| 1. |
- Moore, Edward R.B. 1954, et al.
(författare)
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Applications of MALDI-TOF and SARAMIS analyses for the identification of closely related clinically-relevant bacterial species
- 2009
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Ingår i: Proceedings of the 12th Conference in Genomics and Proteomics of Human Pathogens: Target molecules and biomarkers in the characterisation of microbes in disease and the environment, June 25-26, London, UK.
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Konferensbidrag (refereegranskat)abstract
- Identification of prokaryotes in the complexity of microbial diversity is increasingly problematic for clinical diagnoses. Among the most difficult problems for clinical diagnostics is the identification of bacteria within “complexes” of closely related species, comprising pathogenic and non-pathogenic species with limited differentiating characteristics. However, these species complexes are composed of micro-organisms with different virulence potentials and it is essential to be able to obtain and confirm reliable identifications. A “polyphasic” multi-locus sequence analysis (MLSA) strategy may be applied for the typing and identification (species- and sub-species-levels) of bacteria, including “first-phase” comparisons of partial 16S rRNA gene sequences and subsequent “second-phase” analyses of one or more conserved house-keeping genes, for identification to the species level. These genotypic data are being used to correlate with the data obtained by MALDI-TOF (matrix-assisted laser desorption-ionisation time-of-flight) IC-MS (intact-cell mass-spectrometry) and analysed with the SARAMIS (Spectral Archive And Microbial Identification System) software and database package. The high reproducibility, rapid speed and low cost of MALDI-TOF IC-MS analyses makes the methodology ideal for processing large numbers of microbiological samples sent to the clinical lab for identification. The question of the resolution able to be obtained, using MALDI-TOF IC-MS was addressed by focusing analyses on bacteria comprising clinically-relevant species-complexes. The SARAMIS analysis of the MALDI-TOF IC-MS data was observed to be able to compare spectra from bacteria, yeast and fungi with reference spectra maintained in a database, identify the most closely related bacterial species with statistically-relevant confidence values and type strains to sub-species levels. The analyses are applicable to nearly all micro-organisms with high reproducibility.
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| 2. |
- Moore, Edward R.B. 1954, et al.
(författare)
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Polyphasic phenotypic and genotypic analyses for diagnosing closely related clinically-relevant bacteria
- 2007
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Ingår i: Proceedings of the XXIX Congreso Chileno de Microbología, December 3-5, Viña del Mar, Chile. ; , s. 44-45
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Konferensbidrag (refereegranskat)abstract
- The majority of known pathogenic bacteria have been recognised for more than a century. However, reliable identification of specific pathogens within the increasing complexity of bacterial diversity is becoming more problematic for clinical diagnoses. DNA sequencing and genotyping of bacteria has helped enable recognition of the extensive diversity of microorganisms in the environment and the same approaches are more recently being adopted in clinical microbiology. Genotypic methods are especially suited to the analyses of the “difficult-to-cultivate” organisms, as well as bacteria that pose significant health risks during cultivation. Traditional phenotyping, as well as genotyping by 16S rRNA gene sequence analysis, in many cases are able to provide only good estimations of identifications, although they may be able to provide information on what an isolate is not. In some cases of clinical diagnoses, such information may be useful and adequate. However, such analyses are often not able to provide definitive identifications. Among the most difficult problem for diagnoses in clinical cases, is the identification of organisms within “complexes” of closely related species, e.g., the Burkholderia cepacia “complex”, the Mycobacterium tuberculosis “complex”, the Streptococcus mitis “complex”, etc. These, so called “complexes” are comprised of closely related pathogenic and non-pathogenic species or genomovars, with limited phenotypic and genotypic differentiating characteristics, e.g., the 16S rRNA gene sequence differences among such organisms is less than 1.0%. Because these species “complexes” are comprised of organisms with different pathogenic and virulence potential and different treatment regimen, it is essential to be able to provide reliable identifications to the clinicians. Genotyping of bacteria provides the means for detailed, high-resolution differentiation and identification of organisms, as well as epidemiological monitoring. For identification of clinical isolates, a “multi-locus sequence analysis (MLSA)”, is used, including “first-phasic” comparisons of partial 16S rRNA gene sequences, for identification to the sub-genus level, and subsequent analyses of one or more “house-keeping” genes, for identification to the species level. However, not all house-keeping genes are equally useful for all taxa. Thus, the key to effective identifications, with respect to the speed and cost of analyses, and the resolution and reproducibility of identifications, depends upon the selection of the most applicable house-keeping genes, i.e., those with adequately high levels of resolution among the most closely related species. Protocols and results for species-resolving MLSA, using “house-keeping” genes enable effective differentiation of clinically-relevant closely related species of respective genera of Burkholderia, Mycobacterium, Streptococcus and Staphylococcus, as well as Pseudomonas and Stenotrophomonas. Furthermore, Matrix-Assisted Laser Desorption Ionization Time-of-Flight mass-spectrometry (MALDI-TOF MS), in combination with Spectral Archive And Microbial Identification System (SARAMIS) software analyses, were able to further define species-level differentiation among the most closely-related members of species “complexes”. MALDI-TOF MS, generating cell protein profiles, may exhibit even higher levels of resolution than the most discriminating genotypic analyses for species, as well as strain, identifications. These phenotypic data, as well as traditional phenotypic data, and genotypic MLSA data are correlated in the polyphasic assessment and definition of selected species “complexes”.
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| 3. |
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| 4. |
- Svensson, Liselott A, et al.
(författare)
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New genotypic and phenotypic analyses of clinically-relevant Gram-negative, non-fermenting bacteria: MALDI-TOF MS as a rapid, high-resolution method for identifying and typing microorganisms
- 2010
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Ingår i: 20th European Congress of Clinical Microbiology and Infectious Diseases (ECCMID), Vienna, Austria.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Objectives. Identification of Gram-negative, non-fermenting bacilli, using phenotypic characterization is problematic. Many of the species of this group are frequent nosocomial infectious agents and are ubiquitous in the environment. The aims of this study were to assess the resolving capacities of “house-keeping” gene sequences, including 16S rRNA, atpD, gyrB, recA, rpoB and rpoD, and to compare a multi-locus sequence analysis (MLSA) with matrix-assisted laser desorption time-of-flight (MALDI-TOF) mass spectrometry analyses for identifying and typing strains of Achromobacter, Bordetella, Burkholderia, Pseudomonas, and Stenotrophomonas species. Methods. Genotypic analyses. Type strains of the focus genera and species-complexes, other well-characterised reference strains and selected clinically-relevant strains representing a range of phenotypic and genotypic similarities were included in this study. Partial genes, 16S rRNA, atpD, recA, gyrB and rpoD recA were amplified by PCR and sequenced. DNA-DNA hybridisation analyses were carried out on selected strains for confirmation of species designations. MALDI-TOF analysis. Bacterial biomass were prepared from cultures on agar medium and analysed by MALDI-TOF MS, in the positive mode, using the SARAMIS software for analysis (1) Results. MLSA, using the respective house keeping genes were able to differentiate and identify the most closely related species of the analysed taxa and cluster analyses showed similarities of branching order between species that correlated well between different genes. However, different genes were not equally effective in differentiating species of the different genera. The MALDI-TOF analyses were effective in differentiating the most closely related species of the respective genera. Good correlation was observed between the results of MALDI-TOF MS and MLSA data. Conclusion. In most cases, clinically-relevant isolates and strains of Gram negative, non-fermenting bacilli exhibited good agreement between the methods of this study. In some cases, strains previously defined as given species were observed to be genotypically more similar to other species, as well as some strains with highly aberrant phenotypes were almost genotypically identical to the type strain. MALDI-TOF identification was very well correlated to the MLSA results, and is a much less expensive and effectively able to reduce identification times by 24-48 hours. (1) Vanlaere E et al. J. Microbiol. Meth. 75: 279-286 (2008).
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| 5. |
- Svensson, Liselott A, et al.
(författare)
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Stenotrophomonas species differentiation by MLSA of gyrB and rpoD and identification of isolates from clinical and environmental samples
- 2009
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Ingår i: FEMS 2009 - 3rd Congress of European Microbiologists.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Background: Stenotrophomonas species are commonly observed in clinical and environmental samples. S. maltophilia is associated with respiratory infections, is recognized as the third most common nosocomial Gram-negative, non-fermenting bacterium and is isolated from a range of environmental ecosystems, often exhibiting biodegradation and other biotechnological potential. Identification of Stenotrophomonas spp. is famously problematic, due to lack of phenotypic markers, as well as limited 16S rRNA gene sequence differentiation. Objectives: The aim of this study was to determine the applicability of “house-keeping” genes gyrB and rpoD as biomarkers for species-level differentiation within Stenotrophomonas. Methods: Selected regions from gyrB and rpoD of Stenotrophomonas spp., including all type strains, were amplified by PCR and sequenced, for comparative analyses. Results: Comparative sequence analyses of targeted regions of gyrB and rpoD enabled effective resolution of all species in Stenotrophomonas. Dissimilarities between the sequences of the type strains of S. maltophilia and other Stenotrophomonas spp. ranged from 10.2 – 14.7 % (gyrB) and 13.0 – 21.7 % (rpoD). Inter-species sequence dissimilarities of gyrB and rpoD ranged from 7.2 % --18.0 % and from 5.5 % - 22.8%, respectively. Compared with Stenotrophomonas spp. 16S rRNA gene sequence dissimilarities, (0.9 – 3.1 %), both house keeping genes exhibit better capacity for reliably differentiating Stenotrophomonas species. Clinical isolates identified as S. maltophilia were analysed by gyrB and rpoD sequence analyses. These data elucidated high levels of sequence dissimilarities among isolates, bringing into question the identifications determined by traditional methods, as well as the resolution of the taxonomy of Stenotrophomonas. These questions are being addressed in more detail. Conclusions: Analyses of house-keeping genes gyrB and rpoD exhibited effective species-level differentiation for Stenotrophomonas, which can be applied, in combination with 16S rRNA gene sequence analyses to provide an MLSA strategy for genotypic analyses for reliable identifications of isolates from clinical and environmental samples.
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| 6. |
- Mihaylova, Sashka A, et al.
(författare)
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Analyses of clinically-relevant isolates of Stenotrophomonas spp.
- 2007
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Ingår i: Proceedings of the 26th Annual General Meeting of the European Culture Collections’ Organization; A0264.
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Konferensbidrag (refereegranskat)abstract
- Objectives: The identification of clinical isolates of Stenotrophomonas spp. to the species level, using a polyphasic approach of phenotypic and genotypic methods, and the determination and comparison of the in vitro responses to various classes of antimicrobial agents by these isolates. Previous studies have suggested a marked diversity among clinical, as well as environmental, isolates of Stenotrophomonas spp. and they represent a significant threat within hospital settings, particularly for patients in ICUs and other immunocompromised conditions. Isolates characterised as putative Stenotrophomonas spp. from Pleven University Hospital were analysed in detail. The predominant source of the isolates was respiratory tract and two thirds of patients were admitted to ICUs. Isolates were examined, using the VITEK 2 compact system, CCUG phenotypic characterisation panels and sequencing and analyses of the genes for 16S rRNA and gyrB. The in vitro responses of isolates to 10 antibiotics (azlocillin, piperacillin, ceftazidime, imipenem, meropenem, ampicillin-sulbactam, piperacillin-tazobactam, gentamicin, amikacin and ciprofloxacin) were evaluated, using the disc diffusion method, according to guidelines of the Clinical and Laboratory Standards Institute. The commercial VITEK 2 system defined all isolates as S. maltophilia, with probabilities of identifications varying from 92 to 99%, and “good”, “very good” or “excellent” confidence. Results from conventional phenotypic testing confirmed identifications for isolates as S. maltophilia or S. rhizophila. DNA sequencing and analyses further defined the species affiliations of isolates of the genus Stenotrophomonas. Previous reports have indicated that Stenotrophomonas spp. exhibit resistance to a broad range of antibiotics. More than 90% of examined isolates were observed to be resistant to imipenem, meropenem and ampicillin-sulbactam, more than 80% were resistant to azlocillin, piperacillin and piperacillin-tazobactam. The only beta-lactam antibiotic with an indication of better activity was ceftazidime. Among the ten antimicrobial agents tested, ciprofloxacin demonstrated the highest activity, as evaluated by diameter zone of growth inhibition. Conclusions: The VITEK 2 compact system identified all isolates to the genus level. However, conventional phenotypic testing may be more effective for species differentiation. Analysis of DNA gene sequencing allows further precise taxonomic identification. Ciprofloxacin could be a drug of choice for therapy of infections caused by Stenotrophomonas spp.
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| 7. |
- Mihaylova, Sashka A, et al.
(författare)
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Emergence of clinical strains of Stenotrophomonas maltophilia resistant to trimethoprim/sulfamethoxazole in a Bulgarian university hospital
- 2008
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Ingår i: Journal of Biomedical and Clinical Research. - 1313-9053. ; 1, s. 18-25
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Tidskriftsartikel (refereegranskat)abstract
- Clinical strains of Stenotrophomonas maltophilia, identified by a commercial system as resistant to trimethoprim/sulfamethoxazole (TMP/SMX), were analysed in detail, to confirm their taxonomic positions, to determine their susceptibilities to various classes of antibiotics and to assess this information with respect to the epidemiological relevance. The majority of strains were isolated from respiratory and wound specimens from patients admitted to intensive care units. Multi-locus sequence analyses (MLSA) of 16S ribosomal RNA (rRNA) and gyrase subunit B (gyrB) genes were applied for genotypic-based characterisation. The minimal inhibitory concentrations (MICs) of ten antimicrobial agents were determined, using the E-test method. The MIC values of TMP/SMX for the clinical isolates of S. maltophilia were greater than 32 mg/L, which confirmed their preliminary assessment as resistant. Minocycline, levofloxacin and ciprofloxacin exhibited the lowest MICs. All strains were observed to be susceptible to minocycline and levofloxacin. The emergence of clinical strains of S. maltophilia resistant to TMP/SMX is increasingly problematic as this antimicrobial agent is accepted as the “drug of choice” for treating infections caused by this bacterium. However, minocycline and levofloxacin demonstrated excellent in vitro activities and could be considered as alternative options to counter TMP/SMX-resistant strains of S. maltophilia.
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| 8. |
- Mihaylova, Sashka A, et al.
(författare)
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In vitro activity of antimicrobial agents against extended-spectrum-beta-lactamase-producing enteric bacteria isolated from urine specimens
- 2009
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Ingår i: FEMS 2009 - 3rd Congress of European Microbiologists.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Objectives: To apply detailed phenotypic and genotypic analyses on extended-spectrum-beta-lactamase (ESBL)-producing clinical isolates. To determine in vitro activities of 14 antimicrobial agents and access these data with respect to treatment choice. Methods: One hundred strains were collected from urine samples of patients from Pleven University Hospital, Bulgaria, from January to December 2008. Patient ages: one month to 85 years. Two thirds were male. At the time of sampling, 50 patients were in Urology department, 23 were in Nephrology department, 8 were in Pediatric department, and 19 were in other departments. Double disc and Combination disc tests were applied for ESBL confirmation. ESBL-types were determined, using a multiplex PCR assay for CTX, TEX, SHV and OXA types. In vitro activities of fosfomycin, imipenem, meropenem, ertapenem, amoxicillin/clavulanic acid, piperacillin/tazobactam, gentamicin, amikacin, tigecycline, colistin, nalidixic acid, nitrofurantoin, ciprofloxacin and levofloxacin were determined by the disk diffusion method, according to guidelines of the CLSI. Results: All strains were confirmed to be members of the Enterobacteriaceae and to be ESBL producers. All strains were susceptible to the three carbapenems tested. Eighty-eight percent of the strains were susceptible to tigecycline, 74% were susceptible to amikacin, 64% were susceptible to piperacillin/tazobactam and 63% were susceptible to nitrofurantoin. More than 70% of the strains tested were resistant or intermediately susceptible to amoxicillin/clavulanic acid, gentamicin, nalidixic acid, ciprofloxacin and levofloxacin. Conclusions: Correlation was found between phenotypic and genotypic methods for detection of ESBL-producing bacteria. Carbapenems demonstrated excellent in vitro activity against strains of ESBL-positive enteric bacteria isolated in a Bulgarian university hospital. Tigecycline, amikacin, piperacillin/tazobactam, nitrofurantoin should also be considered as treatment options.
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| 9. |
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| 10. |
- Moore, Edward R.B. 1954, et al.
(författare)
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Microbial systematics and taxonomy: relevance for a microbial commons.
- 2010
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Ingår i: Research in microbiology. - : Elsevier BV. - 1769-7123 .- 0923-2508. ; 161:6, s. 430-8
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Forskningsöversikt (refereegranskat)abstract
- The issues of microbial taxonomy and potential interactions with a microbial commons are discussed, with emphasis on three components: characterization; classification; and nomenclature. The current state of technology and the spectrum of methods that are used for phenotypic and genotypic characterization of prokaryotes, classification at different taxonomic levels and points of prokaryote nomenclature are reviewed. While all taxonomic ranks comprise a cohesive systematic framework for microorganisms, the prokaryotic genus and species provide the "working unit" of taxonomy. Since 2004, the number of validly published genera and species has increased by approximately 50%. Extensive development of technology will continue to enable ever higher resolution characterization and more refined classification of microorganisms. Characterization and classification at the species level may be most relevant for bacterial taxonomy, although reproducible differentiation at the strain level will probably prove to be more relevant for a microbial commons. A dynamic microbial taxonomy, albeit with well-founded and stable guidelines for defining microorganisms, provides an efficient organizational system for dealing with the enormous spectrum of microbial diversity.
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