Exploring internal features of 16S rRNA gene for identification of clinically relevant species of the genus Streptococcus

<p>Abstract</p> <p>Background</p> <p><it>Streptococcus </it>is an economically important genus as a number of species belonging to this genus are human and animal pathogens. The genus has been divided into different groups based on 16S rRNA gene sequence similarity. The variability observed among the members of these groups is low and it is difficult to distinguish them. The present study was taken up to explore 16S rRNA gene sequence to develop methods that can be used for preliminary identification and can supplement the existing methods for identification of clinically-relevant isolates of the genus <it>Streptococcus</it>.</p> <p>Methods</p> <p>16S rRNA gene sequences belonging to the isolates of <it>S. dysgalactiae, S. equi</it>, <it>S. pyogenes</it>, <it>S. agalactiae</it>, <it>S. bovis</it>, <it>S. gallolyticus</it>, <it>S. mutans</it>, <it>S. sobrinus, S. mitis</it>, <it>S. pneumoniae</it>, <it>S. thermophilus </it>and <it>S. anginosus </it>were analyzed with the purpose to define genetic variability within each species to generate a phylogenetic framework, to identify species-specific signatures and <it>in-silico </it>restriction enzyme analysis.</p> <p>Results</p> <p>The framework based analysis was used to segregate <it>Streptococcus </it>spp. previously identified upto genus level. This segregation was validated using species-specific signatures and <it>in-silico </it>restriction enzyme analysis. 43 uncharacterized <it>Streptococcus </it>spp. could be identified using this approach.</p> <p>Conclusions</p> <p>The markers generated exploring 16S rRNA gene sequences provided useful tool that can be further used for identification of different species of the genus <it>Streptococcus</it>.</p

Use of pyrosequencing to identify streptococci and to detect mutations causing antimicrobial resistance

Rapid identification and resistance determination of pathogens in clinical specimens is vital for accurate treatment and monitoring of infectious diseases. Antimicrobial drug resistance is increasing globally and healthcare settings are facing this cost-intensive and even life-threatening problem. The incidence of resistant pathogens in Finland has remained relatively steady and manageable at least for the time being. DNA sequencing is the gold standard method for genotyping, mutation analysis, and identification of bacteria. Due to significant cost decrease in recent years, this technique is available to many research and clinical laboratories. Pyrosequencing technique, a rapid real-time DNA sequencing method especially suitable for analyzing fairly short stretches of DNA, was used in this study. Due to its robustness and versatility, pyrosequencing was applied in this study for identification of streptococci and detection of certain mutations causing antimicrobial resistance in different bacteria. Certain streptococcal species such as S. pneumoniae and S. pyogenes are significantly important clinical pathogens. S. pneumoniae causes e.g. pneumonia and otitis media and is one of the most important community-acquired pathogens. S. pyogenes, also known as group A streptococcus, causes e.g. angina and erysipelas. In contrast, the socalled alpha-haemolytic streptococci, such as S. mitis and S. oralis, belong to the normal microbiota, which are regarded to be non-pathogenic and are nearly impossible to identify by phenotypic methods. In this thesis, a pyrosequencing method was developed for identification of streptococcal species based on the 16S rRNA sequences. Almost all streptococcal species could be differentiated from one another by the developed method, including S. pneumoniae from its close relatives S. mitis and S. oralis . New resistance genes and their variants are constantly discovered and reported. In this study, new methods for detecting certain mutations causing macrolide resistance or extended spectrum beta-lactamase (ESBL) phenotype were developed. These resistance detection approaches are not only suitable for surveillance of mechanisms causing antimicrobial resistance but also for routine analysis of clinical samples particularly in epidemic settings. In conclusion, pyrosequencing was found to be an accurate, versatile, cost-effective, and rapid DNA sequencing method that is especially suitable for mutation analysis of short DNA fragments and identification of certain bacteria.Siirretty Doriast

Molecular characterization and population dynamics of lactic acid bacteria during the fermentation of sorghum

Ting is a cooked fermented sorghum food that is popular amongst southern Africans for its sour taste and unique flavour. However, major challenges are associated with large-scale production and marketing of this spontaneously fermented food due to inconsistent microbiological and sensory quality. The use of starter cultures may circumvent these limitations. Prior to engaging starter cultures, detailed knowledge of the microbial diversity and dynamics during fermentation is important. Therefore, the aim of this study was to investigate microbial diversity and dynamics during sorghum fermentations, and to clarify the role of starter cultures regarding the microbiological safety and consumer acceptance of sensory characteristics of fermented ting. A culture-independent approach, based on the use of PCR-denaturing gradient gel electrophoresis (DGGE), revealed that Lactococcus lactis, Lactobacillus curvatus, Weissella cibaria and some Enterobacteriaceae were predominant at the end of spontaneous sorghum fermentations. Culture-dependent methods indicated that Lb. fermentum, Lb. plantarum, Lb. rhamnosus, E. faecalis, E. mundtii, W. cibaria and L. lactis were predominant at the end of fermentation. These results not only indicated the predominant bacteria during sorghum fermentation, but also indicated that a combined approach is required to reveal microbial diversity and dynamics during spontaneous sorghum fermentations. Based on the above results, L. lactis, Lb. fermentum, Lb. plantarum and Lb. rhamnosus were evaluated as starter cultures for production of ting. All the starter cultures were able to ferment sorghum, but the lowest pH and highest lactic acid was produced in naturally fermented sorghum inoculated with L. lactis. This fermentation showed an increase in the number of lactic acid bacteria and yeasts, whilst pathogen counts decreased. Ting from this fermented gruel, in contrast to naturally fermented sorghum, had sensory properties preferred by panelists. The results indicated that the use of L. lactis in starter cultures may result in ting with consistent and acceptable attributes.Thesis (PhD)--University of Pretoria, 2011.Microbiology and Plant Pathologyunrestricte