 min at 13,000 rpm.

8 Direct testing of positive blood cultures by MALDI-TOF 0.5 μl pellet μl 70% formic acid 10 ml rpm 5 min. 1.5 ml rpm 1 min. Wash twice with saline solution

9 AREAS for IMPROVEMENT Detection of mechanisms of resistance, for instance: β-lactamases Typing Detection of virulence factors Direct detection of microorganisms in clinical samples

10 MALDI-TOF as a tool to detect mechanisms of resistance Bacterial inoculum MacFarland or loop Intact or lysed Antibiotic Type Concentration Incubation Time Matrix Which one (HCCA, DHB) Solvent Criteria for interpretation Hydrolysed forms Intact forms Ratio Automated Shaking * Calibration is an important point

11 Detection of carbapenemases by MALDI-TOF Hydrolysis assay conditions Antibiotic: 50 µl of meropenem (0.5 mg/ml in 20 mm Tris-HCl buffer, ph 6.8) supplemented with 0.01% sodium dodecyl sulfate (SDS) Bacteria: 1 µl inoculation loop or pellet obtained from 1 ml of positive blood culture Incubation: at 37 C for 2-4h without agitation Matrix solution: 10 mg/ml of DHB (2,5-dihydrobenzoic acid) diluted in 50% ethanol Analysis of spectrum: Visual 50 µl 2-4h 14,000 rpm for 5 min 1 µl of supernatant + 1 µl of DHB

12 Detection of meropenem peaks after incubation with susceptible strains P. aeruginosa E. coli E. aerogenes 428.5

13 Detection of hydrolysed meropenem peaks after incubation with resistant strains E. coli (NDM-1) E. cloacae (VIM-1) K. oxytoca (VIM-1)

14 Detection of ESBLs by MALDI-TOF Hydrolysis assay conditions Antibiotic: 50 µl of cefotaxime (0.5 mg/ml in water) supplemented with 0.01% sodium dodecyl sulfate (SDS) with and without clavulanic acid (0.05 mg/ml) Bacteria: 1 µl inoculation loop or pellet obtained from 1 ml of positive blood culture Incubation: at 37 C for 2h without agitation Martix solution: 10 mg/ml of HCCA (α-cyano-4-hydroxycinnamic acid) Analysis of spectrum: Visual 50 µl 2h 14,000 rpm 1 µl of + 1 µl of for 5 min supernatant HCCA

15 Cefotaxime susceptible K. pneumoniae Cefotaxime resistant K. pneumoniae (ESBL) Hydrolized product Low intensity peak of hydrolized product Cefotaxime peak Cefotaxime peak +Disappearancee of cefotaxime peak Cefotaxime peak Only cefotaxime Cefotaxime with clavulanic acid Only cefotaxime Cefotaxime with clavulanic acid

16 AREAS for IMPROVEMENT Detection of mechanisms of resistance, for instance: β-lactamases Typing Detection of virulence factors Direct detection of microorganisms in clinical samples

17 MALDI for typing FlexAnalysis and BioTyper software Gel View PFGE Looks familiar?

18 Thirty-six MRSA clinical isolates, belonging to 4 different Clonal Complexes (CC) defined by Multilocus sequence typing (MLST): CC5 (n=15): ST 146 (n=5), ST125 (n=5), ST 228 (n=5) HA- MRSA clones CA- MRSA METHODS CC8 (n=11): ST8 (n=5), ST 247(n=4), ST1819 (n=2) CC22 (n=5): ST 22 CC398 (n=5): ST398 (livestock-associated clone)

19 METHODS MALDI-TOF PROCEDURE: Ethanol/Formic acid+acetonitrile For each strain, 30 mass spectra were obtained.

20 METHODS DATA ANALYSIS: With FlexAnalysis 3.0 software the m/z values obtained were exported as an Excel file.

21 DATA ANALYSIS: - A consensus peak profile was established for each strain. The discriminatory peaks were included if their presence was consistent within all 30 spectra - Peak intensity was not taken into account - The presence/absence of a given m/z value, was codified as a binary code for clustering analysis with SplitsTree software v4.11

22 Discrimination of the Clonal Complexes (CC) was achieved using reproducible spectrum differences at nine m/z values. The combination of the presence/absence of these peaks was associated with a specific CC. The peaks were located between 2,500 and 7,820 m/z values. RESULTS

23 CC22 (SplitsTree software v4.11) CC5 CC398 CC8

24 OTHER TYPING APPLICATIONS Clostridium difficile Reil et al. EJCM (2011) 30:1431 Enterococci Griffin et al. JCM (2012) 50: 2918 Listeria monocytogenes Barbuddhe et al. AEM (2008) 74: Salmonella Dieckmann and Malorny. AEM (2011) 77: 4136 STEC Karger et al. AEM (2011) 77: 896 Yersinia enterocolitica Stephan et al. J.Microbiol. Methods (2011) 87:150.

25 AREAS for IMPROVEMENT Typing Detection of mechanisms of resistance, for instance: β-lactamases Detection of virulence factors Direct detection of microorganisms in clinical samples

26 Detection of toxins Toxin detection Comparison toxigenic versus nontoxigenic strains Detection of toxin activity Toxin detection

27 Bittar et al: MALDI-TOF for rapid detection of staphylococcal Panton-Valentine leukocidin International Journal of Antimicrobial Agents 2009; 34: 467 The Panton-Valentine toxin is a cytotoxin which comprises two components with Mw of 32 and 38 kda Their presence is an indicator of virulence, 4448 da which has been associated with necrotizing skin lesions and mucous membranes, including necrotizing hemorrhagic pneumonia Subinh. [Oxa] can induce the production of PVL which is inhibited by the presence of RIF or LNZ Strain PVL+ Strain PLV -

28 Carbonelle et al: Detection of Panton-Valentine toxin in Staphylococcus aureus by mass spectrometry directly from colony: time as not yet come International Journal of Antimicrobial Agents 2010; 36: 187

29 Gagnaire et al: Detection of Staphylococcus aureus deltatoxin production by whole-cell MALDI-TOF Mass Spectrometry PLoS one 2012; 7: e40660 Delta toxin Agr (accessory gene regulator) - Global Regulator III RNA, small RNA that regulates the expression of various genes and also expresses the delta hemolysin Agr deficiency:» Associated with persistent bacteremia» Associated with increased mortality» Increase in biofilm formation» Reduced susceptibility to glycopeptides

30 Gagnaire et al: Detection of Staphylococcus aureus deltatoxin production by whole-cell MALDI-TOF Mass Spectrometry PLoS one 2012; 7: e40660 Strains with peak Strains with peak No peaks - Purified delta toxin - Strain Delta + - Strain Delta - Strain Delta - MALDI-TOF MS 139/168 12/168 17/168 Synergistic hly 139/139 10/10 0/17 hld gene sequence NT 1 G10S /1 9 wt /9 MALDI-TOF-TOF 1 wt /1 1 G10S /1 NT Northern blot RNAIII 1/1 NT 0/17

31 Toxins of clinical interest Detection of heat labile and heat stable toxins of enterotoxigenic Escherichia coli Detection of heat labile toxin of Vibrio cholerae Verotoxin detection of enterohemorrhagic E. coli or Shigella dysenteriae

32 AREAS for IMPROVEMENT Typing Detection of mechanisms of resistance, for instance: β-lactamases Detection of virulence factors Direct detection of microorganisms in clinical samples

33 Direct detection of bacteria from a clinical sample Protocol to prepare bacteria to be identified by MALDI-TOF MS. >1 ml rpm 5 min. 1 ml rpm 1 min. Two washes with sterile destilled water 0.5 μl pellet μl 70% formic acid

34 Direct detection of bacteria from a clinical sample SAMPLE GRAM STAIN CONVENTIONAL CULTURE DIRECT MALDI-TOF MS ID Amniotic fluid GNB Capnocytophaga sputigena Capnocytophaga sputigena Amniotic fluid GPB Listeria monocytogenes L. monocytogenes Amniotic fluid GPB Listeria monocytogenes L. monocytogenes Cerebrospinal fluid Not performed Pseudomonas aeruginosa P. aeruginosa Cerebrospinal fluid GNB Pseudomonas aeruginosa P. aeruginosa Cerebrospinal fluid GNB Pseudomonas aeruginosa P. aeruginosa Amniotic fluid GNB Proteus mirabilis P. mirabilis Amniotic fluid Streptococci Streptococcus agalactiae S. agalactiae Amniotic fluid Streptococci Streptococcus agalactiae, Prevotella bivia S. agalactiae Amniotic fluid Staphylococci Staphylococcus aureus S. aureus Synovial fluid Streptococci Streptococcus pneumoniae S. pneumoniae Amniotic fluid GNB Escherichia coli E. coli Bile Streptococci Enterococcus faecium E. faecium Amniotic fluid Staphylococci Staphylococcus epidermidis S. epidermidis Amniotic fluid Streptococci Streptococcus oralis S. oralis Amniotic fluid Streptococci Streptococcus anginosus, Veilonella parvula Veilonella Cerebrospinal fluid No microorganisms Staphylococcus epidermidis NP Peritoneal fluid No microorganisms Acinetobacter pittii NP Amniotic fluid Yeast cells Candida albicans NP

35 Direct detection of bacteria from a clinical sample Drawbacks: - Low volume of sample (!LCR) - Samples with high density - thick (peritoneal, joint, ascetic fluids) - Low concentration of bacteria (Negative Gram stain or low count of microorganisms by microscopic field) - Polymicrobial infections Samples with higher profitability: Amniotic fluid and urine (High volume and inoculum, not so much thick)

36 Proposal of a new protocol for rapid bacterial identification and AST directly from urine samples Urine culture Contaminated Positive Negative 675 samples Bacterial count (bacteria/ml) by UF-500i <5X10 3 >5X10 3 <5X10 4 >5X10 4 <5X10 5 >5X10 5 <5X10 6 >5X10 6 >10 7 >10 8 Negative % Negative 90,4 83,3 69,7 54,5 36,0 21,4 6,6 0,0 0,0 0,0 Positive % Positive 0,0 6,9 11,6 14,5 18,0 26,2 45,9 80,0 96,8 100,0 Contaminated % Contaminated 9,6 9,7 18,7 30,9 46,0 52,4 47,5 20,0 3,2 0,0 Total

37 Urine Detection of the microorganism by MALDI-ToF Directly from urine samples Culture Determine the count of bacteria (Flow cytometry) Centrifugation low speed Centrifugation High speed Antimicrobial susceptibility

38 Proposal of a new protocol for rapid bacterial identification and AST directly from urine samples Routine protocol Urine sample Urine culture MALDI-TOF MS Identification (colony) <14 ml <5x10 6 bacteria/ml No valid ID Study protocol Urine sample ( 14 ml) Flow cytometry screening (4 ml) 5x10 6 bacteria/ml Direct MALDI-TOF MS Identification (10 ml) Valid ID AST No sufficient sample Direct AST Results in 48 h Results in < 24 h

39 Proposal of a new protocol for rapid bacterial identification and AST directly from urine samples Urine culture Contaminated Positive Negative 1029 samples Bacterial count (bacteria/ml) by UF-500i <5X10 3 >5X10 3 <5X10 4 >5X10 4 <5X10 5 >5X10 5 <5X10 6 >5X10 6 >10 7 >10 8 Negative % Negative 94,4 84,4 73,9 48,1 37,9 21,4 17,1 7,5 0,0 0,0 Positive % Positive 1,6 4,6 9,7 15,6 21,1 32,9 43,6 72,5 91,8 100,0 Contaminated % Contaminated 4,0 11,0 16,4 36,4 41,0 45,7 39,3 20,0 8,2 0,0 Total

40 Proposal of a new protocol for rapid bacterial Identification and AST directly from urine samples 3 negatives 2 NP 1 NRI: Lactobacillus (-) 163 samples (>5x10 6 ) 18 contaminated - 6 no sufficient sample - 1 NP - 4 NRI - 7 with valid ID: 1 Aerococcus urinae 2 E. coli 2 K. pneumoniae 1 K. oxytoca 1 E. cloacae/c. koserii/salmonella 142 positives

41 6 NP: - 2 E. coli - 1 K. pneumoniae - 1 P. mirabilis - 1 P. aeruginosa + M. morganii - 1 P. aeruginosa + E. faecalis 11 NRI: - 4 E. coli - 1 E. faecalis - 1 M. morganii - 1 K. pneumoniae - 1 E. cloacae - 1 K. pneumoniae + E. faecalis - 1 E. coli + E.faecalis - 1 E. coli + C. freundii 142 positives 17 no sufficient sample for MALDI-TOF ID 125 positives 17 w/o ID (13,6%) 108 with ID Maldi: 68 E. coli Culture: 59 E. coli 9 E. coli + other Maldi: 22 K. pneumoniae Culture: 16 K. pneumoniae 6 K. pneumoniae + other Maldi: 5 P. mirabilis Culture: 3 P. mirabilis 2 P. mirabilis + other Maldi: 4 K. oxytoca Culture: 4 K. oxytoca Maldi: 3 P. aeruginosa Culture: 1 P. aeruginosa 2 P. aeruginosa + other Maldi: 1 M. morganii Culture: 1 M. morganii Maldi: 1 E. cloacae Culture: 1 E. asburiae Maldi: 1 S. agalactiae Culture: 1 S. agalactiae Maldi: 1 S. aureus Culture: 1 S. aureus Maldi: 1 E. faecalis Culture: 1 E. faecalis Maldi: 1 C. perfringens Culture: C. perfringens

42 19 samples with 2 m/o: 6 no sample for AST (5 E. coli and 1 C. perfringens) 3 AST of GPC performed 99 AST of GNB performed Fully concordant AST 1) 5 with Enterococcus spp. Results of direct AST corresponded to GNB 2) 14 with other GNB 11 direct AST corresponded to one of isolated GNB 3 direct AST resulted uninterpretable 108 positives with ID 102 with ID and AST 80 samples with monomicrobial GNB infection: - 54 E. coli - 16 K. pneumoniae - 4 K. oxytoca - 3 P. mirabilis - 1 M. morganii - 1 P. aeruginosa - 1 E. cloacae Average score of direct ID of 108 identified samples: 2,122 (min 1,753; max 2,42) Fully concordant AST

43 CONCLUSIONS Detection of ESBLs and carbapenemases Typing of different microorganims Detection of virulence factors Direct detection of microorganisms in clinical samples, mainly in urine and amniotic fluid

44 Other potential future applications Detection of biomarkers Detection of virus and parasites