| 1. |
- Helldal, Lisa, et al.
(författare)
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Shifts in Extended-Spectrum Beta-Lactamase types with increasing prevalence of Escherichia coli producing ESBL
- 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: Contrary to other multidrug-resistant pathogens, the prevalence of bacteria producing extended-spectrum beta-lactamase (ESBL) is increasing rapidly in Sweden. In Europe, ESBL of CTX-M-, TEM-, OXA- and SHV-types are generally associated with E. coli infections, CTX-M being the most predominant. We have investigated how the prevalence of these types has changed during the last five years in the low endemic setting of western Sweden. Methods: Yearly resistance in urinary (approximately 10,000 isolates/year) and blood (approximately 250 isolates/year) E. coli during 2004-2008 were determined. Cephalosporin-resistant isolates were screened for ESBL, using a double-disk assay with clavulanic acid as the inhibitory agent. All ESBL-E. coli isolated in the region during the periods Sept 2003-April 2005 (n=46) and April 2008-March 2009 (n=256) were typed by multiplex-PCR, detecting CTX-M, TEM, OXA and SHV. CTX-M-positive isolates were sub-typed by real time Q-PCR for CTX-M-1, CTX-M-2 and CTXM-9 groups. Results: During 2004-2008, ESBL-producing E. coli strains increased from 0.3-1.5% in urinary and 0-1.4% in blood isolates. Resistance to quinolones and trimethoprim was observed in 60-80% of strains, as compared to less than 8% in non-ESBL-producing E. coli. The majority of the ESBL-E. coli strains possessed the CTX-M gene-type, increasing from 78% (36/46) in 2003-2005 to 93% (238/256) in 2008-2009. Between these time-periods, a marked shift occurred in the distribution of CTX-M types, in that strains with the CTX-M-9 group decreased from 42% (15/36) of isolates to 21% (51/238, p=0.01) and, simultaneously, strains with the CTX-M-1 group increased from 58% (21/36) to 78% (185/238, p= 0.02). Furthermore, strains of CTX-M-type exhibiting also TEM- and/or OXA increased to comprise 86% of cases, as compared to 75% previously. Similar trends were seen for community and hospital detected isolates and with no differences associated with age in affected patients. Conclusion: A steady increase in multidrug-resistant ESBL-E. coli, possessing the genes for multiple ESBL-types, was observed in western Sweden, contrary to the patterns of other multidrug-resistant bacteria. As ESBL has increased during the five-year study period, we detected a shift in the prevalence of ESBL-types, currently dominated by the CTX-M-1 group. These observations suggest that a novel ESBL-producing E. coli clone may have emerged in the area, which will be further investigated and presented.
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| 2. |
- Hernández, Marcela, et al.
(författare)
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Isolation and characterisation of a novel simazine-degrading bacterium from agricultural soil of central Chile, Pseudomonas nitroreducens MHP41
- 2009
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Ingår i: FEMS 2009 - 3rd Congress of European Microbiologists.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Background: s-Triazine herbicides, such as simazine, are used in America in agriculture and forestry. This herbicide is persistent in soils, as well as in aquifers. Chemical and biological processes are involved in the dissipation of simazine in soil. However, microbial degradation is probably the main mechanism for removing s-triazine from soils. Objectives: 1. Isolation, metabolic and systematic characterization of bacterium MHP41 from herbicide-contaminated agricultural soil in Chile. Methods: Bacterial strains were isolated by enrichment in minimal medium with simazine as the sole nitrogen source. Simazine degradation was analysed by resting cell assays and analyses of atz genes were assessed by PCR and DNA sequencing. Strain MHP41 was characterised by ARDRA, using restriction endonucleases, MspI and HhaI, phenotypic characterisation, cell fatty acid profiling and multi-locus sequence analysis (MLSA), using 16S rRNA gene, IGS-1, gyrB and rpoB sequencing. Results: 1. Strain MHP41 grew in minimal medium, using simazine as the sole source of nitrogen. 2. Resting cells of strain MHP41 degraded more than 80% of simazine within 60 min. 3. The atzA, atzB, atzC, atzD, atzE and atzF degradation genes were detected by PCR and characterised by sequence analysis. 4. Strain MHP41 was identified initially as a Pseudomonas sp. and comparative MLSA determined MHP41 to be a strain of P. nitroreducens. Conclusions: 1. The isolation and characterization of a simazine-degrading bacterium MHP41, which is capable of growing with simazine as the sole nitrogen source. 2. P. nitroreducens MHP41 possesses all atz genes of the upper and lower catabolic pathways for simazine degradation. 4. MHP41 is the first known strain of P. nitroreducens capable of degrading simazine and represents an interesting bacterium for studies on the bioremediation of s-triazine-contaminated soils.
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| 3. |
- Karami, Nahid, 1959, et al.
(författare)
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Investigation of an outbreak of CTX-M-15-producing Escherichia coli of sequence types 131 and 1441 in a neonatal surgical ward: comparison of typing methods
- 2010
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Ingår i: 20th European Congress of Clinical Microbiology and Infectious Diseases (ECCMID), Vienna, Austria.
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Konferensbidrag (refereegranskat)abstract
- Objectives: In a surgical ward caring mainly for newborns spread of CTX-M-15-producing E.coli had been ongoing at least since September 2008 when we finally recognized the outbreak in late December. We have compared various typing methods with pulsed-field gel electrophoresis (PFGE) to verify the actual outbreak and subsequently to determine number of affected children. Methods: In addition to clinical sampling 125 children hospitalized between Sept-Dec were screened for extended-spectrum beta-lactamase (ESBL)-producing bacteria in stool during Dec-Feb. From Jan-June 2009 newly admitted children were screened at admission and twice weekly. Fifty-one E coli isolates with ESBL from 27 children were found. These isolates have been typed with PFGE, multiple-locus-variable number tandem repeat analysis (MLVA), a mini multiple-locus-sequence typing (MLST) method (dnaJ, purA and fumC genes) as well as with the Phene Plate (PhP) biochemical fingerprinting system. Results: When the outbreak was revealed five children had developed infections with ESBL-producing E. coli that were of two PFGE-types (A and B) later considered to be the outbreak strains. One or both were spread to 21 children. Six children had multiple types. Altogether 38 isolates (20 children) were of type A (ST 131), 7 isolates (5 children) of type B (ST 1441). In addition E coli of six distinct PFGE-types (C-?) were found in one child each. MLVA gave identical discriminatory results as PFGE for all isolates tested. Mini-MLST could not differentiate ST 131 isolates of to distinct PFGE-types (type A and C) but accurately predicted the ST-types of each PFGE-type when confirmed with standard MLST according to http://mlst.ucc.ie/mlst/dbs/Ecoli. By comparing resistance pattern we thus missed the outbreak by a month. PhP indicated that all initial isolates were singletons and there was hardly any correlation with PFGE. Conclusion: If transmission has been ongoing for a long time several types of ESBL-producing bacteria may be found in an outbreak and all isolates including repeat and screening isolates need to be typed to identify affected patients. Only genetic typing, gave satisfactory results in this outbreak. MLVA gave identical results to PFGE and is thus attractive being faster, cheaper and easier to communicate. Our mini-MLST was somewhat less discriminatory but despite using only three house keeping genes accurately predicted the ST-types.
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| 4. |
- Karami, Nahid, 1959, et al.
(författare)
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Multi-locus sequence typing of extended-spectrum β-lactamase-producing Escherichia coli based on dnaJ, purA and fumC and correlation with ESBL types
- 2009
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Ingår i: FEMS 2009 - 3rd Congress of European Microbiologists.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Background: Four extended-spectrum β-lactamase (ESBL) types: CTXM; TEM; OXA; and SHV; are associated with most ESBL Escherichia coli infections in Europe, CTXM being the most predominant. Prevalence of ESBL-E. coli has increased dramatically in hospital- and community-acquired infections in the past 5 years in Sweden. Objectives: Adapt a multi-locus sequence typing (MLST) analysis for ESBL-E. coli isolates and to correlate MLST data to the prevalence of ESBL types. Methods: Forty-eight isolates from urinary tract samples were collected from November 2007 to March 2008 at Sahlgrenska University Hospital, Göteborg. E. coli were identified by standard methods and screening for ESBL was performed by the double disk assay and ESBL Etest strips. The diversity and prevalence of ESBL-types: CTXM; TEM; OXA; and SHV; were determined by a multiplex PCR assay. CTXM-positive isolates were further typed by PCR, for CTXM sub-types: CTXM-1; CTXM-2; and CTXM-9. MLST of ESBL-E. coli, using house-keeping gene sequences for dnaJ, encoding heat shock protein 40, as well as purA (adenylosuccinate dehydrogenase) and fumC (fumarate hydratase) sequences. Results: Among all isolates analysed, 94% of ESBL-E. coli strains were CTXM-positive. Combinations of ESBL-genes were observed in 90% of strains: CTXM+TEM (36%); and CTXM+OXA (33%). Among CTXM-positive E. coli, 76% were observed to be CTXM-1 sub-type. MLST of strains detected eighteen different MLST sequence types, with 38% of strains belonging to one sequence type. Among strains of the most prevalent MLST sequence type, 46% exhibited CTXM+TEM and 39% exhibited CTXM+OXA combinations. Conclusions: A clear predominance of CTXM-ESBL in combination with TEM or OXA was observed in ESBL-E. coli recovered from Swedish patients. No clear correlation could be determined between MLST sequence types and ESBL types, although two MLST-ESBL types were observed to be distinctly prevalent among ESBL-E. coli analysed.
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| 5. |
- 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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| 6. |
- 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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| 7. |
- Moore, Edward R.B. 1954, et al.
(författare)
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Nucleic acid sequencing for identification in clinical and environmental microbiology
- 2008
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Ingår i: Proceedings of the XXX Congresso Chileno de Microbiología. December 4-6, Concepción, Chile.
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Konferensbidrag (refereegranskat)abstract
- Systematic analyses in the complexity of microbial diversity are increasingly problematic for clinical diagnoses and studies of environmental microbiology. DNA sequence-based analyses and genotyping have enabled the detection and identification of microorganisms in the environment and have been adopted as routine in clinical analyses. Comparative 16S rRNA gene sequence analyses are able to estimate identifications, although it is recognised that such analyses are not able to provide definitive diagnoses at the species level. Among the most difficult problems for clinical cases is the identification of infectious agents within “complexes” of closely related species, often comprising pathogenic and non-pathogenic species or genomovars, with limited differentiating characteristics, e.g., 16S rRNA gene sequence dissimilarities among such organisms is often much less than 1.0%. Genotyping of bacteria enable high-resolution differentiation and identifications, as well as epidemiological monitoring. Furthermore, these approaches provide the means for establishing new criteria for defining bacterial species. For the identification of clinical or environmental isolates, a polyphasic multi-locus sequence analysis (MLSA) can be established, including “first-phase” comparisons of 16S rRNA gene sequences, for identification to the sub-genus level, and subsequent, “secondary-phase” analyses of conserved house-keeping genes, for identification to the species level. An average nucleotide index (ANI), based upon the overall nucleic acid sequence similarities among homologous house-keeping genes, can be applied to establish the expected gene sequence similarity “cut-offs” that differentiate the species of any given taxon. Thus, the key to effective bacterial identification depends upon the selection of conserved genes with levels of resolution able to differentiate the most closely related species.
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| 8. |
- Moore, Edward R.B. 1954, et al.
(författare)
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Nucleic acid sequencing for the evaluation of bacterial species and systematics: applications for identification
- 2009
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Ingår i: Proceedings zur VAAM-Jahrestagung 2009, March 8-11, Bochum, Germany, FGE03. - 0947-0867.
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Konferensbidrag (refereegranskat)abstract
- Identification of prokaryotes in the complexity of microbial diversity is increasingly problematic for clinical diagnoses and environmental microbiology. DNA sequence-based analyses of bacteria have enabled the identification of microorganisms in the environment and are being adopted as routine in clinical analyses. DNA-based methods are suited to analyses of fastidious organisms, as well as those presenting health risks during cultivation. Comparative 16S rRNA gene sequence analyses are able to estimate phylogenetic relationships, although it is recognised that such analyses are not able to provide definitive species identifications. Among the most difficult problems for clinical diagnostics is the identification of organisms within “complexes” of closely related species, comprising pathogenic and non-pathogenic species with limited differentiating characteristics, e.g., 16S rRNA gene sequence dissimilarities among such organisms are often less than 1.0%. However, these species complexes are comprised of organisms with different pathogenic and virulence potential and it is essential to be able to obtain reliable identifications. A “polyphasic” multi-locus sequence analysis (MLSA) strategy can be established for the identification of bacteria, including “first-phase” comparisons of partial 16S rRNA gene sequences, for identification to the sub-genus level, and subsequent, “second-phase” analyses of one or more conserved house-keeping genes, for identification to the species level. However, it is recognised that house-keeping genes are not equally useful for all taxa. An average nucleotide index (ANI), based upon sequence similarities among house-keeping genes, may be applied for establishing expected gene sequence “cut-offs” that differentiate the species of a given taxon. Thus, a potential key to effective bacterial identification depends upon the selection of conserved genes with levels of resolution high enough to differentiate the most closely related species.
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| 9. |
- 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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| 10. |
- Yarza, P., et al.
(författare)
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Sequencing orphan species initiative (SOS): Filling the gaps in the 16S rRNA gene sequence database for all species with validly published names
- 2013
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Ingår i: Systematic and Applied Microbiology. - : Elsevier BV. - 0723-2020. ; 36:1, s. 69-73
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Tidskriftsartikel (refereegranskat)abstract
- High quality 16S ribosomal RNA (rRNA) gene sequences from the type strains of all species with validly published names, as defined by the International Code of Nomenclature of Bacteria, are a prerequisite for their accurate affiliations within the global genealogical classification and for the recognition of potential new taxa. During the last few years, the Living Tree Project (LTP) has taken care to create a high quality, aligned 16S and 23S rRNA gene sequence database of all type strains. However, the manual curation of the sequence dataset and type strain information revealed that a total of 552 “orphan” species (about 5.7% of the currently classified species) had to be excluded from the reference trees. Among them, 322 type strains were not represented by an SSU entry in the public sequence repositories. The remaining 230 type strains had to be discarded due to bad sequence quality. Since 2010, the LTP team has coordinated a network of researchers and culture collections in order to improve the situation by (re)-sequencing the type strains of these “orphan” species. As a result, we can now report 351 16S rRNA gene sequences of type strains. Nevertheless, 201 species could not be sequenced because cultivable type strains were not available (121), the cultures had either been lost or were never deposited in the first place (66), or it was not possible due to other constraints (14). The International Code of Nomenclature of Bacteria provides a number of mechanisms to deal with the problem of missing type strains and we recommend that due consideration be given to the appropriate mechanisms in order to help solve some of these issues.
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