RUO

Innovation with IntegrityMALDI-TOF

Changing MicrobiologyFor research use only. Not for use in clinical diagnostic procedures.

MALDI Biotyper®

Subtyping Module

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Geotrichum Fusobacterium Lodderomyces Anaerococcus Colletotrichum Edwardsiella Comamonas Pigmentiphaga Moellerella Nesterenkonia Listonella Udeniomyces Erysipelothrix Escherichia Streptomyces Bartonella Hafnia Terrimonas Pseudoxanthomonas Corynebacterium Streptococcus Aerococcus Saccharothrix Facklamia Schizosaccharomyces Tetragenococcus Lechevalieria Clavibacter Shimwellia Brachybacterium Leclercia Providencia Trabulsiella Xanthobacter Emericella Gardnerella Sporothrix Leuconostoc Pseudoclavibacter Alkalibacillus Debaryomyces Turicella Roseomonas Ruminococcus Scedosporium Dysgonomonas Staphylococcus Peptostreptococcus Paenibacillus Balneatrix Solibacillus Prototheca Cupriavidus Geobacillus Aspergillus Arthrobacter Mesorhizobium Acholeplasma Filobasidium Propioniferax Azohydromonas Chromobacterium Curtobacterium Kloeckera Austwickia Hyphomicrobium Cryptococcus Rummeliibacillus Budvicia Aquincola Enterobacter Sporobolomyces Brevundimonas Capnocytophaga Tatlockia Neisseria Salinivibrio Pullulanibacillus Arcanobacterium Tissierella Eggerthella Methanomonas Mucor Mobiluncus Caulobacter Helcococcus Psychrobacillus Campylobacter Blastomonas Wohlfahrtiimonas Thermoactinomyces Herminiimonas Tsukamurella Mycobacterium Bordetella Pichia Vibrio Iodobacter Tenacibaculum Listeria Plesiomonas Haloarcula Shewanella Paecilomyces Thauera Viridibacillus Yokenella Malassezia Novosphingobium Ornithobacterium Epidermophyton Oligella Paracoccus Aureobasidium Eubacterium Dietzia Salimicrobium Klebsiella Mycoplasma Variovorax Samsonia Schizophyllum Scopulariopsis Odoribacter Anaerotruncus Abiotrophia Burkholderia Sodalis Empedobacter Sphingopyxis Lactococcus Sphingobium Microsporum Peptoniphilus Vagococcus Beauveria Morganella Pasteurella Cedecea Bifidobacterium Micrococcus Propionimicrobium Starkeya Prevotella Histophilus Sphingomonas Acetobacter Francisella Photobacterium Propionibacterium Aneurinibacillus Arthrographis Aromatoleum Pediococcus Phoma Xenorhabdus Methylobacillus Fusarium Wolinella Bacteroides Zygosaccharomyces Cellulosimicrobium Helicobacter Rhizobium Terrabacter Ralstonia Butyricimonas Microsporum Castellaniella Borrelia Microbacterium Rheinheimera Wautersiella Saccharopolyspora Rahnella Nocardioides Gluconobacter Sphingobacterium Mannheimia Cohnella Aggregatibacter Cronobacter Lecythophora Riemerella Chaetomium Atopobium Rhizopus Acidovorax Rothia Kytococcus Chryseobacterium Alishewanella Gemella Methylobacterium Haemophilus Adlercreutzia Buttiauxella Weeksella Alloiococcus Bacillus Arxiozyma Halococcus Rhodotorula Pseudochrobactrum Leminorella Candidatus Xanthomonas Pectobacterium Brevibacterium Arthroderma Slackia Trueperella Inquilinus Brevibacillus Brachyspira Porphyromonas Aurantimonas Actinomyces Eikenella Kitasatospora Magnusiomyces Psychrobacter Acidiphilium Amycolatopsis Lactobacillus Marinibacillus Megamonas Dermatophilus Grimontia Acinetobacter Lysinibacillus Hanseniaspora Parvimonas Moesziomyces Legionella Aliivibrio Dermacoccus Exiguobacterium Virgibacillus Raoultella Gordonia Dialister Parabacteroides Cardiobacterium Stenotrophomonas Sporobolomyces Coprobacillus Sporosarcina Brenneria Rathayibacter Arsenophonus Penicillium Pseudomonas Rubrivivax Bilophila Alloscardovia Nocardia Halomonas Rhodococcus Bergeyella Malikia Actinocorallia Aeromonas Micromonospora Alcaligenes Alistipes Pannonibacter Dickeya Kocuria Ochrobactrum Agrococcus Gracilibacillus Chromohalobacter Yersinia Oerskovia Gallibacterium Erwinia Agromyces Filifactor Devosia Pragia Massilia Collinsella Finegoldia Phenylobacterium Methyloarcula Jonesia Pantoea Elizabethkingia Leifsonia Pseudozyma Streptosporangium Macrococcus Veillonella Sporolactobacillus Moraxella Clostridium Pandoraea Flavobacterium Halobacterium Taylorella Delftia Sinomonas Carnobacterium Myroides Exophiala Sporopachydermia Nocardiopsis Avibacterium Blautia Salmonella Weissella Herbaspirillum Ideonella Kingella Kluyvera Enterococcus Janthinobacterium Kerstersia Luteibacter Photorhabdus Proteus Arcobacter Actinobaculum Alternaria Citrobacter Dichelobacter Achromobacter Candida Ewingella Trichophyton Granulicatella Leptothrix Suttonella Dermabacter Hydrogenophaga Cellulomonas Azoarcus Trichosporon Tatumella Rhodospiridium Acidaminococcus Actinobacillus Serratia Halomonas Rhodococcus Bergeyella Malikia Actinocorallia Aeromonas Micromonospora Alcaligenes Alistipes Pannonibacter Dickeya Kocuria Ochrobactrum Agrococcus Gracilibacillus Chromohalobacter Yersinia Oerskovia Gallibacterium Erwinia Agromyces Filifactor Devosia Pragia Massilia Collinsella Finegoldia Phenylobacterium Methyloarcula Jonesia Pantoea Elizabethkingia Leifsonia Pseudozyma Streptosporangium Macrococcus Veillonella Sporolactobacillus Moraxella Clostridium Pandoraea Flavobacterium Halobacterium Taylorella Delftia Sinomonas Carnobacterium Myroides Exophiala Sporopachydermia Nocardiopsis Avibacterium Blautia Salmonella Weissella Herbaspirillum Ideonella Kingella Kluyvera Enterococcus Janthinobacterium Kerstersia Luteibacter Photorhabdus Proteus Arcobacter Actinobaculum Alternaria Citrobacter Dichelobacter Achromobacter Candida Ewingella Trichophyton Granulicatella Leptothrix Suttonella Dermabacter Hydrogenophaga Cellulomonas Azoarcus Trichosporon Tatumella Rhodospiridium Acidaminococcus Actinobacillus Serratia Sphingobacterium Mannheimia Cohnella Aggregatibacter Cronobacter Lecythophora Riemerella Chaetomium

Fast Microorganism Identification Combined with Instant Typing

Over the past decade, the implementation of Bruker’s MALDI Biotyper in many microbiology labs worldwide has entirely changed microorganism identification. Its high discriminatory power permits the identification of thousands of different species, but some genera are still difficult to differentiate. Bruker has therefore developed the MBT Subtyping Module, allowing for automated differentiation of some species which are typically very difficult to distinguish.

And there’s more - the potential of MALDI-TOF massspectrometry reaches beyond species identificationand the MBT Subtyping Module is the first applicationexploring this potential. It combines the identificationof important pathogens with automated subsequentdetection of specific resistances in one automatedworkflow.

The principle

A prerequisite for the automated typing process is a highconfidence identification of the bacterium in the MALDIBiotyper workflow. For species differentiation, the MBTSubtyping Module then looks for decisive peaks in theidentified mass spectrum. For detection of specific resistances, the software searches for peaks associated with proteins related to antibiotic resistance and, if present, reports the respective bacterium as presumptive resistant.

Seamless and Fast Workflow

Once the samples are directly transferred from the agar to the MALDI target plates, no additional sample preparation is required to obtain a typing result. When the bacterium has been identified, the software automatically performs the typing and result reporting.

Transfer sample directly and add matrix*

Final review and validation

* Only the direct transfer method should be used for resistance marker detection. Preparation procedures including extraction / washing steps risk removal of surface bound peptides / proteins.

Select anisolated colony

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Create project and start measurement

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Automated typing process after high confi dence identification

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Spectrum instantly matched against reference library to perform identification

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Antibiotic resistant bacteria are on the rise and are a major global public health threat. Effective prevention and control are therefore of high importance to reduce the risk of infections associ-ated with antibiotic resistant microorganisms. The MBT Subtyping Module enables fast detection of specific resistances in an automated workflow.

Antibiotic resistant bacteria are troublesome in hospitals, nursing homes and other facilities where immunocompromised patients with open wounds or invasive devices are at great risk of nosocomial infections.

The rise of antimicrobial resistant strains of bacteria is also observed in livestock. Surveillance and epidemiological studies are currently of major concern in food animal production, particularly in dairy cattle.

In many countries a significant increase of carbapenem resistant K. pneumoniae (CRKP) is observed, which is a major concern as infections result in high rates of morbidity and mortality. The most important mechanism of resistance by CRKP is the production of the K. pneumoniae carbapenemase enzyme (KPC), encoded by the blaKPC gene. This plasmid encoded resistance can easily be exchanged between bacteria by horizon-tal gene transfer, making it even more dangerous in healthcare settings.

This mechanism and the fact that CRKP resis-tance could spread rapidly between patients - if not detected in time - demand efficient identification methods for laboratory testing, active surveillance and screening of patients.

Lau et al. (2014) discovered a peak in MALDI-TOF mass spectra of KPC-producing K. pneumoniae, related to the plasmid carrying blaKPC. This specific peak at 11,109 m/z is clearly detectable in bacterial MALDI-TOF mass spectra.

Prerequisite for the automated detection pro-cess with the MALDI Biotyper is the successful identification of the bacterium, i.e. the log(score) ID value must be ≥ 2.0. The MBT Subtyping Module then looks for the blaKPC related peak in the sample spectrum. And, if present, the software will report this sample as a presumptive blaKPC positive one.

Instant Resistance Marker Detection with MALDI Biotyper

Detection of KPC-producing Enterobacteriaceae

Anna F. Lau et al., Journal of Clinical Microbiology 2014:52(8):2804-2812. DOI: 10.1128/JCM.00694-14

Spectra of Klebsiella pneumoniae

ESBL (negative control)

m/z

Example of an identification run of Enterobacteriaceae, with display of the result of the detection of the blaKPC related peak in the Subtype column.

Results can easily be exported in a structured report, see details for positions A1 to A5.

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In addition to the already previously implemented automated detection of the blaKPC related peak in KPC-producing Klebsiella pneumoniae, the automated check for presump-tive presence of KPC has been extended to the following species: Citrobacter freundii Enterobacter aerogenes Enterobacter asburiae Enterobacter cloacae Enterobacter kobei Enterobacter ludwigii Escherichia coli Klebsiella aerogenes Klebsiella oxytoca Klebsiella pneumoniae Klebsiella variicola Serratia marcescens

with blaKPC related peak

Result report for Staph. aureus identification with indication of the presence of the PSM-mec peptide. Note that there is no indication for the ID with score value < 2.00.

Staphyloccocus aureus

MRSA Subtyping

MRSA (Methicillin-Resistant Staphylococcus aureus) strains are genetically distinct from other strains of Staphylococcus aureus, and show multiple drug resistance to beta-lactam antibiotics.

As with the Enterobacteriaceae, successful iden-tification of Staph. aureus is the prerequisite for the subsequent resistance subtyping process. For Staph. aureus the module looks for identifica-tion of the PSM-mec peak. PSM-mec is a sur-face peptide and the proportion of MRSA with

PSM-mec is variable, depending on regionalepidemiology. Absence of this peak does notmean that the respective strain is not an MRSA,but, if detected, it can be reliably considered asan MRSA positivity alert.

Identification results for B. fragilis with indication of cfiA status for the best match

Bacteroides fragilis cfiA Subtyping

An Early Warning System

B. fragilis is the most frequently isolated anaerobic pathogen. Carbapenem resistance in B. fragilis is frequently associated with presence of the cfiA gene encoding for a metallo-beta-lactamase conferring resistance to nearly all beta-lactam antibiotics. As a result, infections with B. fragilis cfiA positive strains are difficult to treat.

After successful identification, the MBT Subtyp-ing Module looks for specific peaks associatedwith the protein expressed by B. fragiliscfiA positive strains. If present, the best matchfor the respective sample will be reported as apresumptive cfiA positive strain.

The MBT Subtyping Module quickly detects blaKPC expressing Enterobacteriaceae, PSM-mec car-rying MRSA and cfiA positive B. fragilis strains. Please note that negative results do not necessarily mean that these strains are susceptible but will require additional confirmation methods.

Subtyping B. fragilis result table

Mycobacterium chimaera very rarely causes infections in humans, but there have been recent reports about contamination of medical equip-ment used in heart surgery. There is a risk that heater-cooler units used in open-heart surgery may be contaminated with M. chimaera and that exposure of patients to these units in the operating theatre might lead to infections that can appear months to years after surgery. Most reported infections are those of prosthetic valves or vascular grafts.

Routine identification using MALDI-TOF mass spectrometry is restricted to the identification of the M. chimaera/intracellulare complex because mass spectra of both species are very similar.

Conventional identification methods suffer from the same limitation.

After successful identification of the M. chimaera/intracellulare complex by the MALDI Biotyper, application of the MBT Subtyping Module allows - for the first time - fast and accurate differentiation of both species by thorough comparison of characteristic mass spectrum peaks as described by Pranada et al. (2017). This differentiation will support further insights into the pathogenic role of M. chimaera and can contribute to epidemiological studies which might improve infection control in future.

Note: Prerequisite for the differentiation is the installed MBT Mycobacteria library version 4.0 or higher.

Accurate Differentiation of Mycobacterium chimaera from Mycobacterium intracellulare

Result report of M. chimaera / intracellulare complex differentiation

Arthur B. Pranada et al., J Med Microbiol 2017:66:670-677. DOI: https://dx.doi.org/10.1099/jmm.0.000469

The genus Elizabethkingia covers mainly three very difficult to distinguish species: E. meningoseptica, E. miricola and E. anophelis, the latter has been described in 2011 only. E. anophelis is an emerging pathogen for which outbreaks have been reported over the last years. As the antimicrobial susceptibility of Elizabethkingia may vary depending on the species, identification to species level is desirable.

In addition to recent improvements of the MALDI Biotyper reference library, the differentiation of the three known Elizabethkingia species has been further strengthened by another application of the MBT Subtyping Module.After successful identification of one of the Elizabethkingia species, the MBT Subtyping Module is automatically activated to start searching for characteristic mass spectrum peaks and will generate a species output.

Elizabethkingia Species Differentiation

Example of an identification run of Elizabethkingia species, with the species differentiation result listed in the Subtype column.

Result report of Elizabethkingia species differentiation

Worldwide, Listeria monocytogenes is a very important foodborne pathogen causing potentially fatallisteriosis. Particularly pregnant women and their newborns, adults aged 65 or older, and people withweakened immune systems are affected by this life-threatening infection.

Listeria spp. are widespread in the environment. Clear differentiation of the pathogenic L. monocytogenes from other Listeria species is of great importance for food business operators and risk managers.

Facilitating Listeria monocytogenes Differentiation

Listeria identification is challenging because of close species relationships on genetic and pro-teomic levels. Bruker is continuously updating the MALDI Biotyper Reference Library with the taxo-nomical evolution and the addition of new spe-cies. Additionally, differentiation is now as easy as it can be by implementing the MBT Subtyping Module into the identification workflow.

With the MBT Subtyping Module comparison of the sample mass spectrum with the Listeria reference spectra is performed by sophisticated bioinformatics. This allows easy-to-apply and fast direct sample transfer methods instead of more labor-intensive workflows.

This enables e.g. food QC laboratories to imple-ment testing on L. monocytogenes in the daily routine workflow directly from culture with mini-mal effort.

The MBT Subtyping Module is an essential part of the bioinformatics of • the AOAC-OMA #2017.10 method for the confirmation and identification of Listeria monocytogenes and Listeria spp., and other gram-positive organisms• the ISO 16140-6 method validated by MicroVal for the confirmation of Listeria spp. and Listeria monocytogenes from various agar plates (Certificate 2017LR75)

Order Information

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Bruker Daltonik GmbH

Bremen · GermanyPhone +49 (0) 421-2205-0

Bruker Scientific LLC

Billerica, MA · USA Phone +1 (978) 663-3660

ms.sales.bdal@bruker.com

www.bruker.com/microbiology

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MALDI Biotyper® is a registered trademark of Bruker Daltonik GmbH in the European Union, Japan and the USA.

Part-No. 1842250The MBT Subtyping Module enables the automated detection of strain specific characteristics.

MBT Compass software (Part-No. 1843241) is prerequisite for the use of the MBT Subtyping Module.

Differentiation of Mycobacterium chimaera from M. intracellulare needs an installed MBT Mycobacteria library version 4.0 or higher.