The use of NGS-based testing of the bacterial microbiota is often impeded by inconsistent or non-reproducible results, especially when applying different analysis pipelines and reference databases. ...We investigated five frequently used software packages by submitting the same monobacterial datasets to them, representing the V1-2 and the V3-4 regions of the 16S-rRNA gene of 26 well characterized strains, which were sequenced by the Ion Torrent™ GeneStudio S5 system. The results obtained were divergent and calculations of relative abundance did not yield the expected 100%. We investigated these inconsistencies and were able to attribute them to failures either of the pipelines themselves or of the reference databases they rely on. On the basis of these findings, we recommend certain standards which should help to render microbiome testing more consistent and reproducible, and thus useful in clinical practice.
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The naming of prokaryotes is governed by the International Code of Nomenclature of Prokaryotes (ICNP) and partially by the International Code of Nomenclature for Algae, Fungi and Plants (ICN). Such ...codes must be able to determine names of taxa in a universal and unambiguous manner, thus serving as a common language across different fields and activities. This unity is undermined when a new code of nomenclature emerges that overlaps in scope with an established, time-tested code and uses the same format of names but assigns different nomenclatural status values to the names. The resulting nomenclatural confusion is not beneficial to the wider scientific community. Such ambiguity is expected to result from the establishment of the 'Code of Nomenclature of Prokaryotes Described from DNA Sequence Data' ('SeqCode'), which is in general and specific conflict with the ICNP and the ICN. Shortcomings in the interpretation of the ICNP may have exacerbated the incompatibility between the codes. It is reiterated as to why proposals to accept sequences as nomenclatural types of species and subspecies with validly published names, now implemented in the SeqCode, have not been implemented by the International Committee on Systematics of Prokaryotes (ICSP), which oversees the ICNP. The absence of certain regulations from the ICNP for the naming of as yet uncultivated prokaryotes is an acceptable scientific argument, although it does not justify the establishment of a separate code. Moreover, the proposals rejected by the ICSP are unnecessary to adequately regulate the naming of uncultivated prokaryotes. To provide a better service to the wider scientific community, an alternative proposal to emend the ICNP is presented, which would result in
names being regulated analogously to validly published names. This proposal is fully consistent with previous ICSP decisions, preserves the essential unity of nomenclature and avoids the expected nomenclatural confusion.
Small-colony variants (SCVs) of Staphylococcus aureus cause persistent and relapsing infections. Relatively little is known regarding infections caused by SCVs of coagulase-negative staphylococci. We ...report two cases of pacemaker electrode infections due to SCVs of Staphylococcus epidermidis and Staphylococcus capitis. Sequence analysis of a portion of the 16S rRNA gene (16S rDNA) confirmed the identity of the staphylococcal species as S. capitis and S. epidermidis. Isolates from cultures of blood obtained over at least a 2-week interval were compared by pulsed-field gel electrophoresis and found to be clonal even though the colony morphology was very different. Analysis for auxotrophism revealed hemin dependencies for all isolated SCVs. The two cases have several clinical and laboratory characteristics (which are also seen with S. aureus SCV infections) and strongly suggest that SCVs of coagulase-negative staphylococci must be actively sought, because they grow very slowly and can be easily missed.
Pasteurella multocida is commonly found in the oral cavity of cats and dogs. In humans it is known as an opportunistic pathogen after bites from these animals. Phenotypic identification of
P. ...multocida based on biochemical reactions is often limited and usually only done on a species level, even though 3 subspecies are described. For molecular taxonomy and diagnostic purposes a phylogenetic analysis of the three subspecies of
P. multocida based on their 16S rRNA (
rrs) gene sequence was therefore carried out. We found
P. multocida subsp.
septica on a distinguished branch on the phylogenetic tree of
Pasteurellaceae, due to a 1.5 % divergence of its
rrs gene compared to the two other, more closely related subspecies
multocida and
gallicida. This phylogenetic divergence can be used for the identification of
P. multocida subsp.
septica by
rrs gene determination since they form a phylogenetically well isolated and defined group as shown with a set of feline isolates. Comparison to routine phenotypic identification shows the advantage of the sequence-based identification over conventional methods. It is therefore helpful for future unambiguous identification and molecular taxonomy of
P. multocida as well as for epidemiological investigations.
Same meat, different gravy Tortoli, Enrico; Brown-Elliott, Barbara A.; Chalmers, James D. ...
The European respiratory journal,
2019, Letnik:
54, Številka:
1
Journal Article
1 Institute of Veterinary Bacteriology, University of Bern, CH-3012 Bern, Switzerland
2 SmartGene, Zug, Switzerland
Correspondence Peter Kuhnert peter.kuhnert{at}vbi.unibe.ch
The genus Campylobacter ...comprises 17 species, some of which are important animal and human pathogens. To gain more insight into the genetic relatedness of this genus and to improve the molecular tools available for diagnosis, a universal sequencing approach was established for the gene encoding the beta-subunit of RNA polymerase ( rpoB ) for the genus Campylobacter . A total of 59 strains, including the type strains of currently recognized species as well as field isolates, were investigated in the study. A primer set specific for Campylobacter species enabled straightforward amplification and sequencing of a 530 bp fragment of the rpoB gene. The 16S rRNA gene sequences of all of the strains were determined in parallel. A good congruence was obtained between 16S rRNA and rpoB gene sequence-based trees within the genus Campylobacter . The branching of the rpoB tree was similar to that of the 16S rRNA gene tree, even though a few discrepancies were observed for certain species. The resolution of the rpoB gene within the genus Campylobacter was generally much higher than that of the 16S rRNA gene sequence, resulting in a clear separation of most species and even some subspecies. The universally applicable amplification and sequencing approach for partial rpoB gene sequence determination provides a powerful tool for DNA sequence-based discrimination of Campylobacter species.
Abbreviations: MLST, multilocus sequence typing
Published online ahead of print on 23 December 2005 as DOI 10.1099/ijs.0.64109-0.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA and rpoB gene sequences of the Campylobacter species determined in this study are shown in Table 1.
Distance matrices for the 16S rRNA and rpoB gene sequences for all the strains investigated in this study are available as Supplementary Tables S1 and S2 in IJSEM Online.
Present address: MCL Medical Laboratories, CH-3186 Düdingen, Switzerland.
Mycobacterium genavense is a recently defined fastidious organism that has been identified as a cause of disseminated infection in patients with AIDS. We report the cases of two patients who had ...advanced AIDS and a clinical syndrome of fever, anorexia, abdominal pain, diarrhea, and weight loss. In addition, splenomegaly and lymphadenopathy were prominent in both cases, and in one patient's case radiographic findings were suggestive of splenic abscesses. Mycobacteria isolated from specimens of blood and bone marrow grew in liquid media but not on solid media. The results of DNA probe tests for Mycobacterium tuberculosis and Mycobacterium avium complex were false-positive for both patients. After treatment of the broth cultures to lyse red blood cells, the results of DNA probe tests were negative for these pathogens. Amplification and sequencing of 16S rRNA with use of the polymerase chain reaction indicated that the mycobacterial isolates from both patients had sequences identical to those previously reported for M. genavense. One patient survived 5 months after diagnosis, the other 2 months after diagnosis; only one patient responded (transiently) to antimycobacterial chemotherapy.
Abstract
Background
Streptococcus pneumoniae
is one of the most frequently encountered pathogens in humans but its differentiation from closely related but less pathogenic streptococci remains a ...challenge.
Methods
This report describes a newly-developed PCR assay (Spne-PCR), amplifying a 217 bp product of the 16S rRNA gene of
S. pneumoniae
, and its performance compared to other genotypic and phenotypic tests.
Results
The new PCR assay designed in this study, proved to be specific at 57°C for
S. pneumoniae
, not amplifying
S. pseudopneumoniae
or any other streptococcal strain or any strains from other upper airway pathogenic species. PCR assays (psaA, LytA, ply, spn9802-PCR) were previously described for the specific amplification of
S. pneumoniae
, but
psaA
-PCR was the only one found not to cross-react with
S. pseudopneumoniae
.
Conclusion
Spne-PCR, developed for this study, and psaA-PCR were the only two assays which did not mis-identify
S. pseudopneumoniae
as
S. pneumoniae
. Four other PCR assays and the AccuProbe assay were unable to distinguish between these species.
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