Professional Documents
Culture Documents
alieria Clavibacter Shimwellia Brachybacterium Leclercia Providencia Trabulsiella Xanthobacter Emericella Gardnerella Sporothrix Leuconostoc Pseudoclavibacter Alkali
omyces Turicella Roseomonas Ruminococcus Scedosporium Dysgonomonas Staphylococcus Peptostreptococcus Paenibacillus Balneatrix Solibacillus Prototheca Cupr
cillus Aspergillus Arthrobacter Mesorhizobium Acholeplasma Filobasidium Propioniferax Azohydromonas Chromobacterium Curtobacterium Kloeckera Austwickia Hyphomic
coccus Rummeliibacillus Budvicia Aquincola Enterobacter Sporobolomyces Brevundimonas Capnocytophaga Tatlockia Neisseria Salinivibrio Pullulanibacillus Arcanoba
ella Eggerthella Methanomonas Mucor Mobiluncus Caulobacter Helcococcus Psychrobacillus Campylobacter Blastomonas Wohlfahrtiimonas Thermoactinomyces Hermini
murella Mycobacterium Bordetella Pichia Vibrio Iodobacter Tenacibaculum Listeria Plesiomonas Haloarcula Shewanella Paecilomyces Thauera Viridibacillus Yokenella Ma
phingobium Ornithobacterium Epidermophyton Oligella Paracoccus Aureobasidium Eubacterium Dietzia Salimicrobium Klebsiella Mycoplasma Variovorax Sa
phyllum Scopulariopsis Odoribacter Anaerotruncus Abiotrophia Burkholderia Sodalis Empedobacter Sphingopyxis Lactococcus Sphingobium Microsporum Pepton
occus Beauveria Morganella Pasteurella Cedecea Bifidobacterium Micrococcus Propionimicrobium Starkeya Prevotella Histophilus Sphingomonas Acetobacter Fra
acterium Propionibacterium Aneurinibacillus Arthrographis Aromatoleum Pediococcus Phoma Xenorhabdus Methylobacillus Fusarium Wolinella Bacteroides Zygosacchar
simicrobium Helicobacter Rhizobium Terrabacter Ralstonia Butyricimonas Microsporum Castellaniella Borrelia Microbacterium Rheinheimera Wautersiella Saccharopo
la Nocardioides Gluconobacter
Kytococcus Chryseobacterium Alishewanella Gemella Methylobacterium Haemophilus Adlercreutzia
ochrobactrum Leminorella Candidatus Xanthomonas Pectobacterium Brevibacterium Arthroderma
imonas Actinomyces
obacter Lysinibacillus
MALDI Biotyper
bacterium Stenotrophomonas
Dear customers To meet the demands of leading microbiology laboratories for even higher productivity and faster turn-
around times, we expanded the MALDI Biotyper product range recently by the introduction of the
Welcome to the 5th edition of our MALDI Biotyper (MBT) Poster Hall, which showcases a selection of MALDI Biotypersmart mass spectrometer system. The MALDI Biotypersmart system features Brukers
scientific publications presented at recent conferences and meetings. proprietary smartbeam solid-state laser technology, which is more than 3 times faster than conventional
nitrogen lasers. Additionally, the smartbeam laser combines the at least one order of magnitude higher
With over 2000 systems in operation, the MALDI Biotyper has become the gold standard for routine lifetime of solid-state lasers with the unmatched MALDI performance of nitrogen lasers used in microbial
identification of microorganisms in clinical laboratories, and its use is steadily increasing for routine identifi- identification.
cation tasks in environmental labs, the pharmaceutical industry, and food and beverage markets.
To complement Brukers established reusable stainless steel MALDI target plates, we enlarged the
Probably the most compelling reasons for the wide acceptance and implementation of the MALDI Biotyper MALDI Biotyper sample preparation portfolio by introducing the disposable MBT Biotarget 96. These
in microbiology labs are its ease of use, its robustness, and broad species coverage which we are con- new MALDI target plates provide increased workflow efficiency by offering 96 sample positions, and by
tinuously expanding. employing Brukers proprietary AnchorChip technology, they enable precise and homogeneous sample
preparations in liquid workflows.
Compared to 2010, the newest reference library contains double the number of reference spectra. One
focus of the additions to the library in the 2016 update is a broad coverage of anaerobe strains. Many of The MBT Biotarget 96 is fully compatible with the paperless and traceable workflow using the MBT Pilot,
these reference spectra were generated in collaboration with the European Network for the Rapid Identifi- which supports guided target preparation based on micro-projection technology, and the MBT Galaxy
cation of Anaerobes (ENRIA). automated MALDI target preparation system, which frees laboratory personal from routine matrix and
formic acid pipetting while ensuring highest preparation quality.
Identification of mycobacteria has been further improved by updating the MBT Mycobacteria Library,
which now covers 159 of 169 known species, and adapted bioinformatics for high-sensitivity and The new MBT Subtyping software module enables the automated analysis of specific resistances that
high-specificity species identification (as illustrated in Dr. Pranadas poster on page 32). are associated with specific bacterial subtypes. In this context, the MBT Subtyping Module supports the
detection of carbapenem resistance associated with cfiA-encoded class B metallo-beta-lactamase in
With sepsis being a potentially life-threatening clinical indication, rapid identification of blood stream Bacteroides fragilis and the analysis of MRSA Staphylococcus aureus.
infections is a prerequisite for initiating adequate antibiotic therapy. The MALDI Biotyper Sepsityper
enables MALDI-TOF mass spectrometry-based proteomic profiling and rapid enrichment and identification Bruker is committed to Innovation with Integrity. A great deal of this innovation for the MALDI Biotyper
of bacteria and yeast directly from positive blood cultures. In Europe, the MALDI Sepsityper Kit is labeled product line is evidenced in this new edition of our Poster Hall.
according to the IVD-Directive 98/79/EC. Compared to conventional workflows, the combination of the
MBT Sepsityper IVD Kit and the MBT Sepsityper IVD software module allows a much earlier microbial As every year, my sincere thanks go to our customers who contributed to this edition and whose scientific
identification from positive blood culture samples derived from patients with a potential sepsis. Following spirit encourages us to develop new applications for the MALDI Biotyper. I am looking forward to reading
the MBT Sepsityper IVD Kit workflow, the sample is submitted to the MALDI Biotyper for identification your future posters and publications!
less than 30 minutes after detection of a positive blood culture. The MBT Sepsityper IVD Module supports
result management for more complex blood culture samples by automatically adapting the peak-picking
mass range and the identification confidence levels, and by optionally allowing mixed culture detection. With best regards,
2
Index of Posters
3
Bruker Analytical Excellence, Acknowledged Expertise and Global Presence
Bruker Offices
Additional information: www.bruker.com/map 4
Bruker Daltonics Facilities
1960 1980 2000 2005 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016
6
MALDI Biotyper References
The MALDI Biotyper system uses a pattern-matching algorithm to compare extracted proteomic fingerprint peak lists with a dedicated library containing an extensive set of reference spectra (main spectra) from
various microorganisms. It has been used successfully for the identification of various groups of microorganisms, as reported innumerous publications.
2016 Chen JHK, Cheng VCC, Wong OY, Wong Karatuna O, Celebi B, Can S, Akyar I, Kilic Magnette A, Huang TD, Renzi F, Bogaerts P,
Andersen KM, Kristoffersen AK, Ingebretsen SCY, So SYC, Yam WC, Yuen KY (2016). The S (2016). The use of Matrix-assisted laser Cornelis GR, Glupczynski Y (2016). Improvement
A, Vikholt KJ, rtengren UT, Olsen I, Enersen importance of matrix-assisted laser desorption desorption ionization-time of ight mass of identication of Capnocytophaga canimorsus by
M, Gaustad P (2016). Diversity and antifungal ionization-time of ight mass spectrometry spectrometry in the identication of Francisella matrix-assisted laser desorption ionization-time of
susceptibility of Norwegian Candida glabrata for correct identication of Clostridium difcile tularensis. Bosn J Basic Med Sci. doi:10.17305/ ight mass spectrometry using enriched database.
clinical isolates. J Oral Microbiol 8:29849 isolated from chromID C. difcile chromogenic bjbms.2016.894 [Epub ahead of print] Diagn. Microbiol. Infect. Dis. 84:1215
agar. J Microbiol Immunol Infect. doi:10.1016/j.
Bernhard M, Zautner, AE, Steinmann J, Weig jmii.2015.12.002 [Epub ahead of print] Kodana M, Tarumoto N, Kawamura T, Saito Marekovi I, Bonjak Z, Jakopovi M, Boras
M, Gro U, Bader O (2016). Towards proteomic T, Ohno H, Maesaki S, Ikebuchi K (2016). Z, Jankovi M, Popovi-Grle S (2016).
species barcoding of fungi - An example using Emami K, Nelson A, Hack E, Zhang J, Green Utility of the MALDI-TOF MS method to Evaluation of Matrix-Assisted Laser Desorption/
Scedosporium/ Pseudallescheria complex DH, Caldwell GS, Mesbahi E (2016). MALDI- identify nontuberculous mycobacteria. J. Infect. Ionization Time-of-Flight Mass Spectrometry in
isolates. Fungal Biol 120:162165 TOF Mass Spectrometry Discriminates Known Chemother. 22:3235 Identication of Nontuberculous Mycobacteria.
Species and Marine Environmental Isolates of Chemotherapy 61:167170
Blosser, SJ, Drake SK, Andrasko JL, Henderson Pseudoalteromonas. Front Microbiol 7:104 Lee WS, Ou TY, Chen FL, Hsu CW, Jean SS,
CM, Kamboj K, Antonara S, Mijares L, Conville (2016). Shewanella putrefaciens bacteremia Ponce-Alonso M, Rodrguez-Rojas L, Del
P, Frank KM, Harrington SM, Balada-Llasat JM, Flores-Gonzlez JC, Guerrero-Lozano I, in a uremic patient receiving hemodialysis. J Campo R, Cantn R, Morosini MI (2016).
Zelazny AM (2016). Multi-Center MALDI-TOF MS Prez-Guerrero JJ, Hernndez-Gonzlez A, Microbiol Immunol Infect 49:159160 Comparison of different methods for identication
Study for the Identication of Clinically-Relevant Galn-Snchez F, Quintero-Otero S, Rubio- of species of the genus Raoultella: report of 11
Nocardia spp. J. Clin. Microbiol. doi:10.1128/ Quiones F, Pantoja-Rosso S (2016). First report Leroy AG, Malandain D, Duchalais , Meurette cases of Raoultella causing bacteraemia and
JCM.02942-15 [Epub ahead of print] of invasive fungal disease by Candida fabianii in G, Corvec S, (2016). Accurate MALDI-TOF mass literature review. Clin. Microbiol. Infect. 22:252257
a non-neonatal paediatric patient. Rev Iberoam spectrometry identication of a colistin-resistant
Buckwalter SP, Olson SL, Connelly BJ, Micol 33:4850 Moellerella wisconsensis strain. Med Mal Infect. Rams TE, Sautter JD, Getreu A, van Winkelhoff
Lucas BC, Rodning AA, Walchak RC, Deml doi:10.1016/j.medmal.2016.01.009 [Epub ahead of AJ (2016). Phenotypic identication of
SM, Wohlel SL, Wengenack NL (2016). Fraser M, Brown Z, Houldsworth M, Borman print] Porphyromonas gingivalis validated with matrix-
Evaluation of Matrix-Assisted Laser Desorption AM, Johnson EM (2016). Rapid identication assisted laser desorption/ionization time-of-ight
Ionization-Time of Flight Mass Spectrometry of 6328 isolates of pathogenic yeasts using Lin WH, Hwang JC, Tseng CC, Chang YT, Wu mass spectrometry. Microb. Pathog. doi:10.1016/j.
for Identication of Mycobacterium species, MALDI-ToF MS and a simplied, rapid extraction AB, Yan JJ, Wu JJ, Wang MC (2016). MALDI- micpath.2016.01.021 [Epub ahead of print]
Nocardia species, and Other Aerobic procedure that is compatible with the Bruker TOF MS Accelerates Pathogen Identication and
Actinomycetes. J. Clin. Microbiol. 54:376384 Biotyper platform and database. Med. Mycol. 54: May Confer Benet in the Outcome of Peritoneal Rodrguez-Snchez B, Ruiz-Serrano MJ, Ruiz
8088. Dialysis-Related Peritonitis. J. Clin. Microbiol. A, Timke M, Kostrzewa M, Bouza E (2016).
Camoez M, Sierra JM, Dominguez MA, doi:10.1128/JCM.03378-15 [Epub ahead of print] Evaluation of MALDI Biotyper mycobacteria
Ferrer-Navarro M, Vila J, Roca I (2016). Kajiwara H (2016). Direct detection of the plant library v3.0 for the identication of non-
Automated categorization of methicillin-resistant pathogens Burkholderia glumae, Burkholderia Lotte R, Lotte L, Ruimy R (2016). Actinotignum tuberculous mycobacteria. J. Clin. Microbiol.
Staphylococcus aureus clinical isolates into gladioli pv. gladioli, and Erwinia chrysanthemi schaalii (formerly Actinobaculum schaalii): a newly doi:10.1128/JCM.02760-15 [Epub ahead of print]
different clonal complexes by MALDI-TOF mass pv. zeae in infected rice seedlings using matrix recognized pathogen-review of the literature.
spectrometry. Clin. Microbiol. Infect. 22:161. assisted laser desorption/ionization time-of-ight Clin. Microbiol. Infect. 22:2836
e1161.e7 mass spectrometry. J. Microbiol. Methods 120:
15
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rhizophila misidentied as Corynebacterium Group Streptococci clinical isolates: MALDI-TOF MS method Klin Mikrobiol Infekc Lek. 21(2):46-50 Ilina EN, Emov BA (2015). Species Diversity of
jeikeium and other errors caused by the Vitek mass spectrometry versus gene sequence-based Bidobacteria in the Intestinal Microbiota Studied
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L (2015). MALDI-TOF mass spectrometry and Cabrolier N, Sauget M, Bertrand X, Hocquet LJ, Sheng WH, Ko WC, Hsueh PR (2015).
Al-Hatmi AM, Normand AC, van Diepeningen blakpc gene phylogenetic analysis of an outbreak D (2015). Matrix-Assisted Laser Desorption Evaluation of the Bruker Biotyper matrix-assisted
AD, Hendrickx M, de Hoog GS, Piarroux R of carbapenem-resistant K. pneumoniae strains. Ionization-Time of Flight Mass Spectrometry laser desorption ionization-time of ight mass
(2015). Rapid identication of clinical members of New Microbiol. 38(4):541-550 identies Pseudomonas aeruginosa high-risk spectrometry system for identication of blood
Fusarium fujikori complex using MALDI-TOF MS. clones. J Clin Microbiol. 53(4):1395-1398 isolates of Vibrio species. J Clin Microbiol.
Future Microbiol. 10:1939-1952 Argemi X, Riegel P, Lavigne T, Lefebvre N, 53(5):1741-1744
Grandpr N, Hansmann Y, Jaulhac B, Prvost Carannante A, De Carolis E, Vacca P, Vella
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Identication by Direct MALDI-TOF MS. PLoS Reveals the Pathogenic Role of Staphylococcus MS) for identication and clustering of Neisseria Acinetobacter ursingii. BMC Infect Dis. 15:400
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A, Al-Bemani A, Joshi S (2015). Microbial Bank S, Sby KM, Kristensen LH, Voldstedl Lai CK, Tang BS (2015). Complementary use of MALDI-TOF MS detection of carbapenemase
enhanced heavy crude oil recovery through M, Prag J (2015). A validation of the Danish MALDI-TOF MS and real-time PCR-melt curve activity in clinical isolates of Enterobacteriaceae
biodegradation using bacterial isolates from an microbiology database (MiBa) and incidence rate analysis for rapid identication of methicillin- spp., Pseudomonas aeruginosa, and
Omani oil eld. Microb Cell Fact. 14:141 of Actinotignum schaalii (Actinobaculum schaalii) resistant staphylococci and VRE. J Antimicrob Acinetobacter baumannii compared against the
bacteraemia in Denmark. Clin Microbiol Infect Chemother. 70(2):441-447 Carba-NP assay. J Microbiol Methods. 111:21-23
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Kmetova M, Kmet V (2015). Extended spectrum (2015). Invasive Bordetella holmesii infections. (2015). Evaluation of the Bruker Matrix-Assisted CC, Chen JH, Chan JF, Tse CW, Lee RA, Lau SK,
beta-lactamases in Escherichia coli from Infect Dis (Lond). 47(2):65-68 Laser Desorption-Ionization Time-of-Flight Mass Woo PC (2015). Gordonia species as emerging
municipal wastewater. Ann Agric Environ Med. Spectrometry (MALDI-TOF MS) System for the causes of continuous-ambulatory-peritoneal-
22(3):447-50 Ghosh AK, Paul S, Sood P, Rudramurthy SM, Identication of Clinically Important Dermatophyte dialysis-related peritonitis identied by 16S rRNA
Rajbanshi A, Jillwin TJ, Chakrabarti A (2015). Species. Mycopathologia. 180(3-4):165-171 and secA1 gene sequencing and matrix-assisted
de Dios A, Jacob S, Tayal A, Fisher MA, Matrix-assisted laser desorption ionization laser desorption ionization-time of ight mass
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Gruenwald M, Rabenstein A, Remesch M, Characterization by Matrix-Assisted Laser H, Zange S, Kiland Granerud B, Drevinek
Dolotabadi S, Kolecka A, Versteeg M, de Kuever J (2015). MALDI-TOF mass spectrometry Desorption Ionization-Time of Flight Mass M, Kokotovic B, Wittwer M, Pger V, Di
Hoog SG, Boekhout T (2015). Differentiation ngerprinting: A diagnostic tool to differentiate Spectrometry and DNA Sequencing and Its Caro A, Stmmler M, Grunow R, Jacob D
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Schaub S, Steiger J, Weisser M, Frei R (2015). by matrix-assisted laser desorption ionization- of Streptococcus parauberis isolates from olive Identication of Weissella species by matrix-
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immunosuppressed hosts. Transpl Infect Dis. Jimnez F, Rojo Martin MD, Miranda Casas CH (2015). Accurate detection of binary toxin Domingo MC, Bekal S, Lefebvre B, Tremblay
17(3):481-487 C, Marin Arriaza M, Navarro Mar JM (2015). producer from Clostridium difcile by matrix- C (2015). A Side by Side Comparison of Bruker
Lactococcus garvieae endocarditis in a native assisted laser desorption/ionization time-of-ight Biotyper and VITEK MS: Utility of MALDI-TOF MS
Erler R, Wichels A, Heinemeyer EA, Hauk G, valve identied by MALDI-TOF MS and PCR- mass spectrometry. Diagn Microbiol Infect Dis. Technology for Microorganism Identication in
Hippelein M, Reyes NT, and Gerdts G (2015). based 16s rRNA in Spain: A case report. New 83(3):229-231 a Public Health Reference Laboratory. PlosOne
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pathogenic in humans. Syst Appl Microbiol. Huang Y, Li J, Gu D, Fang Y, Chan EW, Chen Oh M, Ricke SC, Kim HY (2015). Development Mnsson V, Resman F, Kostrzewa M, Nilson B,
38(1):16-25 S, Zhang R (2015). Rapid Detection of K1 of a Rapid and Accurate Identication Method for Riesbeck K (2015). Identication of Haemophilus
Hypervirulent Klebsiella pneumoniae by MALDI- Citrobacter Species Isolated from Pork Products inuenza Type b isolates by Use of Matrix-
Faron ML, Buchan BW, Hyke J, Madisen N, TOF. Front Microbiol. 6:1435 Using a Matrix-Assisted Laser-Desorption Assisted Laser desorption Ionization-Time of
Lillie JL, Granato PA, Wilson DA, Procop GW, Ionization Time-of-Flight Mass Spectrometry Flight Mass Spectrometry. J Clin Microbiol
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S, Ledeboer NA (2015). Multicenter Evaluation Classication of Rhizobia by Matrix-Assisted Mediavilla-Gradolph MC, De Toro-Peinado
of the Bruker MALDI Biotyper CA System for the Laser Desorption/Ionization Time-Of-Flight Mass I, Bermdez-Ruiz MP, Garca-Martnez Mde
Identication of Clinical Aerobic Gram-Negative Spectrometry. J Proteomics Bioinform. 8:98-107 L, Ortega-Torres M, Montiel Quezel-Guerraz
Bacterial Isolates. PloS One 10(11):e0141350 N, Palop-Borrs B (2015). Use of MALDI-
TOF MS for Identication of Nontuberculous
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M, Matsutani T, Kobayashi M, Iwadate Y, T (2014). Epidemiology of candidemia in Qatar, Appl Microbiol Biotechnol. 98(8):3737-3752 desorption ionization time-of-ight mass
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Clin Chim Acta. 435:59-61 exposure to oxygen on the quality of the MALDI-
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MALDI Biotyper References
Gram-negative bacilli isolated from bronchial aspiration from patients in the Intensive Care
Unit and performance of MALDI-TOF MS for routine identification.
Mara Dolores Guerrero1, Luz Balsalobre1, Cristina Santa Olalla1, Teresa Alarcn1.
1U.H. La Princesa, Madrid, Spain.
RESULTS
Isolates
Microorganisms
To study the frequency of isolates of gram negative bacilli from Number Percentage (%)
bronchial aspirations (BAS) in ICU patients and to analyze the Pseudomonas aeruginosa
71 24.1
breakthrough time of the identification by Matrix-Assisted Laser Non-fermenting Stenotrophomonas maltophilia 5 days 6 days
29 9.8 4 days
Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF Gram-negative bacteria 1,7 % 1,2 %
Acinetobacter 6,4 %
MS) compared with traditional methods. baumannii/haemolyticus 23 7.8
20
MALDI Biotyper Poster Hall 2016 Primary ID
AIM: The aim of this was to use MALDI Biotypers Security-Related (SR) database to identify a possible Brucella species from blood culture growth in a patient with indicative clinical presentation.
BACKGROUND: Brucella spp. are a zoonosis that can cause human infections and can be considered a bioterrorism threat 1. The organism is associated with travel to endemic areas 2 and contact with animals and unpasteurised dairy products 1. MALDI-TOF is a rapid Identification (ID) system 3 used for routine diagnostics in bacteriology at
Coventry and Warwickshire Pathology Services (CWPS) since June 2015 and has a security database which can be used to identify containment level 3 organisms following extraction to render them safe for handling outside of CL3 laboratory.
METHOD: Patient presented with history of high fevers for four weeks since return from Somalia. Blood cultures were taken on admission. A gram stain was performed and showed gram negative cocco-bacilli (Figure 1). Sample was cultured on chocolate and blood agar, incubated at 37oC in CL3 incubator and growth examined at 24 and 48 hrs.
At 24hours, colonies from the plate were used for carrying out the full formic acid extraction method for MALDI-TOF 4,5 and target plate run through the SR database.
RESULTS: MALDI-TOF produced an ID of Brucella melitensis (score 2.423) (Figure 2a). Cultures and serology were sent to the reference laboratory. The patients antibiotic treatment was optimised for brucellosis and they were discharged.
CONCLUSION: Previously, the isolate would have required biochemical testing for provisional ID 1 and sending to reference laboratory for full identification.
MALDI-TOF provided a rapid ID confirming diagnosis of Brucellosis six days before the reference laboratory confirmatory report was received. This resulted in more effective patient management in this case of Brucellosis, as well as efficient management of potentially exposed laboratory staff.
Day 8: Occupational health started prophylaxis for staff considered high-risk of exposure. References
1 Young, E.J. (2010). Brucella Species. In: Mandell, G.L. Bennett J.E. and Dollin,R. eds. Mandell, Douglas and Bennetts Principles and Practice of
Day 13: (6 days post ID by MALDI Biotyper)- reference laboratory Infectious Diseases (7th Edition). Philadelphia: Churchill Livingstone Elsevier, 2921-2925
confirmation of B.melitensis serovar 1; sensitive to tetracycline, rifampicin, ciprofloxacin, 2 Pappas, G. Papadimitriou, P. Akritidis, N.et al. (2006). The Lancet Infectious Diseases, 6(2): 91-99
3 Seng, P. Drancourt, M. Gouriet, F. et al. (2009). Clin. Infect. Dis., 49(4): 543-51
streptomycin, gentamicin. 4 Ferreira,L. Vega Castao, S. Snchez-Juanes, F. et al. (2010). PLoS ONE, 5(12): e14235
5 Marklein, G. Josten, M. Klanke, U. et al. (2009). J. Clin. Microbiol., 47(9): 2912-2917
Day 29: Serology results back from reference laboratory confirming acute brucellosis 6 Hartmeyer, G.N. Jensen, A.K. Bcher, S. et al. (2010). Scandinavian Journal of Infectious Diseases, 42(9): 716-718
Figure 1: Gram stain from patients blood culture showing pleomorphic gram negative cocco-bacilli (IgM >2560, IgG 80, CFT 128) 6 Cunningham, S.A. and Patel, R. (2013).J Clinc.Microbiol, 51(5): 1639-1640
7 Drevinek, M. Dresler, J. Klimentova, J. et al. (2012). Letters in Applied Microbiology, 55: 40-46
21
22
Primary ID
30 spectra obtained per strain were closely analyzed in the flexAnalysis Capnocytophaga
USA 1
Hand wound
program and a minimum of 20 accurate spectra were downloaded to cynodegmi Human
(n = 2)
USA 2
MSP creation standard method. Table 1: CC and CD isolates included in the study (n=104)
Third step: Challenge of the MALDI BioTyper database and the generated database with 45 blind-coded isolates
of CC and CD. Figure 1: Dendrogram of 59 Capnocytophaga strains analyzed, including 8 CD strains and 51 CC strains.
Conclusions MALDI-TOF MS identification of CC and CD challenge strains using the enriched database
The enrichment with our own database by including additional Capnocytophaga canimorsus After including the spectra of 59 extracted CC and CD isolates, the newly constituted database combined with the
isolates significantly improved the performance of identification of CC to the species level and MALDI BioTyper database were challenged with 43 randomly selected CC strains not included in the new
may represent a useful complement to Brukers MALDI BioTyper database for the diagnosis of database
these difficult to identify organisms. Following addition of the homemade database in the BioTyper software, MALDI-TOF MS provided reliable
identification to the species level for all 43 CC strains tested with an average score of 2.33 (range 2.012-2.543).
Correspondance: Dr. Te-Din Daniel Huang. Laboratory of Microbiology CHU Dinant-Godinne UCL Namur, 1 Avenue Dr. G. Therasse, 5530 Yvoir, Belgium.
te-din.huang@uclouvain.be
23
MALDI Biotyper Poster Hall 2016 Primary ID
24
MALDI Biotyper Poster Hall 2016 Primary ID
used. It has been recently described that mass 2 2012 K. oxytoca K. oxytoca R. ornithinolytica (2.52) + - Ampicillin Male 78 Acute cholangitis
spectrometry has an adequate resolution power to 3 2012 K. oxytoca K. oxytoca R. ornithinolytica (2.53) + -
Ampicillin,
Male 60
Renal and hepatic cyst infection
Fosfomycin (hepatorenal polycystosis)
differentiate both genera.
4 2012 K. oxytoca K. oxytoca R. ornithinolytica (2.38) + - Ampicillin Male 77 Acute cholangitis
The aim of this work was to retrospectively re-identify
K. K.
the putative Raoultella isolates that had been reported as 5 2012 R. planticola (2.51) - + Ampicillin Male 67 Fever of unknown origin
pneumoniae pneumoniae
Klebsiella spp. in positive blood cultures of our 6 2013 K. oxytoca K. oxytoca R. ornithinolytica (2.42) + - Ampicillin Male 77 Acute cholangitis
Institution. K. K. Acute cholecystitis with
7 2014 R. ornithinolytica (2.32) + - Ampicillin Male 74
pneumoniae pneumoniae gallblader perforation. Sepsis
Material and Methods. A retrospective search among the
K. K.
8 2014 R. planticola (2.38) - + Ampicillin Male 87 Short febrile illness with unknown focus
blood culture isolates collected in our Microbiology pneumoniae pneumoniae
Post-endoscopic retrograde
Department (2011-2014) was performed, selecting Ampicillin, Cefalotin,
9 2014 K. oxytoca K. oxytoca R. ornithinolytica (1.76) + - Male 33 Cholangiopancreatography, acute
Amoxicillin/Clavulanic
cholangitis. Sepsis
samples initially identified as K. oxytoca or K.
10 2014 K. oxytoca K. oxytoca R. ornithinolytica (2.5) + - Ampicillin Female 81 Acute lithiasic pancreatitis
pneumoniae. A total of 80 isolates were recovered and
submitted to MALDI-TOF re-identification. A subset of 10 Results. Discrepancies between results from the identification methods are shown in the Table. Considering the blaorn and blapla gene sequences as
isolates (12.5%), consistently identified as Raoultella (8 R. the gold-standard method, MALDI-TOF accurately differentiated 8 as R. ornithinolytica and 2 as R. planticola with high scores (median, 2.45). As
ornithinolytica and 2 R. planticola), was further studied expected, all isolates exhibited resistance to ampicillin, with variable susceptibility to other antibiotics.
by MicroScan (EUCAST criteria), API 20E (bioMrieux,
Conclusions. At least 10 isolates of Raoultella spp. have been previously misidentified as Klebsiella spp. in our laboratory in the last 4 years.
France) and 16S rRNA, blaorn and blapla PCR amplifications
MALDI-TOF seems to be a suitable alternative to properly identify these still infrequent isolates. The strong association between bacteraemia
and further nucleotide sequencing. Clinical data were
due to Raoultella and biliary tract-related pathologies justifies the need of a correct identification as it can help clinicians to guide clinical
obtained after patients chart revision.
diagnosis, particularly in those patients with fever of unknown origin.
25
MALDI Biotyper Poster Hall 2016 Primary ID
26
MALDI Biotyper Poster Hall 2016 Primary ID
Yersinia entercolitica 26 27 27 In comparison to PCR the MALDI-TOF algorithm is faster, cheaper and easy to use with improved
Conclusion Phase 2 of the study consisted of prospectively comparing enteric pathogens identified by the incumbent
turn-around-time for patient results, particularly for negative samples and for those organisms which
method to the identification provided by the MALDI-TOF algorithm. The end point of this phase was the Shigella species 20 0 20 MALDI-TOF can reliably identify without supplemental testing. Aside from E.coli, MALDI-TOF is an
MALDI TOF can reliably identify most enteric pathogens with some notable exceptions.
MALDI-TOF accumulation of at least 200 enteric pathogens for comparison accumulated over both phases.
excellent
ll t stool
t l pathogen
th screening
i method.
th d
Limitations can be mitigated through the use of a supplemental PCR based identification EIEC (Enteroinvasive E.coli) 5 0 5
algorithm.
STEC (Shiga-toxin producing E.coli) 5 0 5 With advances in technology and addition of sufficient spectra to the Bruker database it would be no
surprise to see MALDI-TOF be the primary research and diagnostic tool in high volume laboratories
in the near future due to its accuracy, precision and ability to process high volumes of isolates.3
These characteristics allow for accurate, timely and cost-effective results.
Table 2: Accuracy of the MALDI-TOF and PCR
No. (%) correctly identified
Introduction References
MALDI-TOF PCR
A. caviae 10 10 (100) 10(100) 7(70) 3(30) 1. Murray. R.P. What Is New in Clinical MicrobiologyMicrobial Identification by MALDI-TOF Mass
Enteric culture has been a very important tool for understanding gastrointestinal infections.
Spectrometry. A Paper from the 2011 William Beaumont Hospital Symposium on Molecular
I l ti off pathogenic
Isolation th i b
bacteria
t i from
f stool
t l directs
di t antimicrobial
ti i bi l treatment
t t t when
h indicated,
i di t d A. hydrophilia 7 7 (100) 7 (100) 7 (100) Pathology. J Mol Diagn. 2012 Sep; 14(5): 419423.
helps prevent secondary complications and allows for public health agencies to track and
manage gastroenteritis outbreaks to prevent transmission of illness. A. jandaei 3 2 (67) 3 (100) 3 (100)
2. Ying He,Haijing Li, Xuedong Lu,Charles W. Stratton,and Yi-Wei Tang. Spectrometry Biotyper
System Identifies Enteric Bacterial Pathogens Directly from Colonies Grown on Selective Stool
Laboratory identification and isolation of enteric pathogens has traditionally been done via A.media 3 3 (100) 3 (100) 3 (100) Culture Media.,J Clin Microbiol. 2010 Nov; 48(11): 38883892. Published online 2010 Sep 15.
selective media, culture and isolation followed by biochemical identification and serology. doi: 10.1128/JCM.01290-10.
These methods can be costly and time consuming, taking 2 to 4 days to obtain a result. This A. ichthiosmia 1 1 (100) 1 (100) 1 (100) 3. J. Kathleen Lewis, Jing Wei, and Gary Siuzdak. Matrix-assisted Laser Desorption/Ionization
longer turn-around-time can have negative consequences for patient care and treatment. Mass Spectrometry in Peptide and Protein Analysis. Encyclopedia of Analytical Chemistry R.A.
A. salmonicida 1 1 (100) 1 (100) 1 (100)
Providing rapid, accurate, and specific identification of enteric bacterial pathogens is Meyers (Ed.) pp. 5880 5894, John Wiley & Sons Ltd, Chichester, 2000.
considered essential for directing the antimicrobial therapy of diarrheal illnesses.2
A. veronii 12 12 (100) 12(100) 12(100)
The current methodology employed at Lifelabs Medical Laboratory Services (Burnaby site)
C. campy 6 5 (83) 1 (17) 6(100)
uses traditional culture methods to isolate suspected bacteria but instead of biochemical
identification methods, a previously validated in-house multiplex gel-based PCR method is
C. coli 2 2 (100) 2 (100) 2(100)
used for definitive identification of enteric pathogens. Turn-around time for PCR is between 4-
8 hours and is an improvement over traditional biochemical identification.
identification However
However, there C. jejuni 36 19(53) 17(47) 36(100)
exist opportunities for further improvements.
C. upsaliensis 1 1 (100) 1 (100) 1(100)
Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) is a technology that has
been developed that enables rapid identification of microorganisms. Isolated colonies are E. tarda 1 1 (100) 1 (100) 1(100)
spotted on a target plate, covered with matrix, dried and then subjected to a laser pulse. The
laser induces desorption and ionization of the bacteria-matrix layer, which is then separated P. shigelloides 9 8 (89) 1 (11) 9(100)
based on particulate mass/charge ratio1. A characteristic mass spectra is obtained which is
compared to a database of known spectra to definitively identify the microorganism2, Salmonella species 52 52(100) 51(98) 1(2)
Results are typically available within a few minutes of loading a target plate into the mass
V. parahaemolyticus 9 8 (89) 1 (11) 9(100)
spectrometer.
Y. enterocolitica 27 26 (96) 27(100) 26(96) 26(96) 1(4)
MALDI-TOF implementation as a high volume enteric pathogen screening method has the
potential to improve patient result turn-around-time at a high volume community outpatient lab Y. frederiksenii (a) 4 4(100) 4(100)
such as Lifelabs which processes several thousand stool cultures a month.
Shigella species (b) 20 20(100) 20(100)
27
MALDI Biotyper Poster Hall 2016 Primary ID
Case 2
ATCC Strains IND URE GLU MAN LAC SAC MAL SAL XYL ARA GEL ESC GLY CEL MNE MLZ RAF SOR RHA TRE CAT
P. acnes 90 0 99 36 0 1 0 0 0 0 74 0 99 0 100 0 0 18 0 1 95
Figure 6. CT scan of case 3
P. granulosum 0 0 99 43 0 93 31 0 0 0 4 0 99 0 99 12 56 0 4 75 90
Case 2 was a 55-year-old female patient with a known benign brain
P. avidum 0 0 99 32 45 99 72 62 0 0 98 99 99 0 99 60 40 0 0 90 90
tumour had a tumour resection. About 1 month before the operation, she
P. propionicus 0 10 99 96 50 90 64 0 0 14 50 0 60 0 40 0 40 0 0 42 0
had meningeal symptoms: high fever, nausea, vomiting, acute headache.
Strain 1./Case 1. + - + - - - - - - - + - + - + - - - - - -
Intracranial CT showed a 20 mm right-sided parieto-occipital ring-form
Strain 2./Case 2. + - + - - - - - - - + + +/- - - - - - -
plexus with extensive gloves-finger like oedema. This space occupying - -
Strain 3./Case 3. + - + - - - - - - - + + + - - - - - -
lesion might be a metastasis. The pathological opinion was glioblastoma - -
28
MALDI Biotyper Poster Hall 2016 Primary ID
29
MALDI Biotyper Poster Hall 2016 Primary ID of Mycobacteria
30
MALDI Biotyper Poster Hall 2016 Primary ID of Mycobacteria
berrtliche Berufsausbungsgemeinschaft
MVZ Dr. Eberhard & Partner Dortmund
Mycobacteria Identification by MALDI Biotyper System: Evolution of Database Content and Evaluation Criteria
A. B. PrAnAdA1, M. TiMke2, e. WiTT1, M. kosTrzeWA2
ECCMID 2015, Copenhagen
1
MVZ Dr. Eberhard & Partner Dortmund, Department of Medical Microbiology, Dortmund, Germany Poster 1190
2
Bruker Daltonik GmbH, Bremen, Germany apranada@labmed.de
Introduction Materials and Methods Results Comparison Database V2.0 vs. V3.0 and Evaluation of New Threshold Values Conclusion
Identification of mycobacteria by matrix-assisted laser Mycobacterial isolates (n = 1045) were inoculated on Out of 1045 analyses of pure cultures log(score) values Mycobacteria Identifications with Current Threshold Values Mycobacteria Identifications with Proposed New Threshold Values
Every MALDI Biotyper database reference is attributable
desorption/ionisation time-of-flight (MALDI-TOF) mass solid Lwenstein-Jensen medium or in liquid BD BAC- were 2.0 for 82.9 % and between 1.7 and < 2.0 for Library
1.2 %
Library
0.8 % to a strain or an isolate. A higher number of references
spectrometry (MS) requires an optimised preparation TEC MGIT tubes (BD, Heidelberg). 12.1 %, representing the current high and low confidence 94.1 % 4.5 %
2.000
97.3 % 1.9 %
2.000 per species covers potential natural variabilities of a spe-
V3.0 V3.0
method and a corresponding database. In addition, patient material was inoculated in MGIT identification results with database version 2.0. 1.900-1.999
1.800-1.899
1.900-1.999
1.800-1.899 cies.
tubes. Positive cultures (n = 93) were further analysed. Using the adapted values and database version 3.0, Besides this, there can be other influences like human,
1.700-1.799 1.700-1.799
1.600-1.699 1.600-1.699
97.3 % and 1.9 % of high and low confidence level iden- instrument or medium based variability due to practical
1.500-1.599 1.500-1.599
tification were obtained, respectively (Figures 1a and 1b). knowledge, instrument settings or liquid / solid medium,
1.300-1.399 1.300-1.399
Mycobacteria Library 1.0 (Bruker Daltonik, Bremen, Ger- Very few discrepancies were observed (n = 8) using da- High Confidence: 82.9 % Low Confidence: 12.1 % High Confidence: 91.6 % Low Confidence: 5.4 % respectively. All these minor variations have been bal-
many) was released in 2012 and version 2.0 in 2014 with Biomass was collected from solid and liquid media and tabase version 2.0 (Table 2), but only for very closely re- anced by several references per species by the presented
Unreliable: 5.1 % Unreliable: 3.1 %
0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%
173 and 313 references, respectively. processed using the mycobacteria extraction protocol. lated species and of log(score) values below 2.0. Figure 1a: Identification results for 1045 analyses using database V2.0 and V3.0 Figure 1b: Identification results for 1045 analyses using database V2.0 and V3.0 database extension.
with current threshold values. with proposed new threshold values.
An extension by 542 additional references mainly clin- The update of the database eliminated 7 of these dis- As a result, the proportion of high confidence level iden-
ical isolates leads to version 3.0 in this year. Optimised extraction protocol for mycobacteria crepancies. Isolate
Mycobacteria Library 2.0 Mycobacteria Library 3.0 Table 2: Divergences out of 1045 identifications. tifications increased with database version 2.0 and a fur-
The remaining misidentification is likely due to a mix- ID Result log(score) OK? ID Result log(score) OK? ther gain could be observed with version 3.0.
Collect biomass from solid medium or 1.2ml from MGIT M. immunogenum 8608 M. abscessus 1.944 NO M. immunogenum 2.389 OK For identification with database version 2.0
Here we present results obtained for mass spectra com- medium, centrifuge, discard supernatant up of samples: M. nebraskense and M. gordonae usually M. engbaekii 026468 M. hiberniae 1.795 NO M. engbaekii 2.449 OK
n = 8 divergences were observed for closely
Add water, heat inactivation (boiling for 30 min) related species with log(score) values < 2.0.
pared to these three database versions with focus on can be differentiated unambigously from each other by M. kansasii IV 11649 M. gastri 1.753 NO M. kansasii 2.437 OK
Add 900 l ethanol, mix, centrifuge, discard supernatant Increased sensitivity without loss in specificity
sensitivity and specificity. Centrifuge again, discard supernatant MALDI-TOF MS. A comparison of the mass spectra yields M. elephantis 457 M. pulveris 1.743 NO M. elephantis 2.812 OK Database version 3.0 enables the correct identi-
Dry pellet at room temperature M. elephantis 44368 M. pulveris 1.743 NO M. elephantis 2.443 OK fication in these cases.
Add zirconia/silica beads (0.5 mm) to a maximum log(score) value of only 1.24. M. elephantis 125 M. pulveris 1.669 NO M. elephantis 2.828 OK One remaining discrepancy is likely due to a Reliability of identification results is based on log(score)
Resuspend in 10-50 l acetonitrile, vortex for 1 min M. elephantis 101 M. pulveris 1.665 NO M. elephantis 2.853 OK mix-up of samples. values. It was possible to lower threshold values without
Evaluation of adapted threshold values Addition of 70 % formic acid, vortex for 5sec,
centrifuge Improved identification for clinical specimens with M. nebraskense J605 M. gordonae 1.619 NO M. gordonae 1.749 NO risking false species identification results using Myco-
In addition to the investigation of the database versions Transfer 1 l of supernatant to MALDI target newer database versions and adapted thresholds bacteria Library 3.0.
we also have evaluated current threshold values and pro- Clinical Specimens Database Comparison and Evaluation of Threshold Values Both improvements have led to an increased sensitivi-
pose adapted ones for Mycobacterium spp. (Table 1). Out of the 93 enrichment cultures of directly inoculated
ty without a decrease of specificity thus avoiding time
clinical specimens 78.5 % resulted in log(score) values Clinical Specimens and Current Threshold Values Clinical Specimens and Proposed New Threshold Values
Mass spectrometer, databases and software Mycobacteria
Library
Mycobacteria
Library consuming repetitions and additional tests.
2.0 using the current database version 2.0. The new
2.2 %
as high confidence and 5.4 % as low confidence iden- 78.5 % 18.3 % 3.2 % 1.800-1.899
1.700-1.799
94.6 % 5.4 % 1.800-1.899
1.700-1.799
Conflicts of Interest / Disclosures:
The reliability of identifications obtained by MALDI-TOF tifications using database version 2.0. All these species 1.600-1.699 1.600-1.699 A. B. Pranada presents his data in an ECCMID symposium organised by Bruker Dal-
Reference methods for identification 1.500-1.599 1.500-1.599 tonik GmbH and receives speaker fees.
MS is indicated by log(score) values. identifications were correct. E. Witt: None.
V1.0 V1.0
The reference method for study isolates was GenoType The extended database version 3.0 further improved these High Confidence: 62.4 % Low Confidence: 32.3 % High Confidence: 90.3 % Low Confidence: 8.6 %
M. Timke and M. Kostrzewa are employees of Bruker Daltonik GmbH.
In comparison to usual bacterial isolates some myco- Mycobacterium CM (Hain Lifescience, Nehren, Germa- results to 97.8 % for high confidence and 2.2 % at low Unreliable: 5.4 % Unreliable: 1.1 %
Contact:
bacteria show lower log(score) values. Nevertheless, the ny). Sequencing of 16S rRNA gene or ITS sequence was confidence level without any identification in the unre- 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Dr. med. Arthur B. Pranada
Figure 2a: Identification results from clinical specimens (n = 93) in comparison to Figure 2b: Identification results from clinical specimens (n = 93) in comparison to MVZ Dr. Eberhard & Partner Dortmund (BAG)
identification results still seem to be reliable. performed for a few isolates. liable category (Figures 2a and 2b). the different database versions and applying current thresholds. the different database versions and applying adapted thresholds. Balkenstr. 17-19, 44137 Dortmund, Germany apranada@labmed.de
31
MALDI Biotyper Poster Hall 2016 Primary ID of Mycobacteria
D-223
Looking at the Differences: First Report of Possibilities to
Separate Mycobacterium chimaera from Mycobacterium intracellulare by MALDI-TOF MS
A. B. PrAnAdA1, M. TiMke2, e. WiTT1, M. kosTrzeWA2
1
MVZ Dr. Eberhard & Partner Dortmund, Department of Medical Microbiology, Dortmund, Germany
Poster D-223
2
Bruker Daltonik GmbH, Bremen, Germany
apranada@labmed.de
M. intracellulare. MALDI Biotyper (Bruker Daltonik GmbH, gorithm was implemented. The result of this algorithm is Therefore a more reliable method for differentiation of
1.000
Bremen, Germany) groups both species into a complex. a log(IQ) value that indicates which of the two species M. chimaera and M. intracellulare by MALDI-TOF MS was Conclusions
Even the complete 16S rRNA gene differs in only onebase. has been detected. investigated. As a first basic approach the mass spectra
0.800
of the two species were screened for characteristic peaks. Differentiation of M. chimaera and M. intracellulare is dif-
Indeed, several peaks allowing a reliable differentiation 0.600 ficult in routine diagnostics but may be clinically relevant.
Materials and Methods Results Identification at Group Level could be found (Figure 1).
0.400 Here, we present two easy to perform and reliable MALDI-
Intens. [a.u.]
0 -0.400 To our knowledge these are the first methods suitable for
routine to differentiate M. chimaera/intracellulare.
Intens. [a.u.]
32
MALDI Biotyper Poster Hall 2016 Primary ID of Mycobacteria
33
34
Primary ID of Mycobacteria
100%
100%
90% 376,54 (sequencing)) and quick results
80%
80% 70% (daily versus weekly) makes MALDI-TOF MS
60%
60%
50%
a promising first identification method for
40%
40% NTM. Prospective studies on patients must
30%
20% 20% show its practical feasibility. In addition, it is
0%
10% important to use an extensive database and
0%
day 4 day 7 day 10 day 10 day 14 day 21 to carry out the MALDI-TOF MS analysis
rapidly after the initial colony growth.
Laboratorium Microbiologie Twente Achterhoek, Hengelo, Nederland
www.labmicta.nl / info@labmicta.nl
MALDI Biotyper Poster Hall 2016 Primary ID of Mycobacteria
35
36
Primary ID in Urine
Mixed culture 3
CONCLUSION
MALDI -ve 19 15 34 27 15*(8 GNB 42
7 GPB) Unknown reason 2
Totals 27 96 123 27 96 123
MALDI TOF disagreement from +ve culture (n=12)
Sensitivity 84.4% 84.4%
Specificity 70% 100%
Aerobic GNB bacteria able to grow on standard medium
Aerobic GPB bacteria able to grow on standard medium
6
3
Direct use of MALDI-TOF allowed
Monoinfection Polyinfection MALDI Anaerobic GPB bacteria difficult to cultivate 3
bacterial identification from urines
with >10 cfu/ml within 1.5 hours.
found by found by +ve/-ve 5
MALDI MALDI results MALDI TOF +ve results from ve culture (n=8*)
+ve(n=96) 73 8 81/15
-ve culture (n=27) 8 0 8/19
Aerobic bacteria able to grow on standard medium 6
This significantly shorten ed
Anaerobic bacteria difficult to cultivate 2
Mixed culture (n=27) 8 8 16/11
identification time and may aid
Table 1. Positivity/negativity agreement by culture and MALDI-TOF Table 3. Summary of MALDI-TOF: culture disagreement
selection of appropriate therapy.
ACKNOWLEDGEMENT
REFERENCES
1. http://www.cqc.org.uk/file/4346 We gratefully acknowledge all the laboratory staff for support
2. Foxman B. Epidemiology of Urinary Tract Infections Infections: Incidence, Morbidity and Economic Costs. Am J Med 2002;113:5-13 in this research and help in collecting urine samples; also Brker
3. Schmiemann G et al. The Diagnosis of Urinary Tract Infection. Dtsch Arztebl Int 2010;107:361-367 for loan of the MALDI-TOF.
MALDI Biotyper Poster Hall 2016 Primary ID in Urine
37
MALDI Biotyper Poster Hall 2016 Blood Culture
ABSTRACT
RESULTS
Delayed
or
inappropriate
treatment
of
bloodstream
infec3ons
is
detrimental
for
the
prognosis
of
sep3c
pa3ents.
In
addi3on
to
Gram
staining
of
growing
bacteria,
we
tested
whether
a
rapid
bacterial
iden3ca3on
in
blood
culture
broths
(RBI)
based
on
matrix-assisted
laser
desorp3on/ioniza3on
3me-of- Demographic
and
clinical
characteris2c
of
Bacterial
species
directly
iden2ed
in
the
BCBs
Global
view
of
the
changement
of
empirical
an2bio2c
treatment
(EAT1
ight
mass
spectrometry
(MALDI-TOF)
could
improve
empirical
an3bio3c
treatment
(EAT).
pa2ents
N=
180
and
EAT2)
occurred
in
the
studied
popula2on
During
a
6-month
period,
we
prospec3vely
evaluated
for
180
pa3ents
with
monomicrobial
bloodstream
infec3on
and
analyzed
empirical
treatments
started
before
an3bio3c
suscep3bility
tes3ng
(AST).
For
each
pa3ent
we
considered
the
treatment
decisions
made
before
(EAT1)
and
aSer
(EAT2)
RBI,
and
analyzed
their
respec3ve
appropriateness
based
on
the
nal
AST
of
the
isolate.
Onset
of
a
unique
blood
culture
growing
with
a
coagulase
nega3ve
Microbiological
Staphylococcus
(CNS)
was
nally
considered
as
a
contamina3on
(n=40).
diagnosis
procedure
Pa3ents
experiencing
a
bloodstream
infec3on
(140),
Gram
nega3ve
isolates
(56%)
were
mostly
Enterobacteriaceae
(65)
and
non-fermen3ng
bacteria
(11),
and
Gram
posi3ve
isolates
(44%)
consisted
in
S.aureus
(28),
CNS
(11),
Streptococcus
and
Enterococcus
(22).
RBI
in
blood
culture
broths
was
achieved
at
species
level
for
94%
of
isolates.
Before
RBI,
EAT1
was
not
adapted
for
21
BCBs
sampling
not
treated
pa3ents,
and
37
receiving
inadequate
EAT.
ASer
RBI,
EAT2
was
de
novo
introduced
for
23
pa3ents
and
modied
in
24
others.
This
resulted
in
a
nal
op3mal
treatment
for
89%
of
pa3ents
compared
to
57%
before
RBI.
This
improvement
was
mainly
achieved
by
adap3ng
EAT
regimen
against
Rapid
ID
Rapid
Pseudomonas
species
and
other
non
fermen3g
bacteria.
Among
the
140
pa3ents
26
pa3ents
were
documented
with
severe
sepsis
or
sep3c
shock
and
for
5
of
them
EAT1
was
not
adapted
whereas
EAT2
could
be
quickly
modied
according
to
bacterial
iden3ca3on:
P.
aeruginosa
(2),
E.
faecium,
E.
aerogenes,
K.
pneumoniae.
Conv
ID
Conv
Gram
staining
alone
is
no
appropriate
to
predict
the
natural
or
the
expected
All
the
iden3ca3ons
obtained
directly
from
BCBS
were
resistance
prole
to
an3bio3cs.
Taking
into
account
natural
resistance
and
knowledge
of
local
ecology,
RBI
with
MALDI-TOF
is
accurate
enough
to
give
a
conrmed
by
conven3onal
iden3ca3on
of
sub-cultured
one
day
prior
to
the
AST
results
recommenda3ons
to
improve
EAT.
colonies.
We
did
not
observe
any
misiden3ca3on
Adequacy
of
the
adapta2on
of
the
rst
of
second
line
of
EAT
according
to
iden2ed
bacterial
species
Direct
iden2ca2ons
of
bacterial
species
are
at
OBJECTIVES
least
24h
more
rapid
than
conven2onal
Anaerobic
pa3ents.
100 Streptococcus
In
addi3on
to
Gram
staining
of
growing
bacteria,
we
EAT1
adapted
tested
whether
a
rapid
bacterial
iden3ca3on
in
blood
80
S.aureus
EAT2
adapted
culture
broths
(RBI)
based
on
matrix-assisted
laser
desorp3on/ioniza3on
3me-of-ight
mass
spectrometry
60
Enterobacteriace
(MALDI-TOF)
could
improve
empirical
an3bio3c
treatment
40
(EAT).
Non
fermenJng
20
(ICU=Intensive
Care
Unit,
ESBL=Extended
spectrum
beta
lactamase,
0%
20%
40%
60%
80%
100%
MRSA=Methicilin
Resistant
S.aureus,
VRE=
Vancomycin
resistant
METHODS
enterococcus)
0
ConvID RapID Of
pa3ents
Kaplan-Meier
es3mate
survival
likelihood
of
the
studied
1. No-one
of
the
bacteremia
with
non-fermen2ng
microorganism
(
mainly
P.
aeruginosa)
was
adapted.
RapdID=
Iden3ca3on
performed
by
mass
popula3on
at
30
day
was
as
expected
from
other
RBI
allows
quick
adapta2on
of
EAT
for
73%
of
included
pa2ents
During
a
6-month
period,
we
prospec3vely
collected
203
posi3ve
blood
spectrometry
directly
on
BCBs
when
detected
2. When
the
species
S.
aureus
was
iden:ed
and
according
to
the
global
resistance
epidemiology
of
the
culture.
When
Gram
staining
was
polymorphic
and
when
RBI
by
mass
survey
:
posi3ve
care
department,
adapted
EAT
enhances
from
54%
to
96%
spectrometry
failed
the
pa3ent
has
not
been
taken
into
account
n=23
.
Overall
86,5%
ConvID
=
conven3onal
iden3ca3on
of
isolated
3. Dieren:a:on
between
E.
faecalis
and
E.
faecium
by
RBI
signicantly
increased
the
adequacy
of
EAT
Sepsis
88%
from
43%
to
86%
Finally
we
evaluated
180
pa3ents
with
a
unique
posi3ve
blood
culture
bacteria
Sep3c
shock
63,7%
growing
with
a
single
microorganism
and
analysed
empirical
treatments
started
before
an3bio3c
suscep3bility
tes3ng
(AST).
For
each
pa3ent
we
considered
the
treatment
decisions
made
before
(EAT1)
and
aSer
(EAT2)
bacterial
iden3ca3on
in
BCBs.
CONCLUSION
The
appropriateness
of
EAT
was
based
on
its
adequacy
with
the
nal
References
an3bio3c
suscep3bility
tes3ng
(AST)
the
isolate
EAT
is
considered
as
adequate
the
infec3ng
microorganism
was
! Gram
staining
alone
is
not
appropriate
to
predict
the
natural
or
the
expected
resistance
prole
to
an2bio2cs.
Kumar
A,
Ellis
P,
Arabi
Y,
Roberts
D,
Light
B,
Parrillo
JE,
et
al.
Ini3a3on
of
inappropriate
an3microbial
therapy
results
in
a
vefold
reduc3on
of
survival
in
subsequently
found
to
be
suscep3ble
to
the
given
an3bio3c.
! Taking
into
account
natural
resistance
and
knowledge
of
local
ecology,
species
iden2ca2on
by
mass
spectrometry
human
sep3c
shock.
Chest.
nov
2009;136(5):123748
Ibrahim
EH,
Sherman
G,
Ward
S,
Fraser
VJ,
Kollef
MH.
The
inuence
of
RBI
is
considered
to
have
an
impact
if
the
an3bio3c
therapy
was
either
directly
in
BCBs
with
MALDI-TOF
is
accurate
enough
to
give
a
one
day
prior
to
the
AST
results
recommenda2ons
to
inadequate
an3microbial
treatment
of
bloodstream
infec3ons
on
pa3ent
started
or
changed
(spectrum
broadening,
spectrum
streamlining,
outcomes
in
the
ICU
sejng.
Chest.
juill
2000;118(1):14655.
introduc3on
of
empirical
an3bio3c
therapy
or
discon3nua3on)
following
improve
EAT.
Seifert
H.
The
clinical
importance
of
microbiological
ndings
in
the
diagnosis
the
iden3ca3on
and
before
obtaining
an3biogram.
and
management
of
bloodstream
infec3ons.
Clin
Infect
Dis
O
Publ
Infect
Dis
! Pseudomonas
aeruginosa
iden2ca2on
and
Enterococcus
species
dieren2a2on
allow
a
quick
adapta2on
of
the
empirical
Soc
Am.
15
mai
2009;48
Suppl
4:S23845
38
39
Blood Culture
Validation and implementation of MALDI-TOF: a quick and easy method for bacterial identification from clinical samples
Manar Najim Mashhadani M B ChB, MSc, PhD MED MICRO, PGCert Infec Sciences
Department of Microbiology, Northampton General Hospital, United Kingdom
Aims To validate the use of the Bruker Biotyper MALDI-TOF MS system for the routine identification of bacterial and yeast isolates from cultures. To
evaluate whether the conventional identification methods, taken from clinical samples sent to the Department of Microbiology at Northampton
General Hospital from hospital wards and GPs surgeries, warrants replacing.
Background METHODS
The use of the direct examination of both macroscopic and microscopic colony morphology is a v All isolates were recovered from routine examination of clinical specimens submitted to the
traditional approach for the identification of bacterial and yeast isolates from clinical specimens. microbiological laboratory, such as blood, urine, pus, biopsy, and swabs from any site of the
These tests are laborious, involve long incubation periods, require a significant amount of hands- body, cerebrospinal fluid, respiratory tract, and wound specimens. Retrospective isolates
on time, and are subject to human error. Automation tests and molecular-based DNA techniques including previously identified isolates, reference isolates, and quality control isolates, are also
can improve turnaround time and provide results with high sensitivity and specificity, but they used in the evaluation to test a wide range of different pathogens and to cover varieties of
have disadvantages. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass genera and species. The isolates were recovered after aerobic, microaerophilic, or anaerobic
spectrometry, however, is a molecular platform method for automated, rapid, easy to use, and less incubation at 35C on 5% sheep blood agar, chocolate agar, cysteine-lactose electrolyte-
expensive identification of pathogens that provides a viable alternative to the direct examination deficient (CLED) agar and Sabouraud's agar media, Oxford (Perth, UK). A total of 134 isolates
of macroscopic and microscopic colonies and PCR tests. were used validate MALDI -TOF for routine use in Northampton General Hospital (NGH) and 44
positive blood culture bottles used to evaluate MALDI SepsityperTM Kit .
v Conventional Methods used for identification purposes included Gram staining, catalase and
oxidase activities, latex and DNase for staphylococci, serotyping for Streptococci spp.
Results & Summary v Further identification using automated system, vitek2 (bioMrieux) and appropriate cards and
Gram negative species - 98% showed the same genus and species identification. Only one appropriate Phoenix identification panels and Phoenix apparatus (BD)
isolate had discrepancies: the first occurred at genus level with Acientobacter spp being
identified as Lactobacillus johnsonii by MALDI-TOF and Acinetobacter ursingii by Vitek2. v MALDI-TOF (BD-Bruker) and MALDI SepsityperTM Kit were evaluated against the conventional
methods and were used according to the manufacture instructions.
Staphylococci Species - 100% results compatibility of MALDI-TOF to Phoenix and Vitek 2.
Streptococci Species - all tested species were identified correctly by MALDI-TOF, compared
to either Phoenix or Vitek2 in addition to Lancefield grouping results, except one isolate
being identified as Lactococcus greavieae by Vitek2 and Streptococcus angiosus by
MALDI-TOF.
Yeast Isolates - All yeast isolates except one had the same identification compared to
Vitek2. Stephanoascus ciferrii 89% was identified using Vitek2, MALDI gave an excellent
identification of Candida albicans. Further extraction was required for some isolates to
gain high MALDI-TOF scores.
One isolate which was GPB failed to be identified using Phoenix and Vitek2, but API testing
gave a positive identification of Brevibacterium with 83%, while MALDI identified it as
Bacillus pumilus with high score. Colony morphology gave similar classical morphology of
Bacillus spp. on agar plate.
Sepsityper Kit results showed 41/44 isolates had the same identification when comparing
direct extraction form positive blood culture bottles and MALDI-TOF identification from
agar culture method. One sample had no reliable identification by both methods and
thought to be a contamination. Two flagged positive culture bottled failed to give an
identification by the extraction method, but there was no organism seen by Gram staining
and no growth after 48 hour on the culture plates. Both samples had lots of white blood
cells which triggered the flagging. Fig 1 : MALDI-TOF Instrument at NGH and MALDI SepsityperTM Kit.
No. Test/ Phenotypically ID Gram Oxidase Phoenix/ Vitek2 Maldi
GRAM NEGATIVE BACTERIA
1 Alpha haemolysis Streptococci Spp. GPC NoT Aerococcus urinae Aerococcus urinae
2 Gamma (no) haemolysis Streptococci Spp. GPC Group D Eenterococcus faecalis Eenterococcus. faecalis
3 Gamma (no) haemolysis Streptococci Spp. GPC Group D Eenterococcus faecium Eenterococcus. faecium
9 Beta haemolysis Streptococci Spp. GPC Group B Streptococcus agalactiae Streptococcus. agalactiae
5 Beta haemolysis Streptococci Spp. GPC Group G Sterptococcus dysgalactiae Sterptococcus. dysgalactiae
1 Beta haemolysis Streptococci Spp. GPC Group C Sterptococcus equisiminis Sterptococcus. equisiminis
1 Alpha haemolysis Streptococci Spp. GPC NoT Streptococcus lutetiensis Streptococcus. lutetiensis
2 Alpha haemolysis Streptococci Spp. GPC NoT Streptococcus pneumoniae Streptococcus. pneumoniae
7 Beta haemolysis Streptococci Spp. GPC Group A Streptococcus pyogenes Streptococcus. pyogenes Table 2: Comparable isolate identification using MALDI-TOF ID/Kit and MALDI-TOF ID/Culture
1 Gamma (no) haemolysis Streptococci Spp. GPC Group D Streptococcus salivarius Streptococcus. salivarius
1 Alpha haemolysis Streptococci Spp. GPC NoT Lactococcus garvieae Streptococcus. angiosus
No. Test/ Phenotypically ID Gram sts/Latex & Dna Phoenix/ Vitek2 Maldi
Spp.
Staphylococci
20
2
White- gray coolonies Staphylococci Spp.
White- gray coolonies Staphylococci Spp.
GPCL
GPCL
Positive
Negative
Staphylococcus aureus Staphylococcus aureus
Staphylococcus. epidermidis Staphylococcua. epidermidis
Conclusion
2 White- gray coolonies Staphylococci Spp. GPCL Negative Staphylococcus. simulans Staphylococcus. simulans Turnaround times reduced with the use of MALDI-TOF and Phoenix, and
1 White- gray coolonies Staphylococci Spp. GPCL Negative Staphylococcus. saprophyticus Staphylococcus. saprophyticus laboratory workflow adapted.
1 White- gray coolonies Staphylococci Spp. GPCL Positive Staphylococcus.intermedius Staphylococcus.intermedius
MALDI Biotyper Poster Hall 2016
1 White- gray coolonies Staphylococci Spp. GPCL Negative Staphylococcus Lugdunensis Staphylococcus Lugdunensis MALDI-TOF identification and Phoenix AST replaced all Gram staining,
No. Test/ Phenotypically ID Gram Wet prep Vitek2 Maldi
supplementary tests for identification and the use of combi ID/AST Phoenix
Spp.
Candida
13 White- creamy colonies Candida spp. Yeast Yeast Candida albicans Candida albicans
1 White- creamy colonies Candida spp. Yeast Yeast Stephanoascus ciferrii 89% Candida albicans panels.
1 White- creamy colonies Candida spp. Yeast Yeast Candida parapsilosis Candida parapsilosis
1 White- creamy colonies Candida spp. Yeast Yeast Candida krusei Candida krusei MALDI-TOF provide reliable, quick and reproducible results. The current
1 White- creamy colonies Candida spp. Yeast Yeast Candida lusitaniae Candida lusitaniae workflow in the NGH microbiology laboratory has significantly improved the
2 White- creamy colonies Candida spp. Yeast Yeast Candida galbrata Candida galbrata
turnaround times, flexibility and reduced human errors results from multiple
tests and possibility of contamination.
Table 1: The results of 134 isolates identification using conventional methods, Phoenix or
Vitek2 and MALDI-TOF
MALDI-TOF cost around 0.02 ($0.03) to 0.06 ($0.10)
Turnaround times were reduced with the use of MALDI-TOF.
compared to around 3.23 ($5.50) for conventional
methods.
Identification time was cut from 7hrs to >24hrs using cultures, as opposed to
between only 14mins to 16mins using the Sepsityper Kit.
Sepsityper Kit for blood culture samples cost 3.30
($5.57) per sample.
MALDI Biotyper Poster Hall 2016 Blood Culture and Resistance
Performed on Gram-Negative Bacilli Blood Culture is Effective for Sparing the use of Carbapenems
marie-paule.gerlinger@egp.aphp.fr
A. Aubry1, A. Fournier1, H. Pereira2, S. Katsahian2,3, H. Bensekhri1, J-L. Mainardi1,3 and M-P. Fernandez-Gerlinger1,3
1 Unit Mobile de Microbiologie Clinique, Service de Microbiologie, Hpital europen Georges Pompidou, AP-HP, Paris, France; 2 Ple Biostatistique et Sant Publique, Hpital europen Georges Pompidou, AP-HP, Paris, France ;3 Facult de Mdecine Paris Descartes, Universit Paris Descartes, Paris, France
Days
analyzed period (120 days) 25 Strat B
Digestive tract n (%) 11 (9) p=0.04
- Peritonitis 3 Strat C
Antimicrobial optimization is improved by: - Translocation 8 20
1) Rapid identification of the microorganism with Maldi-tof Mass-spectrometry N= 3 Bacteremia due to GNB during the
- Angiocholitis 9
- Cholecystitis 1 15
(MT) analyzed period (120 days) excluded - Liver abscess 1 p=0.6
(anaerobic bacteria and Stenotrophomonas
2) Combined with antimicrobial stewardship (AMS) intervention spp)
- Other 1
10
In terms of length of stay, morbidity, mortality, total hospital costs, in the context Pneumonia n (%) 6 (5)
of GNB bacteremia (2,3), and for patients with antibiotic-resistant Gram-negative Others n (%) 4 (3) 5
N= 128 Bacteremia due to GNB during the - OSI 2
bacteremia (4). analyzed period (120 days) included - Unknown 2
*
Nosocomial: hospital acquired and healthcare associated; ICU: intensive care unit; PAC : Port-a-cath
0
Spare carbapenem Narrow spectrum Avoid inadequat
The purpose of this study was to assess the combination of AMS with rapid implantable central venous access device ;OSI operative site infection. antibiotherapy
identification by MT, and a new rapid biochemical test able to detect ESBL: -
Lacta-test (BLT) (Bio-Rad, Marnes-la-Coquette, France). Table 2: Organism distribution
Pathogens n (%) Agreement Strat.B and -Lacta Test or Maldi-tof MS estimated through Cohens
Patients and methods Enterobacteriaceae
Escherichia coli
100 (84)
62 (50) ESBL carriage and bacteremia kappa
Proteus mirabilis 1
Klebsiella pneumoniae 17 (14) ESBL
All Non ESBL -Lacta Test Maldi-tof MS
Klebsiella oxytoca 3 (2) bacteremia
Bacteremia bacteremia
Enterobacter cloacae 8 (7) N (%)
N (%) N(%)
Enterobacter aerogenes 2 (1)
Prospective observational study (168 days- 24 weeks): Enterobacter sakasakii 1 Spare carbapenem Strat. B
= -0,0465 = -0,0483 not
ESBL known p = 2 .10-13 p=0
-All patients with GNB positive blood cultures during 168 days Serratia marcescens 3 (2) agreement
Pantoea agglomerans 1 carriage
14 (10) 6 (43) 8 (57)
-Analyzed was performed from Monday to Friday morning (120 days). Morganella morganii 1 N (%)
p = 0.001 = 0,85 = 0,82
Citrobacter freundii 1 Avoid inadequate
p=0 p=0
MT and BLT were performed simultaneously on 3h incubated solid medium Nonfermentative 17 (12)
ESBL not OR = 9.65 antibiotherapy Strat.B
Pseudomonas aeruginosa 15 (12)
subcultures Acinetobacter pittii 1
known agreement
carriage 114 (90) 8 (7) 106 (93) = 0,95 = 0,885
Three strategies were compared: Chryseobacterium spp. 1
N (%) Narrow spectrum Strat. B p=0 p=0
Other aerobic 2 (1)
(A) empiric antibiotic therapy initiated by the physician in charge of the patient Salmonella spp. 2 (1)
knowing GNB bacteremia without MT and BLT results Polymicrobial 9 (7)
(B) empiric antibiotic therapy recommended by AMS without MT and BLT results
(C) AMS advice with MT and BLT results.
Distribution between different tests were compared using a Mac Nemar test for Conclusion
paired data. Agreement was estimated through the Cohen's Kappa. We have also
tested whether Kappa coefficient is different from 0.
References 1) Rapid identification with Maldi-tof MS and EBLSE rapid test -Lacta Test associated with antimicrobial stewardship (Strat C) is more efficient than AMS alone to narrow spectrum and avoid inadequate antibiotherapy.
(1) Schwaber et al. JAC 2007 2) Apart, BLT and MT are not in agreement with AMS in term of sparing carbapenem. BLT and MT are in agreement with AMS in terms of narrowing spectrum and avoiding inadequate antibiotherapy.
(2) Clerc et al. CID 2013
(3) Huang et al. CID 2013
3) In this study, ESBL carriage is associated with ESBL bacteremia.
(4) Perez et al. J Infect 2014
40
MALDI Biotyper Poster Hall 2016 Resistance
Introduction and Results Diameter of distribution of temocillin inhibition (30 g, Rosco) for the carbapenemase
enzymes tested in the assay.
Purpose CO2 Structural analysis and experimental
data over temocillin [M], MS spectrum
40
OXA
Group D carbapenemases (especially
Number of isolates
reveal the following mass peaks: IMP
blaOXA-48 isolates) detection represents [M + H]+ ...415 Da 30
a challenge as these enzymes are an NDM
[M + Na]+ ....437 Da
emerging healthcare concern. As the [Mdecarb. + H]+ ......371 Da 20
VIM
carbapenems MICS are low, [Mhydrol/decarb. + H]+.....389 Da KPC
carbapenemases often go undetected H2O
41
MALDI Biotyper Poster Hall 2016 Resistance
Recent evidence suggests that Gram buffer for use in this assay as the 20mM
K. pneumonia 14 >1.0
bla-NDM 5 (1 reference strain) bla-NDM 5 (1reference strain) 5 4
bla-KPC 4 (1 reference strain)
producing from
P. mirabilis bla-NDM 1 >8.0 >8.0 A. baumanii 4
clinical isolates
K. pneumonia 11 >1.0
E. cloacae 5 >1.0 bla-OXA 58 1 0 0
P. aeruginosa 4 >8.0 >8.0
bla-NDM 1 0 0
in 5 hour testing being detected.
This study analyses how these described
E. coli 1 >1.0
Klebsiella sp 1 >1.0 A. indicus bla-OXA 58 1 0 0
clinical laboratory, validated against An ertapenem hydrolysis assay was performed using a MALDI- K. pneumonia 11 0 0 samples. A positive cut-off value for logRQ was set
clinical isolates. It is hoped that ToF mass spectrometer (Bruker Daltronics ). Four variables E. cloacae 5 0 0
at 0.4 for this evaluation, but almost all
P. aeruginosa 4 0 0
Implementation of this methodology will were investigated to optimise methods for translation to a E. coli 1 0 0 non-carbapenemase producing isolates
significantly improve antimicrobial clinical laboratory including: Klebsiella sp 1 0 0
produced logRQ values below 0.25. If
S. marcescens 1 0 0
stewardship and in turn assist in the (i) concentration of ertapenem (1 - 3mg/ml), Enterobacter sp 1 0 0 implemented into a routine microbiology
reduction in the transmission of Multi-Drug (ii) incubation time (3 - 4 hours) Carbapenem Sensitive Isolates 17 0 0
laboratory the positive cut-off value could
Total 16 14
Resistant Organisms (MDROs) causing (iii) buffer (ammonium citrate vs TRIS-HCl) potentially be lowered, resulting in
Healthcare Associated Infections (HCAIs). (iv) -cyano-4-hydroxy-cinnamic acid matrix diluent increased sensitivity without reducing
(0.5 2.5% trifluoroacetic acid). specificity.
Figure 1 Non-carbapenemase producer
Author contact: lee.pitty@imperial.nhs.uk. Acknowledgements: NIHR Health Protection Research Unit in Healthcare Associated Infections and Antimicrobial Resistance at Imperial College London, and NIHR Imperial Biomedical Research Centre.
42
MALDI Biotyper Poster Hall 2016 Resistance
Relative growth
detection. Quantitative MALDI-TOF MS was 1,5
represent sensitive Titration of Tobramycin revealed a minimum
recently applied to detect resistance of 1 strains and red bars inhibitory concentration (ASTRA MIC) for each
Meropenem-resistant K. pneumoniae based on Acquisition Target represent resistant strain. A breakpoint concentration of 32 to 64
Evaluation of MS preparation 0,5
strains. Values > 0.4
bacterial growth. Here, we apply this approach profile
spectra represent growth
g/ml for K. pneumoniae, 16 to 32 g/ml for P.
for detection of Tobramycin-resistant K. 0
2 4 8 16 32 64 128 256 and < 0.4 growth aeruginosa, and 8 g/ml for A. baumannii
pneumoniae, P. aeruginosa, and A. baumannii. Figure 1: Workflow of the MALDI-TOF MS-based resistance inhibition. Red boxes resulted in a separation equivalent to the Etest
test MBT-ASTRA
2
A.baumannii, 3.5 h indicate the results between sensitive and resistant strains
Tobramycin concen-
(Fig. 4).
Methods
1,5
tration(s) resulting
A Susceptible strain
B
Resistant strain
1 in the correct Plotting the ASTRA MIC against the Etest MIC
Fresh overnight cultures of 20 K. pneumoniae, 0,5
classification of showed a direct correlation of both MIC values.
strains (ASTRA Compared to the Etest MIC the ASTRA MIC was
20 P. aeruginosa, and 20 A. baumannii strains breakpoint
0
increased due to the short incubation time of
Spectra number
Spectra number
incubation at 37C (Fig. 1). After growing, the Tobramycin (upper panel) or without Tobramycin (lower panel),
1,2
1
which is in concordance to the Etest MIC
cells were lysed in the presence of an internal
respectively. 0,8 results (Fig. 5B).
10
0,6
standard. 1 l of the lysates is spotted on a
Conclusions
0,4
A
MALDI target and overlaid with HCCA matrix B 0,2
1
after drying. MS profile spectra are acquired on
0
0,01 0,1 1 10 100 1000 0,01 0,1 1 10 100 1000
MBT ASTRA results are available after
a microflex LT/SH benchtop mass spectrometer Etest MIC Tobramycin [mg/mL] Etest MIC Tobramycin [g/mL]
a few hours
(Bruker Daltonik GmbH)(Fig. 2). The relative Figure 5: ASTRA MIC plotted against the respective Etest MIC of each strain (A). Relative growth MBT ASTRA results are concordant to
protein amount was calculated using the values derived at the breakpoint concentration of the MBT ASTRA plotted against the Tobramycin Etest results
internal standard (Fig. 3A). The ratio of the Etest MIC values (B). K.pneumoniae; red dots, P. aeruginosa; green dots, A. baumannii; blue
Species specific assay conditions
dots. The horizontal dotted red, green, and blue lines represent the MBT ASTRA breakpoint
protein content for the BHI plus Tobramycin Figure 3: Evaluation of MBT-ASTRA spectra. Area under the (antibiotic concentration, incubation
concentration of the respective species. The horizontal orange line indicates the threshold of the
setup and for the BHI only setup was curve (AUC) of four different strains each without and with RG of the MBT-ASTRA. The vertical red line indicates the threshold of the MIC for K.pneumoniae, time) have to be optimized and
calculated (relative growth, RG, Fig. 3B). Tobramycin (A). Normalization of growth of antibiotic setups to the vertical green line for P. aeruginosa, and vertical blue line for A. baumannii. applied
the growth in BHI resulted in the Relative Growth(B).
MALDI BiotyperTM
For research use only. Not for use in diagnostic procedures.
43
MALDI Biotyper Poster Hall 2016 Resistance
Norm LogRQ
Norm LogRQ
0.1 mg/mL Ertapenem (ETP) for 2 h at 0,6 0,6
The increasing number of carbapenem 37C in incubation buffer. Stability of Results 0,4 0,4
resistant Gram-negative bacteria is a dissolved IMI without bacteria was 0,2 0,2
challenging health care problem. Well- analyzed by LC-ESI-MS(n). After co- LC-ESI-MS(n) analysis revealed an 0,0 0,0
directed handling of this challenge incubation of antibiotics and bacteria, acceptable in-use stability of dissolved -0,2 -0,2
requires reliable, rapid, and cost efficient cell-free supernatants were spotted onto IMI without bacteria. Within 24 h, only -0,4
0,01 0,1 1 10 100
-0,4
0,01 0,1 1 10 100
assays for detection of carbapenem a MALDI target. Dried spots were 1.3 % of the IMI dissolved in incubation MIC [g/ml IMI]
MIC [g/ml IMI]
resistance. The detection of OXA-48 overlaid with matrix containing an buffer was hydrolyzed (Fig. 2).
positive strains using routine assays internal standard. Spectra were acquired The MALDI analysis using IMI as Fig. 3: Comparison of the MALDI-TOF MS derived results
such as Etest or disc diffusion often fails. on a microflex LT/SH mass spectrometer benchmark antibiotic correctly detected normalized values of the OXA-48 strains (normalized LogRQ values) and the Etest derived MIC
MALDI-TOF MS is an established method (Bruker Daltonik). Fully-automated data all OXA-48 strains after 30 min revealed no difference in the hydrolytic values for IMI (A) and ETP (B). OXA-48 strains are
marked red and susceptible strains are marked green.
in microbiological laboratories. processing based on an intensity ratio incubation. After normalization towards capacity between those strains correctly Above dashed red line: positive; below green dashed
Recently, this technique has also been calculation of internal standard and non- a known negative and a respective detected and those strains which had line: negative.
employed for detection of hydrolyzed IMI or of hydrolyzed and positive control (kpc positive strain), the been misclassified by Etest (Fig. 3A).
carbapenemase activity in non-hydrolyzed ETP was performed normalized values range between 0.43 Using ETP as benchmark antibiotic
Enterobacteriaceae. Here, we using a dedicated software tool and and 1.29. All susceptible strains were required a prolonged incubation time. Conclusions
demonstrate a reliable, rapid, and cost included normalization to respective correctly classified with normalized Nevertheless, 4 of the OXA-48 strains
efficient MALDI-TOF MS-based approach negative and positive control strains (= values below 0.2. Sensitivity and were tested negative and additional
for the detection of OXA-48 expressing NormLogRQ value). Strains with a value specificity were 100% each. In contrast, three strains revealed only an MALDI-TOF MS and
Enterobacteriaceae isolates employing below 0.2 were considered as negative, Etest (IMI) detected 6/20 OXA-48 intermediate hydrolysis of ETP even after
Imipenem (IMI) as reporter substance.
Imipenem provide a
above 0.4 as positive, and between 0.2 and isolates as positive. Comparison of the 2 h. The corresponding Etest with ETP
detected all OXA-48 strains. reliable approach for the
Table 1: Summary of the numbers of isolates correctly detected as resistant and detection of OXA-48 strains
susceptible using IMI as reporter substance, respectively. Total number of resistant and
susceptible strains and specificity and sensitivity of the respecive approach 1,20E+08
IMI_hydro IMI Accelerated procedure
MIC STAR-BL MIC STAR-BL
Gene
IMI IMI (0.5h) ETP ETP (2h)
1,00E+08
(30 min incubation time;
OXA-48 correctly
20 9 20 20 16
8,00E+07
60 min overall time) com-
Area [AU]
found
Susceptible correctly
6,00E+07
pared to the established
72 71 72 72 72 4,00E+07 standard procedures
found
Total resistant 20 10 20 20 16 2,00E+07
44
MALDI Biotyper Poster Hall 2016 Resistance
Bruker Daltonics
resistance or more general resistance standard. Lysates were spotted onto a incubation and application of 4 g/ml to 16
1
detection using heavy amino acids. Very MALDI target and dried. Spots were overlaid g/ml MEM lead to two to four miss-classified
recently, we published a quantitative MALDI- with HCCA matrix. MALDI-TOF MS profile strains and two strains showing an 0,1
0,01 0,1 1 10 100
TOF MS approach facilitating the rapid spectra were acquired on a microflex LT/SH insufficient growth preventing any evaluation. MIC [g/ml]
detection of meropenem resistant K. mass spectrometer (Bruker Daltonik) in the Depending on the MEM concentration and 1000 B
pneumoniae. The principle of this assay is mass range from 2 to 20 kDa. Spectra were the applied cut off value, either some of the
spectra derived either from cells after in calculated for each setup. The ratio of the some of the susceptible strains were
10
incubation with BHI medium or from the AUC for the BHI plus meropenem to the AUC classified as positive strains, respectively.
same cells after incubation in BHI with of the BHI only setup was calculated (relative Increasing the incubation time up to 2 h one 1
antibiotics. Here, we demonstrate the effects growth). Different relative growth cut offs to six misclassifications were observed.
of different assay parameters on the were tested to detect resistant strains (low Table 1: Outcome for the different strains at 4, 8, and 16 g/ml 0,1
0,01 0,1 1 10 100
MEM after 1 and 2 hours incubation (green: correctclassification,
outcome. growth corresponds to a low relative growth red: mis-classification, yellow: invalid due to poor growth of the
MIC [g/ml]
value). Experiments were repeated once and control) Fig. 3: ASTRA breakpoint concentration versus MIC at a
the median was considered for evaluation. Cut off 0.5 4 g/ml 8 g/ml 16 g/ml cut off threshold of 0.5 after 1(A) or 2 (B) hours (Green:
Resistant 10 10 10 susceptible, red: KPC, blue: NDM, and purple: OXA-48)
KPC
Antibiotic
Suceptible 0 0 0 Dashed lines indicate breakpoint concentrations
Invalid 0 0 0 between susceptible and resistant.
BHI
Susceptible strain Resistant strain Resistant 10 8 8
NDM
1 h incubation
Incubation 37C
McF 0.5 x104 x104
Suceptible 0 2 2
Conclusion
1068PEG_CTX 0:D10 MS, BaselineSubtracted
Intens. [a.u.]
BHI only
2.0
1.0 Invalid 0 0 0
1.5
Resistant 9 8 7
Oxa-48
0.8
Suceptible 0 1 2
Lysis reagent
An incubation time of 2 h, a meropenem
1.0 0.6
internal
Resistant 1 0 0
concentration of 8 g/ml, and a relative
ceptible
0.5
standard 0.2
Sus-
Target preparation Suceptible 7 8 8
growth cut off of 0.5 led to the most reliable
0.0 0.0
x104 x104 1068PEG_CTX 0:D10 MS, BaselineSubtracted
2 2 2
20028S_CTX 0:A10 MS, BaselineSubtracted
Invalid
Intens. [a.u.]
Intens. [a.u.]
BHI MEM
1.5
Resistant 10 10 10
detection of meropenem resistant K.
1.5
KPC
1.0
Suceptible 0 0 0
pneumoniae strains. Further validation with
1.0
Invalid 0 0 0
Resistant 10 10 9
additional strains will be necessary.
0.5
NDM
2 h incubation
0.5
Suceptible 0 0 1
Invalid 0 0 0
Optimization of the protocol for other species
0.0
Acquisition of 4000 5000 6000 7000 8000 9000 10000 11000 12000 13000 14000
0.0
4000 5000 6000 7000 8000 9000 10000 11000 12000 13000 14000
MS profile spectra
m/z m/z
Resistant 10 10 10
Oxa-48
Fig. 1: Preparation scheme for the analysis of antibiotic resistant strain after incubation in BHI medium and BHI
Sus-
Lange C, Schubert S, Jung J, Kostrzewa M, Sparbier K. ;Quantitative matrix-assisted laser desorption ionization-time Copyright 2015 Katrin Sparbier, katrin.sparbier@bruker.com
of flight mass spectrometry for rapid resistance detection. J Clin Microbiol. 2014 Dec;52(12):4155-62.
45
MALDI Biotyper Poster Hall 2016 Resistance
Background Results
In 2013, the CDC classified the growing CRE (carbapenem-resistant enterobacteriaceae) threat as Meropenem: 8g/ml Ceftriaxone: 64g/ml Ceftriaxone: 128g/ml Cefepime: 64g/ml Cefepime: 128g/ml
urgent, the highest threat level1 1.6 1.6 1.6 1.6 1.6
- considered an immediate public health threat that needs urgent and progressive action
- estimated that Klebsiella spp. made up 11% of CRE healthcare-associated infections1 1.4 1.4 1.4 1.4 1.4
- approximately 7900 cases in the US leading to 520 attributable deaths annually1
Almost half of all patients with carbapenem-resistant enterobacteriaceae bacteremia will die1 1.2 1.2 1.2 1.2 1.2
- up to 40% of bacteremia pt receive inadequate initial antibiotic treatment2
- mortality increases by 7.6% for each hour that appropriate treatment is delayed3
Relative Growth
Relative Growth
Relative Growth
Relative Growth
Relative Growth
1 1 1 1 1
Thus there is an ever growing need to determine antibiotic susceptibility to improve patient care and for 0.2 0.2 0.2 0.2 0.2
infection control to limit its spread.
0 0 0 0 0
0.1 1 10 0.1 1 10 100 0.1 1 10 100 0.1 1 10 100 0.1 1 10 100
Introduction MIC [g/ml] MIC [g/ml] MIC [g/ml] MIC [g/ml] MIC [g/ml]
Relative Growth vs. MIC (as determined by BD Phoenix). Vertical light green lines denote the MIC based definition of resistance according to CLSI. Horizontal dark green lines
While current methods commonly require overnight incubations, the recently developed Mass denote the Relative Growth cutoffs used.
Spectrometric-Antibiotic Susceptibility Test Rapid Assay (MS-ASTRA) can determine an organisms
susceptibility profile in less than 2 hours depending on the organisms doubling time. In this study, we
have adapted the MS-ASTRA technique to determine meropenem, ceftriaxone, and cefepime resistance Meropenem Ceftriaxone Cefepime
in Klebsiella spp. clinical isolates. Concentration (ug/ml) 8 64 64 128 64 64 128
RG cutoff 0.4 0.4 0.5 0.4 0.4 0.5 0.4
Methods Category Agreement
matching results
98.3% 89.8% 93.2% 98.2% 91.5% 94.9% 94.2%
-reproducibility studies to confirm precision blood cultures and the high accuracy of the gram stain report. J. Clin.
Microbiol. 45 (2007)
-increase sample set to include more Klebsiella spp. isolates 3Kumar A, et al., Duration of hypotension before initiation of effective
-will need to increase study to other organisms and antibiotics antimicrobial therapy is the critical determinant of survival in human
septic shock. Crit Care Med. 34 (2006)
46
MALDI Biotyper Poster Hall 2016 Resistance
Objectives: MALDI-TOF MS, a common tool in routine laboratories, was Data acquisition and processing. Fully automated data acquisition and
1.0
evaluated for ESBL detection and characterization in Enterobacteriaceae Hydrolyzation Inhibition data pre-processing was performed using a benchtop MALDI-TOF MS and
utilizing a research approach. 0.8 the MBT STAR-BL Module (both Bruker Daltonik GmbH, Bremen,
0.6 CTX > 0.3 > 0.1 Germany) by calculating the ratio of hydrolyzed and intact antibiotic peak
Results intensities (normalized to controls).
logRQ
0.4 CPO > 0.3 > 0.1
MALDI-TOF MS sensitivity and specificity of individual antibiotics was The obtained hydrolysis results were subsequently processed and
comparable to the combined Disc Diffusion (DD) test. 0.2 evaluated manually according to the CA inhibition to detect an ESBL
Results varied depending on bacterial species and antibiotic/inhibitor CPM > 0.3 > 0.14 production. Cut-off values for inhibition results were estimated on an
0.0
used in the research assay setup empirical basis.
Negative Positive CAZ > 0.04 > 0.05
The simple and easy to use (semi-) automated workflow allows robust + CA - CA + CA - CA + CA - CA Results were directly compared to the disk diffusion test using the same
Control Control
and very rapid ESBL determinations. Isolate No. 1 Isolate No. 2 Process Control antimicrobial agents, except CPO. In order to determine the accuracy of
ESBL detection, results were also compared to a variety of phenotypical
Figure 1 Example of MBT-STAR BL-Assay results for CTX as presented in Table 1 Estimated normalized logRQ cut-off values
Conclusion used for subsequent manual data analysis and molecular methods that had been used in a previous study [1] including
the software after automated data acquisition and processing
MALDI-TOF MS is a promising and fast tool for ESBL detection isoelectric focusing, targeted PCR and sequencing.
In future, optimized assay conditions, fully automated and dedicated data
Bacterial strains. A total of 146 previously characterized
acquisition and data processing workflows will aid the fast, lean and MALDI-TOF MS DD test MALDI-TOF MS DD test MALDI-TOF MS DD test
CPM CPM CTX CTX CAZ CAZ Enterobacteriaceae isolates - including 96 ESBL producers and 50 non-
accurate detection of ESBLs in routine laboratories
pos neg pos neg pos neg pos neg pos neg pos neg
ESBL produces such as hyper-producers of chromosomal AmpC, Koxy, or
SHV enzymes, and wild-type strains (Escherichia coli (n=61), Klebsiella
agreement agreement agreement agreement agreement agreement
INTRODUCTION MALDI- MALDI- MALDI-
pneumoniae (29), K. oxytoca (16), Enterobacter cloacae (16), E. aerogenes
TOF MS 100% 100% 55.9% 86.4% TOF MS 100% 100% 83.9% 75.5% TOF MS 100% 100% 64.2% 78.5% (5), Citrobacter freundii (5), C. farmeri (1), Morganella morganii (5), Serratia
In recent years, multidrug resistance has emerged among the Gram- CPM CTX CAZ
marcescens (1), Proteus mirabilis (6) and P. vulgaris (1)) - were included in
negative bacteria. Rapid and accurate detection of prevalent resistance DD test
90.5% 45.8% 100% 100%
DD test
85.7% 72.7% 100% 100%
DD test
78.8% 63.8% 100% 100%
CPM CTX CAZ the study.
mechanisms, such as extended-spectrum beta-lactamase (ESBL) activity in
E. coli ATCC 25922, K. pneumoniae ATCC 700603, and Acinetobacter
Enterobacteriaceae, is crucial for timely guidance and application of
Tables 2 4 Correlations between MALDI-TOF and DD test for all bacterial strains (n=146). CPO was not available as DD test baumanii (laboratory strain) were used as controls.
appropriate antimicrobial therapies.
Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass Disk Diffusion Test. The disk diffusion test was conducted according to
Sensitivity Specificity
spectrometry (MS), so far primarily used for species identification in routine 100% CLSI guidelines using disks with 30g of CTX, CPM or CAZ with and
laboratories, has been proposed for the detection of resistance without 10g of clavulanic acid. CPO was not available as DD test.
80%
mechanisms. Only recently, it has been commercialized for fully-automated
SUMMARY & CONCLUSION
detection of a present -lactamase activity. This study was conducted to 60%
evaluate the potential of MALDI-TOF MS to detect and characterize ESBL MALDI-TOF MS results of this research study were in good agreement
in Enterobacteriaceae. 40% with results obtained with the Disc Diffusion (DD) test. Accuracy was also
in good correlation with other methods applied in routine (data not
20%
shown).
MATERIALS AND METHODS
0% A high correlation of results was found for individual bacterial strains,
Cefepime Cefpodoxime Ceftazidime Cefotaxime Disc Diffusion Test
Study design. A modified MBT STAR-BL assay, a dedicated workflow to dependent on individual antibiotics used.
detect a present -lactamase activity in Enterobacteriaceae by analyzing Figure 2 Specificity and Sensitivity of ESBL detection for all bacterial strains (n=146) based on cut off values from Table 1 Appropriate benchmark antibiotics (e.g. a combination CPO and CTX) in
the enzymatic hydrolyzation of the antibiotics -lactam ring, was developed combination with optimized assay conditions implementing fully
and evaluated for ESBL detection [2]. Briefly, bacterial strains were automated MALDI-TOF MS data acquisition and data processing may
CPM CTX CAZ CPM CTX CAZ CPM CTX CAZ CPM CTX CAZ CPM CTX CAZ
incubated 1 4 h with different cephalosporins (cefotaxime (CTX), finally lead to rapid and enhanced detection of ESBL production in
ceftazidime (CAZ), cefepime (CPM), or cefpodoxime (CPO)) with and Escherichia coli Klebsiella spp. Enterobacter spp. Proteus spp. (n=12) Citrobacter sp. (n=6) Enterobacteriaceae superior to currently applied conventional methods
(n=61) (n=45) (n=21) M. morganii (n=5) S.marescens (n=1)
without the presence of the -lactamase inhibitor clavulanic acid (CA), like DD or molecular methods.
respectively. Suitable incubation times and CA concentrations were MALDI / DD pos. agreement 85.4 88.6 74.4 48.5 76.5 75 60 100 36.4 37.5 66.7 25 0 100 0
evaluated in preliminary studies on an individual basis. REFERENCES
MALDI / DD neg. agreement 69.2 82.4 77.3 83.3 54.5 66.7 100 84.6 90 100 100 85.7 100 50 100
1: Wiegand, I., H. K. Geiss, D. Mack, E. Strenburg, H. Seifert. 2007. Detection of
DD / MALDI pos. agreement 88.6 92.9 85.3 89.5 83.9 72 100 80 80 100 100 50 57.1 25 71.4 Extended-Spectrum Beta-Lactamases among Enterobacteriaceae by Use of
Semiautomated Microbiology Systems and Manual Detection Procedures. J Clin Microbiol.
ACKNOWLEDGEMENTS 45(4): 1167-1174.
DD / MALDI neg. agreement 34.6 73.7 55.6 38.5 57.1 70 73.3 100 56.3 44.4 75 66.7 nd 100 nd
2: Bruker Corporation. March 2015. Standard operational procedure for MBT STAR-BL
This study was supported in part by Bruker Daltonics.
Table 5 Correlations between MALDI-TOF and DD test for selected Enterobacteriaceae (n=146). CPO was not available as DD test. Assay for MBT Compass (RUO).
Wisplinghoff laboratories, Cologne1, Bruker Daltonics, Bremen 2 , Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne, Cologne3, Germany
47
MALDI Biotyper Poster Hall 2016 Resistance and Subtyping
MALDI-TOF mass spectroscopy and blakpc gene phylogenetic analysis: an outbreak of multidrug and carbapenem
resistant K. pneumoniae isolates ?
Silvia%AngeleA%1*,%Giordano%Dicuonzo%1,%Alessandra%Lo%Pres;%2,%Eleonora%Cella%2,%3,%Francesca%Crea%1,%Alessandra%Avola%1,%Massimiliano%Andrea%Vitali%4,%Marco%Fagioni%5,%Lucia%De%Florio%1.%%
%%
1%Clinical%Pathology%and%Microbiology%Laboratory,%University%Hospital%Campus%Bio=Medico%of%Rome,%Italy.%% 22K
24K
2%Department%of%%Infec;ous,%Parasi;c,%and%Immune=Mediated%Diseases,%Epidemiology%Unit,%Reference%Centre%on%Phylogeny,%Molecular%Epidemiology,%and%Microbial%Evolu;on%(FEMEM),%Na;onal%Ins;tute%of%Health,%Rome,%Italy.%% 18K
3(Department%of%Public%Health%and%Infec;ous%Diseases,%La%Sapienza%University%of%Rome,%Italy.%% 16K
12K
4(Hospital%Management,%University%Hospital%Campus%Bio=Medico%of%Rome,%Italy.%%
5%Mass%Spectrometry%Support,%Bruker%Daltonics%Srl%,%Macerata,%Italy.%%
* 8K
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I* 2K
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There% are% several% mechanisms% that% may% be% involved% in% 25K
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carbapenems's% resistance% of% Klebsiella( pneumoniae.% 28
~asse 1 (2C)\114 (C1)
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Cluster%II% 45 ~lasse 1 (2C)\34 (C1)
15K
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Among% them,% the% produc;on% of% specic% carbapenem=
38 ~lasse 1 (2C)\36 (C1)
37
39 ~asse 1 (2C)\310 (C1) 9K
~lasse 1 (2C)\66 (C1) 19K
hydrolysing% % lactamases% (carbapenemases)% is% the% most%
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48 ~asse 1 (2C)\112 (C1)
~asse 1 (2C)\111 (C1) 13K
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pneumoniae%carbapenemases%producing)%was%discovered%
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Cluster%I% 49
43
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~lasse 1 (2C)\59 (C1)
~lasse 1 (2C)\77 (C1) * 11K
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in% the% USA% in% 1996% and% have% spread% worldwide% causing% 33
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important% hospital% infec;ons,% increasing% of% length% of% 42
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34 ~e 2 (2C)\336-12 (C2) 0.0005
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hospitaliza;on,% raising% use% of% an;bio;c% therapy% and% 46 ~e 2 (2C)\188-12 (C2)
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48
MALDI Biotyper Poster Hall 2016 Resistance and Subtyping
BACKGROUND: Early detection of epidemic strains of Representative peaks of the nosocomial MRSA complex Berlin
m/z [Da] 3210 3444 4306 4815 5034 5526 6819 6863 6889 9627
methicillin-resistant Staphylococcus aureus (MRSA) is CONCLUSIONS:
essential for preventing MRSA outbreaks in the healthcare MRSA 706 + + + + + + + + + +
MALDI-TOF may be a useful tool to facilitate
setting. Spa-typing and pulsed-field gel electrophoresis
microbiological typing and can be used for efficient MRSA 454 + + 0 + + 0 0 + + +
MRSA strains.
MRSA 1156 + + + + + + 0 0 + +
belonging to the five major hospital acquired MRSA clonal 4236 2.56 Berlin t002 South German no MRSA 902 + + + + + + + + + + + + + + +
1026 2.596 Berlin t110 No result
complexes, i.e. Barnim (5), Rhine Hessen (9), Berlin (5), 2307 2.368 Rhine Hessen t003 Rhine Hessen yes
MRSA 903 + 0 0 0 0 0 + 0 0 0 + 0 0 + +
Southern German (7), Clonal Class III (7) provided the 4242 2.474 South German t1345 No result
MRSA 904 + + + + + + + + 0 + + + + + +
reference mass spectra for the analysis by MALDI-TOF. 2382 2.38 Barnim t032 Barnim yes MRSA 907 + + 0 + + + + 0 0 + + + + + +
MRSA lineages were detected and analyzed. A total of 370 2347 2.39 South German t041 No result MSP peaks characteristic of the major nosocomial MRSA
complex Rhine Hessen
MRSA isolates with different reproducible scores of MALDI- 4325 2.469 Rhine Hessen t002 Rhine Hessen yes
4338 2.501 South German t045 No result
TOF were tested and compared to the results of spa-typing. 4308 2.617 South German t012 No result
Representative peaks of the nosocomial MRSA complex Barnim
Among the five clonal complexes, the results of MALDI-TOF 1014 2.615 Berlin t001 South German no
m/z [Da] 3209 3444 4306 4816 5004 5527 6819 6865 6890 9627
MRSA 232 + + + + + + 0 + + +
MRSA 235 + + + + + + 0 + + +
MRSA 229 + + + + + + 0 + + +
MRSA 952 + + + + 0 + + + + +
MRSA 954 0 + + + + + + + + +
49
MALDI Biotyper Poster Hall 2016 Resistance and Subtyping
Purpose
Results
q Rapid
and
specic
idenAcaAon
to
subspecies
level
and
determinaAon
of
strain
idenAty
are
integral
components
of
q Outbreak
on
neonatal
intensive
care
unit
(Tschudin-Su\er
S
et
al.
Emerging
Infect
Dis
2010)
outbreak
invesAgaAon
and
control.
q ConvenAonal
methods
such
as
pulsed-eld
gel
electrophoresis
(PFGE)
are
labour
intensive
and
require
up
to
one
week
from
sample
collecAon
to
result.
q MALDI-TOF
is
able
to
idenAfy
bacterial
species
and
subspecies
rapidly.
q We
aimed
to
assess
the
validity
of
MALDI-TOF
for
detecAon
of
stain
idenAty
as
compared
to
PFGE.
MALDI-TOF
technology
Target plate Detector
Pulsed laser beam
Figure
3.
PFGE
of
ESBL-producing
E.coli
outbreak
q Eight isolates collected during an outbreak investigation in the neonatal care unit, identifying transmission of
ESBL-producing E.coli from a mother to her newborn twins and subsequent spread to two other neonates and
one healthcare worker, were analyzed by PFGE and MALDI-TOF.
Target plate
Bacterial colony q In comparison to PFGE, PCA-based typing with MALDI-TOF spectra reproduced a very similar dendrogram
- 4 single cultures
- 3 times repeated
Electric field for acceleration of ions with identical clustering of six outbreak isolates. Two isolates of ESBL-producing E. coli not associated with the
Figure
1.
Work-ow
for
MALDI-TOF
typing.
The
protein
signature
is
highly
specic
for
bacterial
species
outbreak were correctly separated.
Corresponding
author
Adrian
Egli
MD
PhD,
Clinical
Microbiology,
University
Hospital
Basel,
Petersgraben
4,
4031
Basel,
Switzerland,
E-mail:
a.egli@usb.ch
50
MALDI Biotyper Poster Hall 2016 Environmental and Food Microbiology
ESKAPE Pathogens
Understanding Nosocomial Risk Factors STENOTROPHOMONAS
The monitoring of water used in care homes and Acinetobacter baumannii ALS Environmental are one of the first UK laboratories to validate and have ALS Environmental are proud to be one of the few
healthcare facilities is covered by several pieces of Health Acinetobacter baumannii is a rapidly emerging pathogen in accredited to ISO 17025:2005 and the Drinking Water Testing Standard (DWTS), laboratories in the UK and Ireland to offer an analytical
and Safety Guidance (HSG). The majority of the guidance the health care system. A. baumannii is usually introduced accredit the rapid identification of positive Microbiological samples by Matrix speciation service for Stenotrophomonas species, which
is on the monitoring of Legionella and is supported by the into a hospital by a colonised patient. Due to its ability to Assisted Laser Disportion and Ionistation by Time of Flight Mass Spectrometry are of particular concern to healthcare facilities as they are a
Approved Code of Practice for Legionella (ACoPL8) and survive on artificial surfaces and resist desiccation it can (MALDI-ToF MS). The ground breaking identification technique employed by common water-borne organism. Although infection by this
Health Technical Memorandums (HTM); however, there are survive and potentially infect new patients for some time. It ALS Environmental, known as MALDI-ToF, allows us to remove the need for opportunistic bacteria is rare, cases of Stenotrophomonas
a range of other risk factors that need to be considered is suspected that A. baumannii growth favours nosocomial presumptive data for bacteriological analysis, with all data reported as Colony maltophila (S. maltophila) are potentially lethal. Following
in nosocomial scenarios. The ESKAPE pathogens are settings due to the constant use of antibiotics by patients Forming Units (CFU). The impact of MALDI-ToF confirmation on the ESKAPE a request from one of our established clients we have
emerging pathogens of concern. ALS Environmental are in the hospital and causes a wide range of infection pathogens is highlighted in the table below. developed a rapid technique to confirm and identify the
able to offer rapid identification of these bacteria using including bacteremia, pneumonia, meningitis, urinary tract colonies of Stenotrophomonas.
our revolutionary MALDI-ToF confirmation technique. The infection, and wound infection. The organisms ability to Bacteria Incubation Confirmation: Confirmation:
MALDI-ToF Saving
ESKAPE Pathogens are: survive under a wide range of environmental conditions, Time Traditional MALDI-ToF An aquatic, ubiquitous, opportunistic organism, S. maltophila
and to persist for extended periods of time on surfaces, Enterococci 2 days 1 day Minutes 1 day may be a particular burden to healthcare facilities due
Enterococcus faecium make it a frequent cause of outbreaks of infection and an Staphylococcus aureus 2 days 1 day Minutes 1 day to the susceptibility of immuno-compromised patients. ALS Environmental are employing our latest technology
Enterococcus faecium; formerly known as Streptococcus Although it has no natural infection route to humans S. in order to produce rapid species level identifications,
endemic, health careassociated pathogen. Klebsiella pneumoniae 1 day 1 day Minutes 1 day
faecium until its re-categorization in 1984, is a human maltophila infection can be facilitated by contaminated this means that we do not report presumptive results,
Acinetobacter baumannii 1 day 1 day Minutes 1 day
pathogen that causes nosocomial bacteremia, surgical Pseudomonas aeruginosa prosthetics such as catheters and intravenous lines. S. but instead provide a full list of the Stenotrophomonas
Pseudomonas aeruginosa 2 day 1 day Minutes 1 day
wound infection, endocarditis, and urinary tract infections. The monitoring for Pseudomonas is covered in HTM04- maltophilas resitance to most antibiotics has proved fatal species present in the sample. The full species breakdown
The bacteria can survive for long periods of time inside 01 Addendum. Serious infections of P. aeruginosa usually Enterobacteriaceae 1 day 1 day Minutes 1 day
to severely immunocompromised hosts. for Stenotrophomonas is important as not all species are
hospitals on a variety of surfaces as well as in soil and occur in the immunocompromised. Infections of the Stenotrophomonas 1 day 1 day Minutes 1 day
pathogenic to humans.
sewage. Growth temperatures range from 10oC to 45oC in blood, pneumonia, and infections following surgery can
basic or acidic environments, and in environments which lead to severe illness and death in these people. The Legionella 10 days 2 days Minutes 2 days
ALS Environmentals Microbiology Operations Manager,
are isotonic or hypertonic. Ent. faecium can be highly highly susceptible nosocomial patients include those on E-coli 1 day 1 day Minutes 1 day Pervinder Johal comments
drug resistant. The spread of the disease occurs between breathing machines, premature babies and patients with Coliforms 1 day 1 day Minutes 1 day
patients in hospitals due to transfer of the pathogen by wounds from surgery or from burns. Additionally, healthy Clostridium Perfringens 1 day 1 day Minutes 1 day When our customers first approached us to analyse
hands or medical instruments. Also, antibiotic use can people can also develop mild illnesses with Pseudomonas Salmonella 4 days 2 days Minutes 2 days for Stenotrophomonas we began our research and
decrease the number of other intestinal bacteria that are aeruginosa, especially after exposure to water. Ear development into validating a new method for this genus
Listeria 4 days 2 days Minutes 2 days
susceptible to the antibiotic and decrease competition for infections, especially in children, and more generalised group. By utilising the latest technology we were able to
the drug resistant Ent. faecium. skin rashes may occur after exposure to inadequately The instant confirmation of the ESKAPE bacteria allows infection control and provide a full speciation of Stenotrophomonas in under a
chlorinated hot tubs or swimming pools. water treatment to make rapid decisions on any potential remedial works that month.
Staphylococcus aureus may need to be undertaken. The MALDIToF can be used to identify any positive
The carriage of Staphylococcus aureus is an important Enterobacter species
bacteria, including Legionella. Species of the Stenotrophomonas genus are common in
source of nosocomial infection and community-acquired The genus Enterobacter is a member of the coliform
methicillin-resistant Staph. aureus (MRSA). Staph. aureus is group of bacteria. Enterobacter species, particularly E. Legionella the environment and human infection is rare. However,
Traditional testing for Stenotrophomonas involves a
common and often found in the nose or on the skin. Most cloacae and E. aerogenes, are important nosocomial The MALDI-ToF confirmation of Legionella removes the presumptive stage; general screen that produces presumptive results for infection risk of opportunistic pathogens such as the
of the time these bacteria do not cause any symptoms. pathogens responsible for various infections; including meaning that ALS Environmental report positive confirmed Legionella on the the prescence of members of the genus. This is usually maltophila species of Stenotrophomonas can be elevated
The ability of the nasal passages to harbour Staph. aureus bacteremia, lower respiratory tract infections, skin original read days (currently days 3, 7 and 10). This is 40% quicker than the followed by biochemical tests which indicate whether S. in healthcare settings and other environments where
results from a combination of a compromised host immune and softtissue infections. Risk factors for nosocomial traditional approach and is fully ISO 17025:2005 accredited. ALS Environmental maltophila is present. immuno-compromised patients, such as in care homes
system combined with the bacterias ability to evade a Enterobacter infections include hospitalisation of greater have one of the worlds largest Legionella species libraries held within our MALDI- may be exposed. Despite the low infection rate, nomo-
hosts innate immunity. The spectrum of Staphylococcus than 2 weeks, invasive procedures in the past 72 hours, ToF. With only 3 known species unidentifiable, two of these are Viable But Not social outbreaks of S. maltophila are of increasing concern
infections can range from skin abscess to life-threatening treatment with antibiotics in the past 30 days, and the Culturable (VBNC) and the final species is being sourced by our laboratory. in modern infection control because it can be difficult to
infections such as septicaemia or endocarditis. presence of a central venous catheter. Specific risk factors treat effectively as it is resistant to most broad-spectrum
for infection with nosocomial multidrug-resistant strains antibiotics.
Klebsiella pneumoniae of Enterobacter species include the recent use of broad-
In nosocomial settings, Klebsiella bacteria can be spread spectrum cephalosporins or aminoglycosides and ICU care. LEGIONELLA SPECIES TABLE
through person-to-person contact or by contamination of
the environment; it is important to note that the bacteria Stenotrophomonas Maltophilia Name of species Number of serotypes Linked to humaninfection Geographic origin Common Matrix Type
are not spread through the air. Patients in healthcare Stenotrophomonas maltophilia is an organism of low Legionella anisa YES USA Process, Drinking, Surface and Recreational
settings may be exposed to Klebsiella when they are virulence which can frequently colonise fluids used in the
Legionella adelaidensis Unknown Adelaide in South Australia Process Water
on ventilators, or have intravenous catheters or wounds. hospital setting (eg, irrigation solutions, intravenous fluids)
Unfortunately, these medical tools and conditions may and patient secretions (eg, respiratory secretions, urine, Legionella beliardensis Unknown Montbeliard in France Process Water
allow Klebsiella to enter the body and cause infection; wound exudates). S. maltophilia usually bypasses normal Legionella birminghamensis YES Birmingham, Alabama, USA Process Water
which can be fatal in the immuno-compromised. hosts defenses to cause human infection. The growth of Legionella bozemanii (bozemanae) 2 YES Named after F.Marilyn Bozeman Clinical Isolation
S. maltophilia from sites which would normally be sterile
Legionella brunensis Unknown Brno, Czech Republic Process Water
(e.g., blood) usually represents true infection; growth of
S. maltophilia in microbiological cultures of respiratory Legionella busanensis Unknown Busan in Korea Process Water
or urinary specimens is therefore sometimes difficult to Legionella cardiaca YES Northwestern University, Chicago, USA Clinical Isolation
interpret and not always a proof of infection. Legionella cherrii Unknown Minnesota, USA Process Water
Legionella cincinnatiensis YES Cincinnati, Ohio, USA Clinical Isolation
Legionella donaldsonii Unknown Process, Drinking, Surface and Recreational
The ability to reduce confirmation times is as a result of Legionella fallonii Unknown Cruise ship in international waters Process Water ESKAPE Pathogens Understanding
MALDI-TOF being able to be performed on colonies from Legionella feeleii YES Process Water and Clinical Isolation Nosocomial Risk Factors Part 1
selective agar. This removes the need for sub culturing and
incubation, which is the stage that incurs additional time.
Depending on the organism being tested, this additional
time can be anything from 4 hours to 5 or more days.
The reduction in confirmation times removes the risk Legionella micdadei -L. pittsburghensis YES USA Clinical Isolation and Drinking Water
associated with acting (or not acting) on presumptive Legionella monrovica YES USA Unknown
data by providing an accredited, confirmed result within Legionella moravica Unknown Moravia, Czech Republic Process Water
minutes of the presumptive result being generated. The Legionella nagasakiensis YES Aomori, Japan Clinical Isolation, Recreational and Drinking Water
added benefit is that in addition to a rapid confirmation
Legionella nautarum Unknown London , UK Drinking and Process Water
result, in the majority of cases, species-level identification
will also be available. ALS will provide this additional Legionella oakridgensis YES Oak Ridge, Tennessee , USA Clinical Isolation and Process Water
information as part of the standard service. Legionella parisiensis YES Paris, France Clinical Isolation, Process and Surface Water
Legionella pittsburghensis Unknown USA Clinical Isolation
If you would like more information on specific organisms Legionella pneumophila 15 YES USA Clinical Isolation, Surface and Recreational Water
that we are able to identify, please contact our customer
Legionella pneumophila ssp fraseri YES Los Angeles, USA Clinical Isolation
services department.
Legionella pneumophila ssp pascullei YES Clinical Isolation
Legionella pneumophila ssp pneumophila YES Philadelphia , USA Clinical Isolation
ALS SERVICE OVERVIEW Legionella quateirensis Unknown Quarteira, Portugal Process and Drinking Water
Contaminated Land Legionella and Microbiology Waste Management Drinking Water If you have any questions on our services or would like more information on
We understand the time pressures of large scale Being members of the Legionella Control Association (LCA) By working closely with some of the largest companies We are one of only a handful of commercial laboratories ALS Environmental please call the Coventry laboratory on 02476 42 12 13 or
Remediation and Brownfield projects and are a member we understand the emphasis placed on laboratory analysis in this sector we are able to offer unrivalled analytical to have a dedicated Drinking Water Testing Specification visit our website.
of the AGS. Our Coventry laboratory utilises state of the for the Control of Legionella. With 3 methods for testing and administration services to ensure that your samples (DWTS) accredited laboratory, based in Wakefield, Yorkshire.
art analytical equipment with the backup of our sister Legionella (including rapid PCR) and an understanding and are processed swiftly and in line with the UKAS Deviating We are able to supply analysis to the Public and Private ALS Environmental Limited
laboratories across Europe to ensure that we deliver your appreciation the implications of ACoP L8, HSG 274 and Sample Guidance. Drinking Water Regulations T +44 (0)24 7642 1213
projects on time every time HTM04-01 we are your ideal analytical partner for all of F +44 (0)24 7685 6575
your water hygiene monitoring requirements. E info.ukenviro@alsglobal.com
52
53
Environmental and Food Microbiology
Fig. 4. Three strains of L. helveticus were differentiated successfully. Fig. 5. Strain CCM 7010 was discriminated from the remaining two indistinguishable
Fig. 3. Dendrogram separated closely related Lactobacillus spp. correctly. Equivalent results were obtained also for L. jensenii and L. delbrueckii strains. strains. Equivalent results were obtained also for L. amylovorus strains.
CONCLUSIONS: REFERENCES:
edo et al., Mass Spectrom. Rev. 2011: 30(3), 417-434.
MALDI-TOF MS was found as a suitable tool for identification Dukov et al., Int. J. Food Microbiol. 2012: 159(2), 107-114.
of bacterial isolates from dairy products. Kamenk et al., Acta Vet. Brno 2013: 82(2), 181-186.
edo et al., Rapid Commun. Mass Spectrom. 2013: 27(24), 2729-2736.
Extension of the database of reference spectra is needed for improved
performance of the method for identification of isolates from meat products.
The method can serve to discriminate strains of the same species, ACKNOWLEDGEMENTS: This work was supported by the project
CEITEC (Central European Institute of Technology, CZ.1.05/1.1.00/02.0068)
however, in the standard arrangement, some strains remain indistinguishable. funded from the European Regional Development Fund and by the
European Social Fund (CZ.1.07/2.3.00/20.0189).
55
Veterinary Microbiology
56
MALDI Biotyper Poster Hall 2016 New Applications
D-1146
Bacterial Antibiotic Resistance Determination by Metal Oxide-Catalyzed MALDI MS Fatty Acid Profiling
1310 Maple St., Golden, CO 80401
303-384-2493
crcox@mines.edu
New approaches that combine ID and resistance profiling into a single test would drastically reduce turnaround
and improve treatment of drug resistant infections. We investigated MALDI MS fatty acid analysis as a means
of simultaneous ID and antibiotic resistance profiling. The energy inherent to the MALDI laser was used to
drive in situ metal oxide-catalyzed lipid fragmentation into taxonomically viable fatty acids using conventional
MALDI instruments already widely in clinical use. With a CeO2 catalyst in place of a traditional matrix, we
achieved strain-level ID and where able to rapidly and accurately differentiate antibiotic resistance among a
diverse collection of methicillin resistant and susceptible Staphylococcus aureus (MRSA/MSSA).
Experimental Design
Lipids from five replicates each of six MRSA and eight MSSA were extracted and fatty acid profiles obtained by
CeO2-catalyzed metal oxide laser ionization (MOLI) MS. Differentiation of resistant strains was achieved by
assembly of a database of spectral profiles followed by principal component analysis and K-Nearest Neighbor Figure 2. A) Differentiation of MRSA/MSSA by MOLI-MS fatty acid profiling. B) Fatty acid classification by FuRES
pattern recognition. Classifications were confirmed by leave-one-out cross validation and verified by Biotyper discriminant weights analysis; 95% confidence interval. Negative weights correspond to larger features in MRSA; positive
protein profiling. Resistance was confirmed by disc diffusion. weights to larger features in MSSA.
Results
MOLI MS fatty acid profiling resulted in 100% correct differentiation of MRSA/MSSA. Odd-numbered fatty
acids (C15:0, C17:0) were more prevalent in sensitive isolates, while a shift to even-numbered fatty acids
(C14:0, C14:1, C16:0, C16:1) was observed in resistant strains. A separate study of 160 samples
encompassing 32 strains from 14 Staphylococcus species yielded accuracies of 98% and 96% at the species
and strain level, respectively.
Conclusions Figure 5. Representative CeO2-catalyzed fatty acid profiles. Chain length and degree of unsaturation
MOLI MS lipid fragmentation readily produced unique species and strain-level staphylococcal fatty acid indicated for all peaks.
profiles. Results from a 160-sample study of 32 strains resulted in highly accurate differentiation of resistant
isolates. Importantly, this allowed for simultaneous ID and resistance determination with a single test without STRAIN-LEVEL MOLI-MS FATTY ACID PROFILING CONCLUSIONS
the need for secondary methods or additional culturing.
Novel CeO2-catalyzed MALDI-TOF MS provides a more accurate alternative to protein-based methods.
By coupling the catalytic activity of CeO2 with MALDI laser energy, we demonstrated in situ decomposition
INTRODUCTION of staphylococcal lipids into taxonomically useful FA constituents. This allowed for highly accurate ID and
differentiation of MRSA from MSSA.
Diagnostic bacterial ID by MALDI-TOF MS protein analysis has gained acceptance by the clinical and
research communities following U.S. FDA and European Commission CE Mark approval of commercial
Supervised and unsupervised learning techniques yielded accuracies of 98% and 96% at the species and
systems.
strain level, respectively.
However, a significant drawback exists when using protein profiling to differentiate closely related bacterial
MRSA and MSSA were differentiated with 100% accuracy.
phylotypes. Moreover, this approach does not allow for determination of antimicrobial resistance.
The primary advantages of this new diagnostic technique are the avoidance of misidentification of closely
To address the problem of accurate ID of closely-related isolates, we previously reported the use of rapid,
related phylotypes, greatly improved accuracy, and simultaneous ID and determination of antimicrobial
fatty acid (FA) profiling by CeO2-catalyzed MALDI-TOF (MOLI-MS).1-3
resistance in a single rapid test, which can improve therapeutic management and infection control.
By focusing on bacterial lipids as diagnostic biomarkers rather than proteins, and by exploiting the unusual
catalytic propensity of the rare-earth lanthanide CeO2 to cleave lipids to FAs, we obtained highly accurate and METHODS
reproducible species- and strain-specific bacterial FA profiles. Bacterial strains and growth conditions.
Figure 3. MOLI-MS fatty acid-based differentiation of staphylococci at (A) species and (B) strain level. Species Staphylococci were streaked to isolation on BHI agar and incubated for 18 hr at 37o C.
We hypothesize that this was achieved through rapid, in situ conversion of bacterial lipids into FAs by the 4+ represented by color; strains by shape.
Lipid extraction.
reactive state of CeO2 using the laser energy inherent to MALDI-TOF MS. Individual colonies were suspended in 100 L of 2:1 (Vol/Vol) chloroform/methanol followed by addition of an equal volume PBS as previously
described.1
While cerium has been used in a wide range of biomedical applications,4 to our knowledge, its capacity as a Metal oxide-catalyzed mass spectrometry.
biocatalyst for in situ conversion of bacterial lipids into taxonomically viable FAs using MALDI-TOF laser 100 mg of CeO2 was added to one mL of 2:1 (Vol/Vol) chloroform/methanol. One L was then removed from the bottom of the resulting slurry
energy is novel. and spotted on a stainless steel MALDI plate. Two L aliquots of bacterial lipid extracts were spotted directly onto CeO2. Negative controls
were run on SBA-15 to ensure FA spectra were the result of CeO2 catalysis and not thermal lipid desorption. All data was obtained as
described.1-3 Briefly, biological and technical replicate FA spectra were obtained in negative-ion reflectron mode with a grid voltage of 50.3%,
By exploiting the strain-level classification capabilities of MOLI-MS, here we demonstrate simultaneous delayed extraction of 120 ns, and a sampling frequency of 1kHz using a 355 nm Nd:YAG laser.
bacterial ID and antimicrobial resistance determination in a single rapid test using conventional MALDI-TOF
Data analysis.
MS instrumentation, which is increasingly utilized in clinical diagnostics worldwide. 23 FA spectral peaks were selected, centroided, assigned nominal masses, and compiled using software written in-house. Data were imported
into the R Statistics package (Ver. 3.0.2) for PCA and leave-one-out cross-validation (CV). The prcomp function was used to calculate PCA
scores. PCA scores were plotted using the plot function. CV was conducted using the lda function from the Modern Applied Statistics with S
GENERALIZED WORKFLOW (MASS) package.5 Results from CV are reported as percentages of correct assignments divided by total measurements.
Processed FA profiles were analyzed with MATLAB 2014a. Generalized prediction rates were measured using three Latin partitions and 100
bootstraps.6 Two classifiers were evaluated: a fuzzy rule-building expert system (FuRES)6 and partial least squares discriminant analysis
(PLS-DA).7 The PLS-DA algorithm used two Latin partitions and ten bootstraps to calculate average pooled prediction errors.7 The number of
components (latent variables) that minimized error was selected and used to build a model from the training data, which was then used as a
prediction set. Training data consisted of a set of profiles used to build the classifiers; the test data were the set of profiles used to evaluate the
performance of these classifiers. Hierarchical cluster analysis was used to generate dendrograms and graphically illustrate Euclidean linkage
distances obtained from an agglomerative algorithm. Distances were between pairs of profiles or between averages of profiles from
subclusters.
ACKNOWLEDGEMENTS
This work was supported in part by an NIH Career Development Award through Rocky Mountain Regional
Center of Excellence for Biodefense and Emerging Infectious Diseases Research NIAID grant U54 AI065357,
and NSF MRI GRANT CHE-1229156.
LITERATURE CITED
1. Cox, C.R et al. 2015. Strain-level bacterial ID by CeO2-catalyzed MALDI-TOF MS fatty acid analysis and comparison to commercial protein-based methods. Nature
Scientific Reports. 5:10470.
2. Voorhees, K.J. et al. 2015. Comparison of metal oxide catalysts for pyrolytic MOLI-MS bacterial ID. J. Anal. Appl. Pyrolysis. 113:78-83.
3. Voorhees, K.J. et al. 2013. Modified MALDI MS fatty acid profiling for bacterial identification. J. Mass Spectrom. 48, 850-855
Figure 1. Overview of MOLI MS for diagnostic bacterial ID. A) Bacterial sampling followed by enrichment on nutrient Figure 4. Phylotypic classification of staphylococci by MOLI-MS fatty acid profiling. A) Species- and B) Strain-level 4. Xu, C. & Qu, X. Cerium oxide nanoparticle: a remarkably versatile rare earth nanomaterial for biological applications. Nature Publishing Group Asia Mater 6, e90 (2014).
agar and lipid extraction. B) In situ CeO2-catalyzed lipid conversion to profilable FAs by laser irradiation in contact with dendrograms constructed using average linkages and Euclidean distances. 5. Venables, W.N. & Ripley, B.D. 2002. Modern Applied Statistics with S. Springer.
6. Harrington P.B. 1991. Fuzzy multivariate rulebuilding expert systems: Minimal neural networks. Journal of Chemometrics. 5: 467-486.
the oxide surface using a conventional MALDI laser. C) Generation of strain-specific FA profiles of each colony type. 7. Harrington P.B. et al. 2009. Automated principal component-based orthogonal signal correction applied to fused near infrared-mid-infrared spectra of french olive oils. Anal.
D) Automated comparison of spectra to database for rapid ID and differentiation of drug resistant isolates. Results are Chem. 81: 7160-7169.
generated in tabular form in descending rank order. E) Taxonomic classification and statistical analysis allow for 8. Harrington P.B. 2006. Statistical validation of classification and calibration models using bootstrapped Latin partitions. Trends Anal. Chem. 25: 1112-1124.
57
58
New Applications
Introduction
HL_00357
confused strains and the new group 2nd derivative 800-1300 cm-1 vector
normalization PCA (95% variance)
CV_04888.2
HL_00310
CV_00283 with very few common bands which
Fourier transform infrared
HL_00297
Summary
BR_00111
pathogen. 1 BR_00032
BR_00038
HL_00251
group 11
group 03
group 14
group 36
group 60
group 59
group 48
group 13
group 07 31%
group 11 50%
0%
95%
0%
20%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0% validation with the spectra of the
group 03 19%
group 14 0%
5%
0%
80%
0%
0%
64%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
other labs resulted in mean recall is mainly necessary to quantify
group 36 0% 0% 0% 36% 100% 0% 0% 0% 0% 0% partially confused at HL
data preprocessing:
rates of 98.7%, 96.9% and
group 60 0% 0% 0% 0% 0% 70% 0% 0% 0% 0% due to high variance:
group 59 0%
group 48 0%
0%
0%
0%
0%
0%
0%
0%
0%
30%
0%
100%
0%
0%
100%
0%
0%
0%
0%
2nd derivative 800-1300 cm-1 vector normalization BR_00045 & BR_00078 discriminatory power.
94.2%.
group 13 0% 0% 0% 0% 0% 0% 0% 0% 100% 0%
100% 100% 100% 100% 100% 100% 100% 100% 100% 100%
59
chia Streptomyces Bartonella HafniaTerrimonas Pseudoxanthomonas Corynebacterium Streptococcus Aerococcus Saccharothrix Facklamia SchizosaccharomycesTetrageno
alieria Clavibacter Shimwellia Brachybacterium Leclercia Providencia Trabulsiella Xanthobacter Emericella Gardnerella Sporothrix Leuconostoc Pseudoclavibacter Alkali
omyces Turicella Roseomonas Ruminococcus Scedosporium Dysgonomonas Staphylococcus Peptostreptococcus Paenibacillus Balneatrix Solibacillus Prototheca Cupr
cillus Aspergillus Arthrobacter Mesorhizobium Acholeplasma Filobasidium Propioniferax Azohydromonas Chromobacterium Curtobacterium Kloeckera Austwickia Hyphomic
coccus Rummeliibacillus Budvicia Aquincola Enterobacter Sporobolomyces Brevundimonas Capnocytophaga Tatlockia Neisseria Salinivibrio Pullulanibacillus Arcanoba
ella Eggerthella Methanomonas Mucor Mobiluncus Caulobacter Helcococcus Psychrobacillus Campylobacter Blastomonas Wohlfahrtiimonas Thermoactinomyces Hermini
murella Mycobacterium Bordetella Pichia Vibrio Iodobacter Tenacibaculum Listeria Plesiomonas Haloarcula Shewanella Paecilomyces Thauera Viridibacillus Yokenella Ma
phingobium Ornithobacterium Epidermophyton Oligella Paracoccus Aureobasidium Eubacterium Dietzia Salimicrobium Klebsiella Mycoplasma Variovorax Sa
phyllum Scopulariopsis Odoribacter Anaerotruncus Abiotrophia Burkholderia Sodalis Empedobacter Sphingopyxis Lactococcus Sphingobium Microsporum Pepton
occus Beauveria Morganella Pasteurella Cedecea Bifidobacterium Micrococcus Propionimicrobium Starkeya Prevotella Histophilus Sphingomonas Acetobacter Fra
acterium Propionibacterium Aneurinibacillus Arthrographis Aromatoleum Pediococcus Phoma Xenorhabdus Methylobacillus Fusarium Wolinella Bacteroides Zygosacchar
simicrobium Helicobacter Rhizobium Terrabacter Ralstonia Butyricimonas Microsporum Castellaniella Borrelia Microbacterium Rheinheimera Wautersiella Saccharopo
la Nocardioides Gluconobacter Sphingobacterium Mannheimia Cohnella Aggregatibacter Cronobacter Lecythophora Riemerella Chaetomium Atopobium Rhizopus Acid
Kytococcus Chryseobacterium Alishewanella Gemella Methylobacterium Haemophilus Adlercreutzia Buttiauxella Weeksella Alloiococcus Bacillus Arxiozyma Halococcus Rhod
ochrobactrum Leminorella Candidatus Xanthomonas Pectobacterium Brevibacterium Arthroderma Slackia Trueperella Inquilinus Brevibacillus Brachyspira Porphyro
imonas Actinomyces Eikenella Kitasatospora Magnusiomyces Psychrobacter Acidiphilium Amycolatopsis Lactobacillus Marinibacillus Megamonas Dermatophilus Gr
obacter Lysinibacillus Hanseniaspora Parvimonas Moesziomyces Legionella Aliivibrio Dermacoccus Exiguobacterium Virgibacillus Raoultella Gordonia Dialister Parabact
bacterium Stenotrophomonas Sporobolomyces Coprobacillus Sporosarcina Brenneria Rathayibacter Arsenophonus Penicillium Pseudomonas Rubrivivax Bilophila Allosc
ia Halomonas Rhodococcus Bergeyella Malikia Actinocorallia Aeromonas Alcaligenes Dickeya Kocuria Ochrobactrum Agro
bacillus Chromohalobacter
Bruker Daltonik Yersinia
GmbH Oerskovia
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Daltonics Inc.
Bruker Daltonics is continually improving its products and reserves the right
niaspora Parvimonas Moesziomyces Legionella Aliivibrio Dermacoccus Exiguobacterium Virgibacillus Raoultella Gordonia Dialister Parabacteroides Cardioba