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      Rapid identification of Burkholderia mallei and Burkholderia pseudomallei by intact cell Matrix-assisted Laser Desorption/Ionisation mass spectrometric typing

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          Abstract

          Background

          Burkholderia (B.) pseudomallei and B. mallei are genetically closely related species. B. pseudomallei causes melioidosis in humans and animals, whereas B. mallei is the causative agent of glanders in equines and rarely also in humans. Both agents have been classified by the CDC as priority category B biological agents. Rapid identification is crucial, because both agents are intrinsically resistant to many antibiotics. Matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-TOF MS) has the potential of rapid and reliable identification of pathogens, but is limited by the availability of a database containing validated reference spectra. The aim of this study was to evaluate the use of MALDI-TOF MS for the rapid and reliable identification and differentiation of B. pseudomallei and B. mallei and to build up a reliable reference database for both organisms.

          Results

          A collection of ten B. pseudomallei and seventeen B. mallei strains was used to generate a library of reference spectra. Samples of both species could be identified by MALDI-TOF MS, if a dedicated subset of the reference spectra library was used. In comparison with samples representing B. mallei, higher genetic diversity among B. pseudomallei was reflected in the higher average Eucledian distances between the mass spectra and a broader range of identification score values obtained with commercial software for the identification of microorganisms. The type strain of B. pseudomallei (ATCC 23343) was isolated decades ago and is outstanding in the spectrum-based dendrograms probably due to massive methylations as indicated by two intensive series of mass increments of 14 Da specifically and reproducibly found in the spectra of this strain.

          Conclusions

          Handling of pathogens under BSL 3 conditions is dangerous and cumbersome but can be minimized by inactivation of bacteria with ethanol, subsequent protein extraction under BSL 1 conditions and MALDI-TOF MS analysis being faster than nucleic amplification methods. Our spectra demonstrated a higher homogeneity in B. mallei than in B. pseudomallei isolates. As expected for closely related species, the identification process with MALDI Biotyper software (Bruker Daltonik GmbH, Bremen, Germany) requires the careful selection of spectra from reference strains. When a dedicated reference set is used and spectra of high quality are acquired, it is possible to distinguish both species unambiguously. The need for a careful curation of reference spectra databases is stressed.

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          Most cited references34

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          Matrix-assisted laser desorption ionization-time of flight mass spectrometry for fast and reliable identification of clinical yeast isolates.

          The clinical impact of severe infections with yeasts and yeast-like fungi has increased, especially in immunocompromised hosts. In recent years, new antifungal agents with different and partially species-specific activity patterns have become available. Therefore, rapid and reliable species identification is essential for antifungal treatment; however, conventional biochemical methods are time-consuming and require considerable expertise. We evaluated matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for the rapid routine identification of clinical yeast isolates. A total of 18 type collection strains and 267 recent clinical isolates of Candida (n = 250), Cryptococcus, Saccharomyces, Trichosporon, Geotrichum, Pichia, and Blastoschizomyces spp. were identified by MALDI-TOF MS. The results were compared with those obtained by conventional phenotyping and biochemical tests, including the API ID 32C system (bioMérieux, Nürtingen, Germany). Starting with cells from single colonies, accurate species identification by MALDI-TOF MS was achieved for 247 of the clinical isolates (92.5%). The remaining 20 isolates required complementation of the reference database with spectra for the appropriate reference strains which were obtained from type culture collections or identified by 26S rRNA gene sequencing. The absence of a suitable reference strain from the MALDI-TOF MS database was clearly indicated by log(score) values too low for the respective clinical isolates; i.e., no false-positive identifications occurred. After complementation of the database, all isolates were unambiguously identified. The established API ID 32C biochemical diagnostic system identified 244 isolates in the first round. Overall, MALDI-TOF MS proved a most rapid and reliable tool for the identification of yeasts and yeast-like fungi, with the method providing a combination of the lowest expenditure of consumables, easy interpretation of results, and a fast turnaround time.
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            Melioidosis: an important cause of pneumonia in residents of and travellers returned from endemic regions.

            Melioidosis is endemic in South East Asia, Asia and northern Australia. Infection usually follows percutaneous inoculation or inhalation of the causative bacterium, Burkholderia pseudomallei, which is present in soil and surface water in the endemic region. While 20-36% of melioidosis cases have no evident predisposing risk factor, the vast majority of fatal cases have an identified risk factor, the most important of which are diabetes, alcoholism and chronic renal disease. Half of all cases present with pneumonia, but there is great clinical diversity, from localised skin ulcers or abscesses without systemic illness to fulminant septic shock with multiple abscesses in the lungs, liver, spleen and kidneys. At least 10% of cases present with a chronic respiratory illness (sick > 2 months) mimicking tuberculosis and often with upper lobe infiltrates and/or cavities on chest radiography. As with tuberculosis, latency with reactivation decades after infection can also occur, although this is rare. Confirmation of diagnosis is by culture of B. pseudomallei from blood, sputum, throat swab or other samples. Microbiology laboratories need to be informed of the possibility of melioidosis, as those not familiar with it can misidentify the organism. Antibiotic therapy is initial intensive therapy with i.v. ceftazidime or meropenem or imipenem +/- cotrimoxazole for > or = 10 days, followed by eradication therapy with cotrimoxazole +/- doxycycline +/- chloramphenicol (first 4 weeks only) for > or = 3 months. Melioidosis has been increasingly recognised in returning travellers in Europe and recently melioidosis and colonisation with B. pseudomallei have been documented in cystic fibrosis patients visiting or resident in endemic areas.
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              Melioidosis: the tip of the iceberg?

              For nearly 80 years clinical melioidosis has been considered a rare disease. This bacterial infection is caused by Pseudomonas pseudomallei, a saprophyte found in soil and surface water of endemic areas. Consequently, those who have most contact with soil, the rural poor, are likely to be at greatest risk of infection. Since the diversity of clinical manifestations necessitates the isolation and identification of the causative organism for a definitive diagnosis of melioidosis and the population at greatest risk within endemic areas rarely have access to an appropriate level of health care, the disease has probably been underrecognized. Melioidosis is now known to be an important cause of human morbidity and mortality in Thailand, and this may be true throughout Southeast Asia, which is usually regarded as the main endemic area for the disease. In Australia, melioidosis causes a smaller number of human infections, while disease among livestock has important economic and possible public health implications. Sporadic reports of the infection indicate its presence in several other tropical regions: in the Indian subcontinent, Africa, and Central and South America. Clinical melioidosis may be highly prevalent in these areas, but underdiagnosed as a result of a lack of awareness of the clinical and microbiological features of the disease, or simply because of a lack of health care facilities. Furthermore, during the last two decades the importation and transmission of melioidosis within nontropical zones have been documented. The causative organism is not difficult to grow, and modern antibiotics have improved disease prognosis. Further studies are needed to determine the true worldwide distribution and prevalence of melioidosis so that improved therapeutic and preventive measures can be developed and applied.
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                Author and article information

                Journal
                BMC Microbiol
                BMC Microbiol
                BMC Microbiology
                BioMed Central
                1471-2180
                2012
                10 October 2012
                : 12
                : 229
                Affiliations
                [1 ]Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Molecular Biology, Südufer 10, Greifswald-Insel Riems, D-17493, Germany
                [2 ]Bundeswehr Institute of Microbiology, Neuherbergstrasse 11, Munich, D-80937, Germany
                [3 ]Working Group Biomathematics, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Südufer 10, Greifswald-Insel Riems, D-17493, Germany
                [4 ]Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Bacterial Infections and Zoonoses, Naumburger Str. 96 a, Jena, 07743, Germany
                [5 ]Bruker Daltonik GmbH, Fahrenheitstr. 4, Bremen, 28359, Germany
                Article
                1471-2180-12-229
                10.1186/1471-2180-12-229
                3534143
                23046611
                136cea48-390a-4f05-b697-e3a74dcccc57
                Copyright ©2012 Karger et al.; licensee BioMed Central Ltd.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 22 March 2012
                : 25 September 2012
                Categories
                Research Article

                Microbiology & Virology
                Microbiology & Virology

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