Application of MALDI-TOF Mass Spectrometry for Rapid Pathogen Identification in an Era of ‘One Medicine-One Health’ Microbiology T

Poster P-2331 ECCMID, 2012 Application of MALDI-TOF Mass Spectrometry for Rapid Pathogen Identification in an Era of ‘One Medicine-One Health’ Microbiology T. Fritsche1,3, B. Olson2, C. Pike1, M. Stemper1,3, F. Moore1, J. Meece2,3, S. Shukla2,3, T. Novicki1 Marshfield Clinic, Marshfield, WI, USA1. Marshfield Clinic Res. Fndn., Marshfield, WI ,USA2. Univ. of Wisconsin, La Crosse, WI, USA3

REVISED ABSTRACT* INTRODUCTION OBJECTIVES: To evaluate the utility of MALDI-TOF mass The majority of human bacterial and fungal pathogens (up to 60% by some assessments) also produce disease in Table 2. Comparison (%) of genus- and species-level spectrometry (MS) as a single platform for pathogen various animal species. The remainder comprise a variety of species that are more specific pathogens for the particular identifications using MBT and Standard Lab Methods (SLM). identification (ID) in a regional laboratory practice host, and/or that only cross species barriers occasionally (zoonoses). Differences in laboratory practices between routinely performing analyses on human- and animal- human and veterinary testing laboratories relate to the differences in disease-producing agents, identification schema, Human Source Animal Source Overall source clinical specimens. Integration of signature- different antimicrobial agents and different guidance documents. SLM Genus Level ID 94.1 88.2 91.2 based proteomic analyses into medical and veterinary SLM Species Level ID 70.0 51.8 60.9 laboratories may enhance ID performance, improve Despite such differences, the increasing recognition that many of our core pathogens increasingly display similar MBT Genus Level ID 98.8 95.4 97.1 clinical service and epidemiologic research, and antimicrobial resistance phenotypes, and that we readily share these pathogens with our companion and food-source promote cross-disciplinary collaboration promulgated animals, behooves us to consider simultaneously the emergence and significance of these ‘shared’ pathogens in both MBT Species Level ID 94.4 87.2 90.8 by the ‘One Medicine-One Health’ concept (Veterinaria environments. Such recognition of the need for greater integration of human and animal health concerns has been the Italiana 45[1]; 2009). Here we demonstrate that MS can impetus for some years in the promulgation of the ‘One Medicine-One Health’ concept by the medical, veterinary and be as rapid, accurate and cost-effective when testing public health professions (Kaplan, 2009; Kahn, 2009). The lack of opportunity to capitalize on this concept is a practical isolates of veterinary importance as when testing matter; whether the human and animal laboratory testing communities are prepared to assist each other with this CONCLUSIONS human-source isolates in the same laboratory setting. initiative remains to be determined, but the benefits are considerable (Cardiff, 2008). •Use of mass spectrometry-generated signature-based

proteomic analyses for pathogen identification is rapidly METHODS: Clinically significant pathogens (n=1,502) Technology is one area where medical and veterinary professions strongly complement one another. The recent gaining acceptance among human and veterinary from humans (573), and domestic and exotic animals introduction of MALDI-TOF mass spectrometry is displaying great promise for the rapid, accurate and cost-effective diagnostic microbiology laboratories. (929) were analyzed by MS and included identification of bacterial and fungal pathogens. Integration of this proteomic signature-based approach into

(human/animal isolates): staphylococci (112/193), contemporary laboratory practice holds great promise in revolutionizing clinical microbiology, as is evidenced by recent •The rapidity and accuracy of the MBT for human streptococci (61/112), enterococci (54/109), other applications in both human (Bizzini, 2012; Boggs, 2012; Carbonnelle, 2011; van Veen, 2012) and veterinary (Ahrholdt, (98.8%/94.4%, genus-/species-level, respectively) and Gram-positive cocci (36/1), Gram-positive bacilli 2011; Hijazin, 2011; Kuhnert, 2012; Vila, 2012) medicine. The improvement in laboratory turn-around time from 48-96 animal (95.4%/87.2%) pathogen identifications is (18/56), Enterobacteriaceae (147/252), Pseudomonas to 24 hours for pathogen identification can be expected to have real-time benefits in terms of earlier, targeted unsurpassed compared with traditional biochemical aeruginosa (61/69), other nonfermentative bacilli antimicrobial selection, detection of emerging diseases, and permitting of quarantine to be instituted sooner. methods. (56/49), Pasturella spp. (1/51), Actinobacillus spp. (0/9), and other Gram-negative bacilli (27/28). Isolates were In this study we evaluated the utility of MALDI-TOF mass spectrometry as a single platform for rapid pathogen •Infection and antimicrobial resistance health threats in identified by standard biochemical methods, and by MS identification in a regional USA laboratory practice routinely performing analyses on both human and animal-source humans and animals are often shared between these (Bruker Daltonics MALDI Biotyper™ v3.0) according to specimens. populations, and local and regional diagnostic the manufacturer’s recommendations and validation laboratories serve a critical public health sentinel algorithms. Table 1. Identification of 1,502 human- and animal-source isolates (year 2010) tested by MBT (Bruker Daltonics MALDI Biotyper™ v3.0). function in detecting their presence. RESULTS: Overall, MS provided species- and genus-level IDs, respectively, for 90.8% and 97.1% of 1,502 tested % Species Level ID % Genus Level ID % No ID •Utilization of MS for identification of pathogens recovered from human and animal specimens facilitates isolates. Among human and animal-source isolates, Isolate Groups (nspecies) nHuman nAnimal Human Animal Human Animal Human Animal respectively, MS identified 94.4%/87.2% to species- Coagulase-negative Staphylococcus (14) 52 48 88.5 62.5 9.6 37.5 1.9 0 and enhances the likelihood of detecting emerging level, 98.8%/95.4% to genus-level and 1.2%/4.6%, no Staphylococcus epidermidis 29 6 100 100 0 0 0 0 pathogens in both populations, and supports a key Staphylococcus felis 0 13 0 15.4 0 84.6 0 0 component of laboratory infrastructure essential for identification (not in database); compared with MS, Staphylococcus haemolyticus 2 2 100 0 0 100 0 0 promotion of the ‘One Medicine/One Health’ concept. standard ID approaches identified 70.0%/51.8% to Staphylococcus saprophyticus 7 0 71.4 0 28.6 0 0 0 species-level and 94.1%/88.2% to genus-level. Staphylococcus schleiferi 0 22 0 77.3 0 22.7 0 0 Discordant or missing IDs were present in 5.9% of Coagulase-positive Staphylococcus (4) 1 124 100 84.7 0 14.5 0 0.8 Staphylococcus intermedius group 1 124 100 84.7 0 14.5 0 0.8 human and 11.8% of animal source isolates. Staphylococcus aureus 59 21 100 100 0 0 0 0 Enterococcus species (8) 54 109 100 100 0 0 0 0 SELECTED REFERENCES CONCLUSION: Emerging infection health threats are Enterococcus faecalis 47 83 100 100 0 0 0 0 often zoonotic in origin, and local and regional Enterococcus faecium 6 19 100 100 0 0 0 0 Ahrholdt J, Murugaiyan J, Straubinger RK, Jagielski T, diagnostic laboratories serve a critical sentinel function Escherichia coli 49 104 100 100 0 0 0 0 Roesler U. Epidemiological analysis of worldwide bovine, Klebsiella species (3) 21 22 100 100 0 0 0 0 in detecting their presence. Utilization of MS for ID of canine and human clinical Prototheca isolates by PCR Klebsiella oxytoca 6 6 100 100 0 0 0 0 genotyping and MALDI-TOF mass spectrometry proteomic pathogens recovered from human and animal Klebsiella pneumoniae 14 16 100 100 0 0 0 0 specimens facilitates and enhances the likelihood of Proteus mirabilis 10 39 100 100 0 0 0 0 phenotyping. Med Mycol. 2011 Sep 12. [Epub ahead of detecting emerging pathogens in both populations. The Other Enterobacteriaceae (24) 67 87 95.5 92.0 4.5 4.6 0 3.4 print] accuracy and rapidity of MS technology for human Citrobacter species 13 13 92.3 76.9 7.7 7.7 0 15.4 Enterobacter species 15 12 93.3 100 6.7 0 0 0 (94.4%/98.8%, species-/genus-level) and animal (87.2%/ Bizzini A, Greub G. Matrix-assisted laser desorption Morganella morganii 6 7 100 100 0 0 0 0 ionization time-of-flight mass spectrometry, a revolution in 95.4%) pathogen IDs, supports a key component of Providencia species 3 5 100 100 0 0 0 0 laboratory infrastructure essential for promotion of the Salmonella species 10 16 100 93.8 0 6.2 0 0 clinical microbial identification. Clin Microbiol Infect. 2010 ‘One Medicine/One Health’ concept. Pseudomonas aeruginosa 61 69 100 100 0 0 0 0 Nov;16(11):1614-9. Other Non-fermentative GNB (38) 56 49 89.3 71.4 7.1 24.5 3.6 4.1 Achromobacter xylosoxidans 5 2 100 100 0 0 0 0 Boggs SR, Cazares LH, Drake RR. Characterisation of a *Revised to include additional isolates. Acinetobacter species 12 9 91.7 88.9 8.3 0 0 11.1 Other Pseudomonas species 10 14 80.0 57.1 0 42.9 20.0 0 Staphylococcus aureus USA300 protein signature using Stenotrophomonas maltophilia 17 6 100 83.3 0 16.7 0 0 matrix-assisted laser desorption/ionization time-of-flight Pasturella/Actinobacillus spp. (7) 1 60 100 78.3 0 8.3 0 13.3 mass spectrometry. J Med Microbiol. 2012 Feb 9. [Epub Actinobacillus species 0 11 0 72.7 0 9.1 0 18.2 ahead of print] Pasteurella canis 0 19 0 84.2 0 15.8 0 0 Pasteurella multocida 1 22 100 81.8 0 4.6 0 13.6 Gram-positive bacilli (27) 18 56 72.2 42.9 16.7 23.2 11.1 33.9 Carbonnelle E, Mesquita C, Bille E, Day N, Dauphin B, MATERIALS AND METHODS Arcanobacterium pyogenes 0 14 0 92.9 0 7.1 0 0 Beretti JL, Ferroni A, Gutmann L, Nassif X. MALDI-TOF mass Bacterial isolates. Clinically significant pathogens Actinomyces species 0 10 0 10.0 0 50.0 0 40.0 spectrometry tools for bacterial identification in clinical (n=1,502) were recovered prospectively by a USA Bacillus species 1 5 0 80.0 100 20.0 0 0 microbiology laboratory. Clin Biochem. 2011 Jan;44(1):104- Corynebacterium species 7 2 100 100 0 0 0 0 9. midwest laboratory practice serving both human and Other gram-negative bacilli (17) 27 28 88.9 57.1 11.1 17.9 0 25.0 veterinary clients. Isolates originated from humans Aeromonas species 3 9 100 100 0 0 0 0 (573) and domestic and exotic animals (929) during Haemophilus species 14 1 92.9 0 7.1 0 0 100 Cardiff RD, Ward JM, Barthold SW. 'One medicine---one 2010-11 and were archived for subsequent analysis. Neisseria species 2 11 100 45.4 0 36.4 0 18.2 pathology': are veterinary and human pathology prepared? Challenge pathogens included (human/animal isolates): Miscellaneous gram-positive cocci (7) 36 1 91.7 100 5.5 0 2.8 0 Lab Invest. 2008 Jan;88(1):18-26. Aerococcus sanguinicola 13 0 100 0 0 0 0 0 staphylococci (112/193), streptococci (61/112), Aerococcus urinae 15 0 86.7 0 13.3 0 0 0 enterococci (54/109), other Gram-positive cocci (36/1), Aerococcus viridans 1 1 100 100 0 0 0 0 Hijazin M, Hassan AA, Alber J, Lämmler C, Timke M, Gram-positive bacilli (18/56), Enterobacteriaceae Streptococcus species (14) 61 112 90.2 96.4 8.2 0.9 1.6 2.7 Kostrzewa M, Prenger-Berninghoff E, Zschöck M. Evaluation (147/252), Pseudomonas aeruginosa (61/69), other Streptococcus agalactiae 14 4 100 100 0 0 0 0 of matrix-assisted laser desorption ionization-time of flight nonfermentative bacilli (56/49), Pasturella spp. (1/51), Streptococcus canis 4 38 100 97.4 0 0 0 2.6 mass spectrometry (MALDI-TOF MS) for species Streptococcus dysgalactiae 4 16 100 100 0 0 0 0 identification of bacteria of genera Arcanobacterium and Actinobacillus spp. (0/9), and other Gram-negative Streptococcus equi 0 34 0 100 0 0 0 0 bacilli (27/28). Details of species and numbers tested Streptococcus pneumoniae 18 3 100 100 0 0 0 0 Trueperella. Vet Microbiol. 2011 Dec 26. are found in Table 1. Totals 573 929 94.4 87.2 4.4 8.2 1.2 4.6 Kahn LH, Kaplan B, Monath TP. 'One health' in action series: Identification test methods. All isolates were identified nos 1-8. Vet Ital. 2009 Jan-Mar;45(1):195-208. using existing laboratory biochemical algorithms for RESULTS routine processing and included rapid spot tests, •Among all human- (n=573) and animal- (n=929) source isolates tested, MBT identified 97.1% to the genus-level and Kaplan B, Kahn LH, Monath TP. The brewing storm. Vet Ital. biochemical strip tests, traditional tube biochemicals, 90.8% to the species-level (Table 2). 2009 Jan-Mar;45(1):9-18. use of the Phoenix® (human isolates only) and Vitek® Legacy (animal-source isolates only) automated •When separated by pathogen source (human/animal, respectively), MBT identified 98.8%/95.4% to the genus-level Kuhnert P, Bisgaard M, Korczak BM, Schwendener S, instruments, and/or use of the Sherlock Microbial and 94.4%/87.2% to the species-level; compared with MBT results, standard identification approaches identified Christensen H, Frey J. Identification of animal Identification system [MIS; MIDI, Inc.]). 94.1%/88.2% to the genus-level and 70.0%/51.8% to the species-level (Table 2). Pasteurellaceae by MALDI-TOF mass spectrometry. J Microbiol Methods. 2012 Feb 10;89(1):1-7. Isolates were subsequently analyzed by mass •Discordant or missing IDs were present in 5.9% of human and 11.8% of animal-source isolates; MBT database entries spectrometry (Bruker Daltonics MALDI Biotyper™ v3.0) were missing for 1.2% of human- and 4.6% of animal-source isolates (Table 1). Van Veen SQ, Claas EC, Kuijper EJ. High-throughput according to the manufacturer’s recommendations and identification of bacteria and yeast by matrix-assisted laser •Absolute genus/species identification error rates, based upon subset analysis of those isolates with definitive validation algorithms. From the direct transfer target desorption ionization-time of flight mass spectrometry in identifications were, respectively, 0.5/1.0% for human-source isolates (n=420) and 0.4/1.9% for animal-source isolates preparation method, utilizing two target spots conventional medical microbiology laboratories. J Clin (n=462). inoculated with a single colony collection, the MALDI Microbiol. 2010 Mar;48(3):900-7.

Biotyper (MBT) generated organism classification results •MBT provided highly accurate species-level identifications (human- and animal-source isolates) for Staphylococcus that correlated the probability of secure species-and Vila J, Juiz P, Salas C, Almela M, de la Fuente CG, aureus, Enterococcus species, Escherichia coli, Klebsiella species, Proteus mirabilis and Pseudomonas aeruginosa (all Zboromyrska Y, Navas J, Bosch J, Agüero J, de la Bellacasa JP, genus-level IDs by the following score value range and 100%; Table 1). color code: 3.00-2.00 (green-species level identification), Martinez-Martinez L. Identification of clinically relevant Corynebacterium spp., Arcanobacterium haemolyticum and 1.99-1.70 (yellow- genus-level identification) and 1.69- •Human-source MBT identification limitations included non-fermentative bacilli other than P. aeruginosa (3.6%) and 0.00 (red-not in database/unreliable identification). To Rhodococcus equi by MALDI-TOF MS. J Clin Microbiol. 2012 Gram-positive bacilli (11.1%); among animal-source pathogens, Gram-positive bacilli (33.9%), ‘Other’ Gram-negative Feb 15. optimize spectral analysis, a formic acid extraction was bacilli (25.0%), and Pasturella/ Actinobacillus spp. (13.3%) were most problematic. performed on isolates in which the direct transfer procedure resulted in a score value ≤1.9. The MBT ‘best •All Salmonella spp. tested (n=26) were correctly identified to the genus-level by both the MBT and standard ID match’ identification was compared with existing methods. laboratory biochemical algorithms described above. Genus and species-level identification error rates were •MBT identified 77.3% of S. schleiferi and 100% of S. equi; subspecies differentiation validation is ongoing for animal- ACKNOWLEDGEMENTS determined on a subset of human- (420) and animal- source isolates of Staphylococcus schleiferi ssp. schleiferi/ssp. coagulans and Streptococcus equi ssp. Technical support was provided by Bruker Daltonik GmbH. (462) source isolates for which full characterization was equi/ssp.zooepidemicus. available, and included use of 16S sequencing approaches to adjudicate discrepant results. •MBT is a cost efficient and rapid identification system, in which a bacterial isolate can be identified for under US$0.50 in less than 7 minutes (based upon a run of 45 isolates).

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