Microbial Diagnostic Applications of Mass Spectrometry · Microbial Diagnostic Applications of Mass Spectrometry 4th and 5th April 2012 Preface Following a decade of concerted research

Microbial Diagnostic Applications of Mass Spectrometry

Micoteca da Universidade do Minho, Braga, Portugal

ABSTRACT BOOK

London, Colindale 4th and 5th April 2012

Organised by

HPA Microbiology Services, Colindale, England

in collaboration with

Celebrating 15 years providing professional products & services to our customers

_______________________________________________________________

MUM is certified for Deposit, Preservation and Supply of Filamentous Fungi

Microbial Diagnostic Applications of Mass Spectrometry 4

th and 5

th April 2012

Preface Following a decade of concerted research by several laboratories and various mass spectrometry companies, MALDI-

TOF-MS and LC-MS/MS are now at the forefront of a new era of diagnostics in clinical microbiology.

MALDI-TOF-MS has been gaining momentum as a high throughput, low cost,

rapid and accurate diagnostic tool and its applications for direct blood cultures

are increasingly being reported. Although the emphasis has been largely on

bacterial identification, its capability to differentiate viruses, fungi, yeast and

a range of parasites are now under investigation. Consequently, a large number

of databases are being assembled in various laboratories. In manner analogous

to comparative 16S rRNA sequence analysis, it should be possible in the near

future to develop a global database that can be expanded as more laboratories

enter the field.

While the genome dictates the characteristics of a cell, the proteome is a

manifestation of its expression at a given time. Thus, pathogenic determinants

such as toxins, adhesins, antibiotic resistant proteins, signaling molecules etc.

can be elucidated using LC-MS/MS while also providing genus, species and

strain-specific signatures. Throughout Europe clinical laboratories are embracing these technologies and expanding the range of applications.

Success has been driven by continuous developments in both hardware and software, however, improvements in several areas

are in progress and this workshop aims to demonstrate some of these while highlighting some of the hurdles that needs to

be addressed.

Meeting Venue: HPA Microbiology Services

Colindale, 61 Colindale Avenue, London NW9 5EQ, England

Programme Outline: 4th April 2012 13.45 - 17.00: Session 1 Workshop on MALDI-TOF-MS and LC-MS/MS Current Platforms and New developments

17.00 - 19.00: Reception, Trade Show and Poster Session

5th April 2012 10.00 - 12.30: Session 2 Gel-Based and MS-MS Proteomics of Microbes Open presentations

12.30 - 14.00: Lunch

14.00 - 16.30: Session 3 Microbial Application of MALDI-TOF-MS Open presentations

16.30 - 17.00: General Discussion

Registration: £50. This covers refreshments, evening reception (first day) and lunch (second day).

For further information, please contact the organisers:

Professor Haroun N. Shah

Conference April 2012

Head, Molecular Identification Services Unit, DBHT

HPA Microbiology Services, Colindale

61 Colindale Avenue

London NW9 5EQ - England

Tel: (+44) 20 8327 6749

Fax: (+44) 20 8327 7870

E-mail: [email protected]

Dr. Cledir Santos

Conference April 2012

IBB/Biological Engineering Centre

Micoteca da Universidade do Minho (MUM)

Universidade do Minho - Campus de Gualtar

4710-057 Braga – Portugal

Tel: (+351) 253 604 403

Fax: (+351) 253 604 429

E-mail: [email protected]

Celebrating 15 years providing professional products & services to our customers

_______________________________________________________________

MUM is certified for Deposit, Preservation and Supply of Filamentous Fungi

Organised by

HPA Microbiology Services, Colindale, England

in collaboration with

Micoteca da Universidade do Minho, Braga, Portugal

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Microbial Diagnostic Applications of Mass Spectrometry, 4 - 5 April 2012 – HPA Microbiology Services, Colindale, London UK

APPLICATION OF MALDI-TOF MS TO THE IDENTIFICATION OF DIALYSIS CATHETERS ASSOCIATED MICROORGANISMS M. Martins1, C. Santos1, D. Machado1, M. Maciel1, N. Lima1, O. Santos2, A. Rodrigues2, A. Cabrita2, M.J. Carvalho2, H. Silva2, F. Silva2, J. Queirós2, A.M. Gomes3 and R. Oliveira1 1IBB-Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, Universidade do Minho, Campus de Gualtar, Braga, Portugal 2Nephrology Department, Centro Hospitalar do Porto - Hospital Santo António, Largo Abel Salazar, Porto, Portugal 3Department of Nephrology, Centro Hospitalar Vila Nova Gaia/Espinho, Rua Conceição Fernandes, Vila Nova de Gaia, Portugal e-mail: [email protected]

It has been estimated that 80% of the human infections are associated with biofilms. These microbial communities develop associated with surfaces, such as catheters, and contribute to the chronicity and spreading of the infections and device loss. To date, the conclusive diagnosis of catheter-associated infection relies on the removal of the device, culture of associated microorganisms and their subsequent identification. The objective of this work was to evaluate the usefulness of culturomics associated with matrix-assisted laser desorption/ionisation - time of flight mass spectrometry (MALDI-TOF MS) for the detection and identification of dialysis catheter associated microorganisms. A prospective observational study was carried out on catheters explanted from adult patients on renal replacement therapy with peritoneal dialysis (PD) and haemodialysis (HD). During a five-month period different segments of PD and HD catheters were analysed. Specifically, the samples were cultured on blood agar and the subsequent identification of the isolated microorganisms was performed using selective/differential culture media and MALDI-TOF MS. A total of 54 PD and 22 HD catheters were analysed. The frequency of microbial recovery, defined as a positive culture in at least one segment of the catheter, was found to be 44% for PD catheters and 77% for HD catheters. Polymicrobial cultures were only identified on PD catheters (20% of the positive cultures). Seventy-nine isolates were analysed by MALDI-TOF MS: 77.2% were identified at the species level, comprising eight genera and 10 species; 6.3% were identified at the genus level, comprising Corynebacterium and Sphingomonas bacteria; 16.5% were not identified. The non-identified isolates (11 from PD and 2 from HD) included gram-positive (n= 9), gram-negative (n= 2) and fungal (n= 2) species, as determined by presumptive identification. Our results show that MALDI-TOF MS is a powerful method for the identification of catheter-associated bacteria, particularly derived from HD. Acknowledgements: M.M. is grateful for financial support from Fundação para a Ciência e Tecnologia, Portugal through the grants SFRH/BPD/73663/2010 financed by POPH-QREN and co-funded by FSE and MCTES. Part of the research leading to these results received funding from the European Community’s Seventh Framework Program (FP7, 2007e2013), Research Infrastructures Action, under grant agreement No. FP7-228310 (EMbaRC project).

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VALIDATION MALDI-TOF MS FOR RAPID CLASSIFICATION AND IDENTIFICATION OF LACTIC ACID BACTERIA FOCUSING ON ISOLATES FROM TRADITIONAL FERMENTED FOOD IN NORTHERN VIETNAM

Nguyen Thi Lam D.1,, Van Hoorde K.1,2, Cnockaert M.1, De Brandt E.1, De Bruyne K.1, Maarten A.1, Le Thanh B.3,4, Vandamme P.1 1Laboratory of Microbiology, Department of Biochemistry and Microbiology, Faculty of Sciences, Ghent University, K.L. Ledeganckstraat 35, BE-9000 Ghent, Belgium 2Faculty of Applied Bioscience Engineering, University College Ghent, Valentin Vaerwyckweg 1, BE-9000 Ghent, Belgium 3Institute of Biotechnology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Hanoi, Vietnam 4Department of Biochemistry and Food Biotechnology, Faculty of Food Science and Technology, Hanoi University of Agriculture, Trauquy - Gialam, Hanoi, Vietnam.

In the present study, MALDI-TOF MS was used as a powerful, fast, reliable, and cost effective alternative for the identification of lactic acid bacteria (LAB) associated with the production of fermented foods such as fermented meats and vegetables. To determine the efficacy of this method, a total of 158 strains of LAB from Vietnamese fermented meat (Nem chua) were analyzed using both MALDI-TOF MS and repetitive element sequence-based PCR, more specifically (GTG)5-PCR fingerprinting. Cluster analysis and observation of the profiles revealed 7 unique isolates (4.4 %) using both techniques. 86 isolates (54.4 %) could be discriminated with higher discriminatory power using MALDI-TOF MS compared to (GTG)5-PCR, whereas, 65 isolates (41.2 %) were better differentiated by (GTG)5-PCR. Validation of identification was done by pheS sequence analysis of one up to three isolates of each (GTG)5-PCR cluster. As proof of concept of MALDI-TOF MS as identification tool, LAB diversity of one fermented mustard sample was analyzed using MALDI- TOF MS. PheS gene sequencing was used for validation.

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MATRIX-ASSISTED LASER DESORPTION IONIZATION-TIME OF FLIGHT MASS SPECTROMETRY (MALDI-TOF MS) OF MICROBIAL MIXTURES: FIRST IMPRESSIONS OF ITS APPLICABILITY FOR MONITORING THE EFFECT OF FOOD AND FEED ON GASTRO-INTESTINAL MICROBIOTA Van Hoorde K.1,2, Vandamme P.2, Van Landschoot A.1,2 1Faculty of Applied Bioscience Engineering, University College Ghent, Valentin Vaerwyckweg 1, BE-9000 Ghent, Belgium 2Laboratory of Microbiology, Department of Biochemistry and Microbiology, Faculty of Sciences, Ghent University, K.L. Ledeganckstraat 35, BE-9000 Ghent, Belgium2Complete address e-mail: [email protected]

The scientific community is more than ever conscious of the important role played by the gastrointestinal (GI) microbiota and its influence on the health of the host. Still, to date, our knowledge on the subject remains restricted. Not to mention the various effects food or feed might exert on the microbial composition of the GI tract. The introduction of molecular culture-independent techniques already signified an important step forwards in a better understanding of these microbiota, still a large part remains uncharted. In addition, there is also a need for applications which are less labor intensive and much faster to allow more instant analyses. A technique only just emerging in the field of microbiology is matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Its high sensitivity and high throughput made it a very interesting method for characterization and identification of large numbers of microbial isolates. The fact that a given species can be identified through unique biomarker(s) (i.e. mass peaks with a certain m/z ratio in the MS spectrum) opens up new perspectives to be able to discriminate and identify different members of a mixed microbial community. With this, important advantages are the comparative simplicity of the mass spectra for a given species and the high reproducibility of the technique. We investigated the effect of an increasing complexity of a microbial mixture on the quality of the MS profiles and as a consequence the usability of MALDI-TOF for the analysis of more complex microbial communities such as those of the GI tract. In addition, an attempt was made to determine the sensitivity of the technique. For this, mixtures of increasing complexity comprising several taxa known to be associated with the GI tract were subjected to MALDI-TOF MS analysis. In parallel, denaturing gradient gel electrophoresis (DGGE), a commonly applied approach which has proven its usefulness studying GI microbiota, was carried out. Visual and cluster analysis were performed for data interpretation. Complexity of MS profiles increased with growing numbers of species added to the mixture. However, spectral profiles remained of good quality allowing to compare mixtures of different species composition. Mixtures only differing in one species could easily be distinguished. Despite the quality profiles, visual interpretation became more difficult as complexity increased. Current software limitations could not give an answer to this problem. As a result, individual members could not always be retrieved from the community profile, explaining the need for specialized data interpretation software allowing more in-depth analyses. Sensitivity of MALDI-TOF

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MS was at least as high as for DGGE with a detection limit situated around 1%. However, actual sensitivity could be higher as this observation could also be biased by the software and visual restrictions. These preliminary results indicate possibilities for MALDI-TOF MS as a fast and high throughput application for the analysis of more complex microbial communities like encountered in the GI tract and holds certain possibilities for fast paced monitoring of the effect of food and feed, additives, pro-, pre- and synbiotics etc…

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POLYPHASIC IDENTIFICATION OF STAPHYLOCOCCUS SPECIES ISOLATED FROM RAW MILK CHEESES: MATRIX-ASSISTED LASER DESORPTION/IONIZATION-TIME OF FLIGHT MASS SPECTROMETRY AS AN ALTERNATIVE IDENTIFICATION TOOL Van Hoorde K.1,2, Peeters C.2, Vandamme P.2, Van Landschoot A.1 1Faculty of Applied Bioscience Engineering, University College Ghent, Valentin Vaerwyckweg 1, BE-9000 Ghent, Belgium 2Laboratory of Microbiology, Department of Biochemistry and Microbiology, Faculty of Sciences, Ghent University, K.L. Ledeganckstraat 35, BE-9000 Ghent, Belgium

Most species of the genus Staphylococcus are harmless. They are distributed in various environments and can be part of the ‘normal’ microbiota of skin and mucosal surfaces. Some strains are even of interest for the production of fermented food products such as dry fermented meats and certain cheese varieties and as such they are often appointed the GRAS status. However, a few species might constitute a risk for human health. Staphylococcus aureus in particular, is an omnipresent pathogen and is one of the major causes of food poisoning. In addition, over the past few years, coagulase negative staphylococci (CNS), such as Staphylococcus epidermidis en Staphylococcus saprophyticus, have been recognized as emerging pathogens, raising doubts about their ‘presumption of safety’ grade. In this context, even though CNS are predominantly nosocomial pathogens and no cases of CNS food poisoning have been reported thus far, from a food safety point of view, a fast accurate identification of food associated staphylococci is of utmost importance. A technique emerging in the field of microbiology is matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Its high sensitivity, reproducibility and high throughput make it a very interesting method for the characterization and identification of (large numbers) of microbial isolates. To date, the use of MALDI-TOF MS has largely been restricted to the identification of micro-organisms of clinical origin, as is the case for staphylococci. Its successful application in clinical environments, however, opens perspectives for the identification of food related Staphylococcus species. This study consisted of two parts: First, staphylococci retrieved from raw milk Gouda-type cheeses were identified using a polyphasic identification approach combining (GTG)5-PCR fingerprinting and sequencing of the dnaJ and/or 16S rRNA gene. In the next phase, the possibilities of MALDI-TOF MS as an identification tool for cheese related staphylococci were evaluated. A polyphasic approach consisting of (GTG)5-PCR fingerprinting and sequencing of the dnaJ gene or 16S rRNA gene was used for the identification of 217 staphylococcal isolates retrieved from mannitol salt agar (MSA). The formic acid-acetonitrile extraction procedure as developed by Bruker Daltonics was optimized for staphylococci in particular. MALDI-TOF MS analysis was performed on a 4800 Plus MALDI-TOF/TOFTM Analyzer (AB Sciex) and spectral data analysis was done using the BioNumerics software package 5.1 (Applied Maths).A total of nine different species was retrieved from MSA including Staphylococcus aureus, Staphylococcus cohnii, Staphylococcus epidermidis, Staphylococcus equorum,

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Staphylococcus haemolyticus, Staphylococcus pasteuri, Staphylococcus saprophyticus, Staphylococcus sciuri and Staphylococcus xylosus. An additional washing step at 6000 x g prior to the standard protocol whether or not in combination with ultrafiltration by centrifugation using 5 kDa MWCO Vivaspin columns (Sartorius) resulted in optimal MS spectra. Identification of the Staphylococcus isolates via MALDI-TOF MS confirmed the polyphasic identification results. The raw milk cheeses exhibited a rather high staphylococcal diversity of nine different species encompassing several species, e.g. S. aureus, S. epidermidis and S. saprophyticus, that could represent a potential health risk. This large diversity of staphylococci in the raw milk cheeses together with the awareness of a potential negative impact on the consumer’s health emphasizes the need for a fast and accurate identification tool for food related staphylococci. As was largely the case for clinical staphylococci, we were able to demonstrate that also food and more specific cheese related staphylococci could be identified using MALDI-TOF MS, and this in a much faster and also more cost-effective manner. Likewise, this could open perspectives regarding the tracking of potential contamination sources.

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IMPLEMENTING A METHODOLOGY FOR IDENTIFICATION OF Sporothrix COMPLEX ISOLATES BY MALDI-TOF ICMS M.M.E. Oliveira1,2,3, C. Santos2, P. Sampaio3, C. Pais3, N. Lima2 and R.M. Zancopé-Oliveira1 1Laboratório de Micologia, Instituto de Pesquisa Clínica Evandro Chagas, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brazil 2IBB-Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, Micoteca da Universidade do Minho, Braga, Portugal 3Molecular and Environmental Biology Centre (CBMA), Department of Biology, University of Minho, Braga, Portugal e-mail: [email protected]

Sporotrichosis is a subcutaneous mycosis worldwide distributed. However, Latin America, South Africa, India and Japan are areas of high endemicity. Recently, the combination of phenotypic and genotypic features suggested that Sporothrix schenckii should not be considered as a single taxon causing sporotrichosis once the 3 new species S. brasiliensis, S. globosa and S. mexicana have recently been described for the Science. Sporothrix mexicana was related with environmental samples and apparently restricted to Mexico. However, our Research Group has recently described the first case of human sporotrichosis caused by S. mexicana in Portugal [1]. A key identification for the Sporothrix complex species has now been proposed. It includes macro- and micro-morphology and auxonogram analyses using raffinose and sucrose as carbon sources. Nevertheless, identification based on this methodology could be inconclusive due to phenotypic variability within these species. In addition, conclusive species identification is reached only after partial calmodulin gene (CAL) sequence analysis. In order to show the potentiality of Matrix-Assisted Laser Desorption⁄Ionisation Time-Of-Flight Intact Cell Mass Spectrometry (MALDI-TOF ICMS) technique on the identification of Sporothrix complex species the aim of this study was to optimise the MALDI-TOF ICMS methodology for the 4 available Sporothrix isolates related with human sporotrichosis. For that proposal the type strain S. brasiliensis IPEC16490 (CBS 120339) and the reference strains S. globosa IPEC27135, S. schenckii IPEC27722 and S. mexicana MUM11.02 were used. Data obtained from the MALDI-TOF ICMS analyses show that the best spectral profiles and statistic clustering were obtained when the microbial cell were analysed on the yeast phase. Acknowledgement: M.M.E. Oliveira was supported by a grant from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Brazil Reference: [1] Dias, N., Oliveira, M.M.E., Portela, M., Santos, C., Zancopé-Oliveira, R.M., Lima, N. (2011) Human sporotrichosis caused by Sporothrix mexicana in a Portuguese patient. Emerging Infectious Diseases, 17:1975-1976.

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MALDI-TOF MS: IMPROVED METHODS FOR THE IDENTIFICATION/CHARACTERISATION AND AUTHENTICATION OF FUNGAL STRAINS C. Santos, P. Rodrigues, A. Venâncio and N. Lima 1IBB-Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, Micoteca da Universidade do Minho, Campus de Gualtar, Braga, Portugal 2CIMO-Mountain Research Centre, School of Agriculture of the Polytechnic Institute of Bragança, Campus Santa Apolónia, Bragança, Portugal e-mail: [email protected]

The identification of species is an important goal in taxonomic mycology. Information about each fungus (e.g. morphological description, physiological and biochemical properties, ecological roles, and societal risks or benefits) is key element in this process. Identifications can be a long and seemingly never-endeding process with frequent revisions of the taxonomic schemes. These changes make identifications even more complicated for the non-specialised researchers as each taxonomic group has specialised literature, terminology and characters. This occurs to the extent that identifications can only be undertaken by a narrow group of scientists especially skilled in the “art”, which can make the procedures appear to be subjective. Aspergillus is a large fungal genus, with a complex and ever evolving taxonomy. Section Flavi is one of the most significant sections in this genus. Taxonomy and species identification is subject of great interest for scientists aiming to clarify the species concept and limits within the section. Furthermore, this section comprises both toxigenic and non-toxigenic species/strains, with great interest to biotechnology and food industry. In the present study, from 352 isolates of Aspergillus section Flavi obtained from Portuguese almonds and identified based on morphological, biochemical and MALDI-TOF MS profiles, 24 isolates were further characterised through molecular analyses by use of ITS region and calmodulin gene. Molecular results confirm that ITS gene was not able to resolve differences at the species-level on this particular taxonomic group. In contrast, calmodulin gene was a robust and reliable genomic marker for this taxon. In conclusion, the results obtained from MALDI-TOF MS confirm that this technique is as good as calmodulin gene analysis for fungal identification. Another important output of this work was the clear evidence that two putative new species were present among these isolates. Finally, MALDI-TOF MS technique is rapid, reliable and inexpensive in terms of labour and consumables when compared with molecular techniques. At present, it adds an additional step for polyphasic identification which is essential when there is a paucity of characters for defining many fungal species.

Acknowledgements: Part of the research leading to these results received funding from the European Community’s Seventh Framework Program (FP7, 2007e2013), Research Infrastructures Action, under grant agreement No. FP7-228310.

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POLYPHASIC IDENTIFICATION OF Aspergillus ISOLATES BELONGING TO SECTION Nigri WITH CLINICAL RELEVANCE M. Maciel1,2, C. Santos2, N. Lima2 and C. Souza-Motta1

1Centre of Biological Sciences, Department of Mycology, Micoteca URM, Federal University of Pernambuco, Recife, Brazil 2IBB-Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, Micoteca da Universidade do Minho, Campus de Gualtar, Braga, Portugal e-mail: [email protected]

Aspergillosis is the name of a group of diseases in humans and animals caused by opportunist moulds of the genus Aspergillus. The vast majority of infections are caused by A. fumigatus, followed by other species such as A. flavus, A. terreus and A. niger. Among the pulmonary infection, aspergillosis is gaining prominent position not only in immunocompromised patients, but also in immunosuppressed. The absence of a reliable fungal identification system affects the control of systemic fungal infections. Matrix-Assisted Laser Desorption⁄Ionisation Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) is a spectral technique that analysis the chemical molecular mass of the microbial cellular composition providing rapid and discriminatory fingerprints for identification. This technique starts to have application in clinical laboratories.This work aimed to get a reliable identification of Aspergillus isolates from section Nigri deposited at University of Recife Mycology (URM) culture collection. These materials were used as clinical reference strains to attend the great variability (morphology, biochemical, genomics and proteomics patterns) of the Brazilian fungal population. A polyphasic approach based on morphological, biochemical and spectral analysis by MALDI-TOF MS was applied for the characterisation and identification of 74 Aspergillus isolates from section Nigri deposited at URM. In addition, 12 Aspergillus type strains belonging to section Nigri deposited at Micoteca da Universidade do Minho (MUM) culture collection were used as reference for MALDI-TOF MS studies. The data obtained from the polyphasic approach indicates that MALDI-TOF MS results corroborate with those data obtained using classical taxonomy, biochemical and molecular biology analyses. Finally, MALDI-TOF MS technique is rapid, reliable and inexpensive in terms of labour and consumables when compared with other biological techniques. At present, it adds an additional step for polyphasic identification which is essential for the clinical diagnostic. Acknowledgements: Marília Maciel was supported by a grant from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Brazil and Erasmus Mundus External Cooperation Window (EMECW) - Lot 17, Portugal.

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IDENTIFICATION OF BRAZILIAN WILD-TYPE ISOLATES OF Aspergillus fumigatus BASED ON POLYPHASIC APPROACH INCLUDING MALDI-TOF MS TECHNIQUE M. Lima1,2, C. Santos2, N. Lima2 and M.L. Polizeli1 1Laboratory of Microbiology and Cell Biology, Department of Biology, Faculty of Philosophy, Sciences and Letters of Ribeirão Preto, University of São Paulo, SP, Brazil 2 IBB-Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, Micoteca da Universidade do Minho, Campus de Gualtar, Braga, Portugal e-mail: [email protected]

Currently is accpeted that the majority of the unknown species in the world are in the tropics and Brazil possibly holds more than 25% of this biodiversity. In the filamentous fungi case the number of new species described for science in Brazil is very small when compared with the potentiality of the region. In addition, there is expected to find a large biological variability inside of described and new fungal species. Species belonging to the Aspergillus genus are responsible for invasive aspergillosis. This is gaining prominent position because of their capability to affect immunocompromised and immunosuppressed patients and aspergillosis is most commun mould infection more recognised worldwide. A better understanding about the regional fungal traits can help to a fast and effective treatment of patients with aspergillosis. In order to set up a collection containing regional fungal strains with clinical relevance to attend the great variability (morphology, biochemical, genomics and proteomics patterns) of the Brazilian fungal population 13 wild-type Aspergillus isolates from section Fumigati were studied. They were isolated from the five official Brazilian regions (North, Northeast, Centre-east, Southeast and South) and identified based on a polyphasic approach which is. based on morphological, biochemical, molecular biology and spectral analyses by MALDI-TOF MS. After collected, purified and identified all 13 Aspergilli isolates were preserved at Filamentous Fungi Collection of Ribeirão Preto (CFF-RP), São Paulo, Brazil. Data obtained from the polyphasic approach led to a final fungal identification at species level. Overall, MALDI-TOF MS was the fast technique for identify the species. However, molecular biology was the gold standard for the species identification once it was used to validate data obtained from the other techniques mentioned above. Moreover, either MALDI-TOF MS or molecular biology should not be used as a single step for fungal identification. Acknowledgements: M. Lima was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil) and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, Brazil).

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THE IMPORTANCE OF A QUICK IDENTIFICATION OF Aspergillus niger FOR A PROPER MYCOTOXIN RELATED DIAGNOSIS C. Soares1, C. Santos1, M. Maciel1,2, C. Souza-Motta,2, N. Lima1 and A. Venâncio1 1IBB-Institute for Biotechnology and Bioengineering, Universidade do Minho, Campus de Gualtar, Braga, Portugal 2Centre of Biological Sciences, Department of Mycology, Federal University of Pernambuco, Recife, Brazil e-mail: [email protected]

Food safety has become an important food quality attribute within the last decade. Moreover, consumers have a better perception about the food contamination with mycotoxins. These secondary metabolites produced by filamentous fungi can cause acute toxic, mutagenic, teratogenic and carcinogenic effects on animals and humans. Mycotoxins can appear in the food chain and are greatly resistant to decomposition or being broken down in digestion. They remain intact in the food chain in livestock and dairy products and even after temperature treatments, such as cooking and freezing. Aspergillus niger aggregate species are commonly found on soil and are pathogenic to several crops. This group of filamentous fungi is formed by a series of morphologically indistinguishable species. Aspergillus niger is one of the species in the aggregate and, apart from its economic value (it is used for industrial purposes), it is also an important mycotoxin producer, such as ochratoxin A (OTA) and, more recently, was described as fumonisin B2 (FB2) producer. Both mycotoxins were evaluated by the International Agency for Research on Cancer (IARC) as “Group 2B carcinogens”, i.e., probably carcinogenic to humans. The continued exposure to these mycotoxins can cause chronic toxicity which is characterised by low-dose exposure over a long time period, resulting in cancers and other generally irreversible effects. Hence, a proper diagnosis is important, which will allow correct treatment. Fast identification of fungi is, therefore, a must needed necessity. Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) allows rapid and reliable identification of microorganisms, being sensitive and accurate for the discrimination between species and strains of Aspergillus. This work consisted in the identification of fungal isolates belonging to the Aspergillus niger aggregate. Fungal identification was based on a polyphasic approach consisting of macro- and micro-morphology, mycotoxigenic profiles (OTA and FB2) and MALDI-TOF MS. About 250 isolates belonging to Aspergillus niger aggregate were analysed and results obtained were compared with type strains. Final results showed that all isolates were identified as Aspergillus niger, A. lacticoffeatus and A. vadensis. Acknowledgements: C. Soares was supported by a grant from Fundação para a Ciência e Tecnologia, Portugal (reference SFRH/BD/37264/2007). M. Maciel was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Brazil and Erasmus Mundus External Cooperation Window (EMECW) - Lot 17, Portugal.

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NEW INSIGHTS FOR IDENTIFICATION OF CLINICAL ISOLATES OF Trichophyton rubrum USING MALDI-TOF MS L. Pereira1, C. Santos1, N. Dias1,2 and N. Lima1 1IBB-Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, Micoteca da Universidade do Minho, Campus de Gualtar, Braga, Portugal 2CITS-Centro de Investigação em Tecnologias da Saúde, (CITS-IPSN/CESPU), Paredes, Portugal email: [email protected]

Dermatophytoses are the most common fungal infection worldwide with a non-despicable impact in health-care costs. Trichophyton rubrum is an antropophilic dermatophyte species very well adapted to human host causing chronic and slowly progressing disease on keratinised tissues. It is the causative agents of about 70% of all human dermatophytoses. Besides their distribution all over the world this species is by far, the most frequently isolated species on onychomycosis and tinea pedis. Matrix Assisted Laser Desorption Ionisation Time of Flight Mass Spectrometry (MALDI-TOF MS) analysis has already been used as a rapid technique in the identification and classification of microorganisms. It has progressively been incorporated as a technique in the polyphasic approach to improve the accuracy of the microbial identification issue. This technique has also been used as a tool for the fast identification of filamentous fungi with clinical relevance including dermatophytes. In this study twenty clinical dermatophyte isolates were analysed using a polyphasic approach that was based on macro- and micro-morphologies, biochemistry, molecular biology using ITS 1-4 sequencing data and primers M13, (GACA)4 and (AC)10 for typing and, MALDI-TOF MS analyses. Eighteen of these clinical dermatophyte isolates were collected from human nails. The remaining 2 isolates were the reference strain T. rubrum ATCC MYA-4438 that was used as positive control and the strain T. mentagrophytes ATCC MYA-4439 that was used as out-group. Preliminary results based on macro- and micro-morphologies indicated that all isolates were T. rubrum. These results were confirmed by molecular biology and MALDI-TOF MS techniques. For molecular approach 16 T. rubrum isolates were clustered in a single group with 100% of genotypic homology of the ITS1-4 region. Moreover, 2 remaining T. rubrum isolates were found having 98% of homology in this region. Similar clusters were found for M13 and (GACA)4 primers. In contrast, (AC)10 was not discriminative. MALDI-TOF MS analysis corroborates with morphological and molecular identifications. Nine T. rubrum isolates were clustered on a single group evidencing 100% similarity and the remaining T. rubrum isolates were distributed over the MALDI-TOF MS dendrogram showing phenotypic variability. MALDI-TOF MS shown to be as good as molecular biology and, moreover, rapid, low-cost and accurate alternative tool for identification and strain typing of T. rubrum clinical isolates. Analysis of mass spectra profiles provided new insights in the proteomic approach for strain typing of clinical isolates of T. rubrum. As a matter of consequence, MALDI-TOF MS can be suitable as point-of-care diagnostic for dermatophytoses. Acknowledgements: L. Pereira acknowledges the EMbaRC project grant. Part of the research leading to these results has received partial funding from the European Community’s Seventh Framework Programme (FP7, 2007-2013), Research Infrastructures action, under the grant agreement No. FP7-228310 (EMbaRC project).

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ASSESSMENT OF FUNGI STABILITY BY MALDI-TOF ICMS FOLLOWING PRESERVATION ON ALGINATE ENCAPSULATED SAMPLES

M.F. Simões, C. Santos and N. Lima IBB-Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, Micoteca da Universidade do Minho, Campus de Gualtar, Braga, Portugal e-mail: [email protected]

Alginate-encapsulation is a commonly used, simple and cost effective, method to preserve plant samples. Since alginate has proven to protect tissues against physical and environmental damage, minimising dehydration, it presents as a good preservation technique. The application of this method for the preservation of filamentous fungi was intended to present an alternative for the commonly used preservation methods, especially to be used on recalcitrant fungal strains. Matrix-assisted laser desorption⁄ionisation time-of-flight intact cell mass spectrometry (MALDI-TOF) emerged in the late 1980s as a sound technique to investigate the mass spectrometry of molecular high-mass of organic compounds through a soft ionisation of molecules resulting in minimum fragmentation. This technique has demonstrated its high potentiality for identification of filamentous fungi species and, in some specific cases, for strain identification. One of the most interesting advantages of the technique is the possibility of analysing the intact fungal cell generating peptides and proteins profiles. A novel technique was applied on the preservation of Botrytis cinerea (MUM 10.163, 10.165, 10.167), Aspergillus ibericus (MUM 04.68) and Aspergillus brasiliensis (MUM 06.181) using the methodology of alginate encapsulation, in two different conditions: distilled water (I) and a 10% glycerol solution (II), both at 4 ºC for 1 year; the viability of these strains was studied. The assessment was made by comparison with the method of Castellani preservation in water (III), using morphologic and MALDI-TOF ICMS analyses for the analysis of the 3 preservation methods. The encapsulated samples of the strains preserved in distilled water (I), presented lower viability than those preserved in 10% glycerol (II). However, when comparing the growth from the samples preserved in water (III) with the ones encapsulated and maintained in 10% glycerol (II), we noted that the last ones presented a healthier and faster growth. From the MALDI-TOF ICMS analysis, it was observed that the strains were clustered according to species identification, and for Botrytis cinerea all presented small differences (< 5%) in the percentages of similarity, except for MUM 10.167 (>5%) when preserved in water (III). From our evaluation we were able to conclude that the success of the preservation method is strain dependent. Acknowledgements: The research leading to these results has received funding from the European Community’s Seventh Framework Programme (FP7, 2007-2013), Research Infrastructures action, under the grant agreement No. FP7-228310 (EMbaRC project). M.F. Simões acknowledges FCT – Portugal for the scholarship SFRH/BD/64260/2009.

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4 - 5 April 2012HPA Microbiology Services, Colindale

n I am interested in presenting a poster

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Professor Haroun N Shah Mass Spectrometry Workshop April 2012,Head, Molecular Identification ServicesUnit, DBHTHPA Microbiology Services, Colindale61 Colindale AvenueLondon NW9 5EQTel: 020 8327 6749 Fax: 020 8327 7870E-mail: [email protected]


4 - 5 April 2012

HPA Microbiology Services, Colindalein collaboration with Micototeca da Universidade do Minho,Braga, Portugal

Dr. Cledir SantosMass Spectrometry Workshop April 2012, IBB/Biological Engineering CentreMicoteca da Universidade do Minho (MUM)Universidade do MinhoCampus de Gualtar4710-057 Braga – PortugalTel: (+351) 253 604 403Fax: (+351) 253 604 429E-mail: [email protected]

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Following a decade of concerted research by severallaboratories and various mass spectrometry companies,MALDI-TOF-MS and LC-MS/MS are now at the forefront of anew era of diagnostics in clinical microbiology.

MALDI-TOF-MS has been gaining momentum as a highthroughput, low cost, rapid and accurate diagnostic tool andits applications for direct blood cultures are increasinglybeing reported. Although the emphasis has been largely onbacterial identification, its capability to differentiate viruses,fungi, yeast and a range of parasites are now underinvestigation. Consequently, a large number of databases arebeing assembled in various laboratories. In manneranalogous to comparative 16S rRNA sequence analysis, itshould be possible in the near future to develop a globaldatabase that can be expanded as more laboratories enterthe field.

While the genome dictates the characteristics of a cell, theproteome is a manifestation of its expression at a given time.Thus, pathogenic determinants such as toxins, adhesins,antibiotic resistant proteins, signaling molecules etc can beelucidated using LC-MS/MS while also providing genus,species and strain-specific signatures.

Throughout Europe clinical laboratories are embracing thesetechnologies and expanding the range of applications.Success has been driven by continuous developments in bothhardware and software, however, improvements in severalareas are in progress and this workshop aims to demonstratesome of these while highlighting some of the hurdles thatneeds to be addressed.

VENUE: HPA Microbiology Services, Colindale,Wilson Lecture Theatre61 Colindale AvenueLondon NW9 5EQEngland.

PROGRAMME 4 April 2012

12.00 – 13.45 Registration

Session 1: Workshop on MALDI-TOF-MS andLC-MS/MSChair: Haroun N. Shah and Robert Tonge

13.45 - 14.00 Opening Remarks:

14.00 - 14.30 Advantages and Limits of MALDI-TOF for RoutineIdentification? Greub Gilbert, Institut de microbiologie, Lausanne

14.30 - 15.00 New Horizons in the Use of MALDI-TOF for the ClinicalMicrobiology LaboratoryNikos Hontzeas, bioMérieux UK Ltd

15.00 - 15.30 Investigation into Pseudomonas aeruginosa VirulenceFactors Using HDMSE and Quantitative Label-FreeProteomics Robert Tonge, Waters Corporation, Mass SpectrometryTechnology Centre, Manchester

15.30 - 16.00 Recent Developments in High Resolution and AccurateMass Analysis of Proteomic Samples Jenny Ho and Gary Woffendin, Thermo Fisher Scientific

16.00 - 16.30 General Discussion

17.00 - 19.00 Reception and Poster Session

PROGRAMME 5 April 2012

Session 2 Gel-Based and MS-MS Proteomics ofMicrobesChair: Martin Hornshaw and Raju Misra

10.00 - 10.30 Comparative Proteomics Analysis of Clostridium difficileusing Gel-Based, MALDI-TOF-MS and LC- MS-MS AnalysisCaroline Chilton, Saheer E. Gharbia and Haroun N. Shah, DBHT,HPA and Department of Microbiology, Leeds General Infirmary

10.30 - 11.00 Listeria monocytogenes; Mass Spectrometric Analysis of aFood Pathogen to Search for Mechanisms of Adaptationfrom Cold to Ambient Temperatures Martin Hornshaw and Shurene Bishop Simon, Life Science MassSpectrometry Marketing Manager and DBHT, HPA

11.00 - 11.30 Sample Extraction and Comparative MS/MS Analysisfollowing Gel-based and nano-LC Chromatography ofMicrobial Cells Min Fang, Raju Misra, Jenny Ho, Tom Gaulton and RenataCulak. DBHT, HPA

11.30 - 12.00 Evaluation of the diversity of Propionibacterium acnes:mass spectrometry versus gene sequencing Dominique Clermont, P. Bouvet, L. Motreff, K. Anger, and C. Bizet. Institut Pasteur, France

12.00 - 12.30 General Discussion

12.30 - 14.00 LUNCH

Session 3 Microbial Applications of MALDI-TOF-MS Chair: Cledir Santos and Markus Kostrzewa

14.00 - 14.30 Blood Culture Analysis by MALDI-TOF-MS Matthew Ellington, HPA, Cambridge

14.30 - 15.00 Application of MALDI TOF MS in a Routine ClinicalLaboratoryNathan Reading, City Hospital, Birmingham

15.00 - 15.30 MALDI-TOF MS for Subspecies Identification and Typing of Microorganisms Markus Kostrzewa, Bruker Daltonik GmbH, Bremen

15.30 - 16.00 MALDI-MS for fungal identification: how far from clinicalapplications? Arnaud Riat, Stephane. P. Emonet and Jacques Schrenzel, University Hospital, Geneva

16.00 - 16.30 MALDI-TOF MS: Improved methods for the identification/characterisation and authentication of fungal strainsCledir Santos and Nelson Lima, Micototeca da Universidade doMinho, Portugal

Documents

Microbial Diagnostic Applications of Mass Spectrometry · Microbial Diagnostic Applications of Mass Spectrometry 4th and 5th April 2012 Preface Following a decade of concerted research