The possibility of identifying single nucleotide polymorphisms (SNPs) that would be useful for rapid bacterial typing was investigated. Neisseria meningitidis was the organism chosen for modelling the approach since informative SNPs could be found amongst the sequence data available for multi-locus sequence typing (MLST) at http://www.mlst.net. The hypothesis tested was that a small number of SNPs located within the seven gene fragments sequenced for MLST provide information equivalent to MLST. Preliminary investigations revealed that a small number of SNPs could be utilised to highly discriminate sequence types (STs) of clinical interest. Laboratory procedures demonstrated that SNP fingerprinting of N. meningitidis isolates is achievable. Further tests showed that laboratory identification of a defining SNP in the genome of isolates was to be a practical method of obtaining relevant typing information. Identification of the most discriminating SNPs amongst the ever-increasing amount of MLST sequence data summoned the need for computer-based assistance. Two methods of SNP selection devised by the author of this thesis were translated into computer-based algorithms by contributing team members. Software for two computer programs was produced. The algorithms facilitate the optimal selection of SNPs useful for (1) distinguishing specific STs and (2) differentiating non-specific STs. Current input information can be obtained from the MLST database and consequently the programs can be applied to any bacterial species for which MLST data have been entered. The two algorithms for the selection of SNPs were designed to serve contrasting purposes. The first of these was to determine the ST identity of isolates from an outbreak of disease. In this case, isolates would be tested for their membership to any of the STs known to be associated with disease. It was shown that one SNP per ST could distinguish each of four hyperinvasive STs of N. meningitidis from between 92.5% and 97.5% of all other STs. With two SNPs per ST, between 96.7% and 99.0% discrimination is achieved. The SNPs were selected from MLST loci with the assistance of the first algorithm which scores SNPs according to the number of base mismatches in a sequence alignment between an allele of an ST of interest and alleles belonging to all other STs at a specified locus. The second purpose was to determine whether or not isolates from different sources belong to the same ST, regardless of their actual ST identity. It was shown that with seven SNPs, four sample STs of N. meningitidis could, on average, be discriminated from 97.1% of all other STs. The SNPs were selected with the aid of the second algorithm which scores SNPs at MLST loci for the relative frequency of each nucleotide base in a sequence alignment as a measure of the extent of their polymorphism. A third algorithm for selecting SNPs has been discussed. By altering the method of scoring SNPs, it is possible to overcome the limitations inherent in the two algorithms that were utilised for finding SNPs. In addition, the third approach caters for finding SNPs that distinguish members of a complex from non-members.

No description available.

Yogurt is a characteristic fermented dairy product of Turkey and Bulgaria and its popularity has been increasing all over the world. Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus (Lactobacillus bulgaricus) are used together as starter culture in production of yogurt. The objective of this study was to isolate and characterize yogurt cultures from traditionally produced yogurts (i.e. produced without using commercial starter cultures) and to search the genotypic diversity within traditional S. thermophilus isolates. Yogurt cultures were isolated from traditionally produced yogurts collected from different regions of Turkey and identified biochemically. Acidification ability of the isolates was examined and the cultures giving best acidifying rates were further subjected to a selection in terms of their acetaldehyde production ability. Then, phage resistance and proteolytic activity of chosen isolates were tested. Finally, twenty-five L. bulgaricus and twenty-two S. thermophilus isolates were selected as cultures having best technological properties. Furthermore, subtyping studies were carried out to indicate strain diversity among isolates. S. thermophilus was selected as target organism for subtyping in this study. Clustered regularly interspaced short palindromic repeats (CRISPR) loci are highly polymorphic genetic regions, which are composed of partially palindromic direct repeats interspaced by sequences called spacers. In order to characterize S. thermophilus isolates genotypically, CRISPR1 locus of the isolates were analyzed. Additionally, nineteen isolates selected after CRISPR1 analysis were characterized using multilocus sequence typing (MLST). This provided to compare CRISPR1 analysis with MLST as a typing method. According to CRISPR1 analysis S. thermophilus isolates were grouped into 6 main clusters with a total of 15 sub-clusters. MLST results demonstrated an evolutionary relationship among these strains compatible with that derived from the CRISPR1 analysis.

The epidemiology of blastomycosis remains poorly understood in part due to the lack of a robust and discriminatory strain typing method for Blastomyces dermatitidis. Here we describe the development of a multilocus sequence (MLST) method to study the genetic variation and population structure of B. dermatitidis. Eighty geographically diverse clinical and environmental isolates were examined. Thirty-six unique sequence types were identified. With a discriminatory index of 91.4%, MLST identifies significant genetic diversity for the characterization of local and global B. dermatitidis isolates. To test whether this fungus represented a single species throughout its geographic range we performed phylogenetic analyses, applying Genealogical Concordance Phylogenetic Species Recognition (GCPSR). Phylogenetic analysis revealed two distinct clades, with five of the eight gene phylogenies studied supporting the separation of these lineages, which were also geographically partitioned. Based on fulfillment of GCPSR, we propose the current species B. dermatitidis harbors two genetically distinct non-interbreeding phylogenetic species.

The epidemiology of blastomycosis remains poorly understood in part due to the lack of a robust and discriminatory strain typing method for Blastomyces dermatitidis. Here we describe the development of a multilocus sequence (MLST) method to study the genetic variation and population structure of B. dermatitidis. Eighty geographically diverse clinical and environmental isolates were examined. Thirty-six unique sequence types were identified. With a discriminatory index of 91.4%, MLST identifies significant genetic diversity for the characterization of local and global B. dermatitidis isolates. To test whether this fungus represented a single species throughout its geographic range we performed phylogenetic analyses, applying Genealogical Concordance Phylogenetic Species Recognition (GCPSR). Phylogenetic analysis revealed two distinct clades, with five of the eight gene phylogenies studied supporting the separation of these lineages, which were also geographically partitioned. Based on fulfillment of GCPSR, we propose the current species B. dermatitidis harbors two genetically distinct non-interbreeding phylogenetic species.

Methicillin-resistenta Staphylococcus aureus (MRSA) som bär på genen mecA, har förekommit och spridit sig över hela världen, främst i sjukhusmiljö, och orsakat utbrott av vårdrelaterade (så kallade nosokomiala) infektioner. Dessa infektioner kan inte behandlas med stafylokock-penicilliner och MRSA-bakterierna är ofta resistenta även mot flera andra grupper av antibiotika vilket medför att infektionerna ofta är påtagligt svårbehandlade. Under senare år har emellertid allt fler fall beskrivits av samhällsförvärvad MRSA infektion, det vill säga uppträdande av MRSA hos personer som tidigare ej har haft kontakt med sjukhusvård eller behandlats med antibiotika. Det har länge varit oklart om de samhällsförvärvade MRSA [community-acquired (CA-MRSA)] representerar spridning av bakterier från sjukhusmiljön ut till samhället eller om dessa MRSA är spontant uppträdande. Många av dessa stammar har dessutom visat sig bära på sjukdomsrelaterade gener som vanligen inte återfinns hos S. aureus, t.ex. Panton Valentine leukocidin (PVL) som associeras med hudinfektioner och allvarlig lunginflammation med hög dödlighet hos unga och annars friska individer. Denna avhandling beskriver den molekylära epidemiologin hos MRSA med fokus på samhällsförvärvade MRSA som utgjorde mer än hälften av samtliga fall av MRSA i Örebro län och som dessutom ofta producerade PVL toxinet, vars funktion vidare analyserades i detalj. Undersökning av ursprung och släktskap hos samtliga MRSA som isolerats i Örebro län, samt karaktärisering av det genetiskt element som kallas staphylococcal cassette chromosome mec (SCCmec) vilket innehåller genen mecA och ibland även andra resistensgener, visade att CA-MRSA inte är relaterade till de nosokomiala MRSA, och att dessa har uppstått oberoende av varandra. Flertalet MRSA visade sig dessutom bära på SCCmec, och resistensmekanismer, som tidigare inte beskrivits. Troligen har dessa MRSA uppstått genom ett genetiskt utbyte av SCCmec mellan methicillin-resistenta koagulas-negativa stafylokocker (MR-KNS), som utgör huvudparten av normalfloran på huden, och methicillin-känsliga S. aureus som därvid erhåller genen mecA och resistensmekanismer mot samtliga stafylokockantibiotika. I den här avhandlingen framläggs bevis för att ett sådant genetiskt utbyte har skett på Barnkliniken på Universitetssjukhuset i Örebro i slutet på 1990-talet, vilket resulterade i uppkomsten av en ny klon av MRSA som därefter orsakade ett allvarligt utbrott. Kartläggning av DNA-sekvensen hos flertalet unika SCCmec från svenska MRSA gav dessutom en bättre förståelse för hur resistens uppkommer och sprider sig, samt mekanismerna bakom detta. Dessa nya kunskaper kan bidra till en förbättrad diagnostik av MRSA. Detta är framför allt av stor betydelse eftersom nya effektiva kloner av MRSA verkar kunna uppstå ute i samhället med potential att orsaka svårbehandlade infektioner men även att sprida sig bland den friska befolkningen. / Material and methods - During a period of 14 years, around 2000 patients with head injuries were admitted to the emergency ward at Lindesberg County Hospital and Örebro Medical Centre Hospital. Six hundred subjects suffered from skull fracture and/or brain contusion and diagnosis was established using a computed tomography scan (CT). The degree of initial brain injury was estimated using the Swedish Reaction Level Scale (RLS). Sixty-six subjects were investigated with pure tone audiometry in close proximity to the trauma, and this gave an opportunity to study the issue of progress. The investigation took place two to 14 years after trauma, and the results were compared to matched control groups. A battery of different audiological methods was used to investigate peripheral and central auditory function, and a specially designed acoustic environmental room was also utilized. Cognition was investigated using a computer-based test-battery, text information process system (TIPS). Self-assessed hearing, cognition and quality of life were explored using different questionnaires. Results - A high percentage of peripheral and central auditory impairments and also cognitive shortcomings were demonstrated. Progress of SNHL was a common finding, and fracture, high age at trauma and large initial hearing loss predicted progress. Antibody-mediated autoimmunity as a mechanism behind posttraumatic progress of SNHL or clear evidence for sympathetic cochleolabyrinthitis could not be demonstrated. Binaural auditory deficits could be demonstrated when tested in a realistic acoustic environment. Tinnitus, vertigo and memory shortcomings proved to be common sequelae, even in a long-term perspectiveCognitive shortcomings were found in several of these well-rehabilitated subjects.On a group level, there was a good correlation between self-assessments and audiometric results, even if some individuals had a tendency to over- or underestimate their abilities. Conclusion - Auditory and cognitive long-term sequelae of CHI are a common finding even in well-rehabilitated and socially well-functioning subjects, as are vertigo and tinnitus. Vertigo and tinnitus are also common sequelae after CHI, therefore a basic audiovestibular investigation after CHI is recommended, at least in selected cases.Early awareness of the risk for hearing and cognitive sequelae after CHI could lead to measurements taken to prevent tension-related symptoms.Early detection of HI offers an opportunity to try immunosuppressive treatment in cases with a large initial SNHL.

Chlamydia trachomatis is an obligate intracellular bacterium of major human health concern, causing urogential chlamydia infections, lymphogranuloma venereum (LGV) and trachoma. Chlamydia is one of the most common sexually transmitted infections worldwide and can cause infertility. In the first four papers described herein we used a high resolution multilocus sequence typing (MLST) system to investigate the epidemiology of C. trachomatis, and showed that MLST is superior to conventional ompA genotyping with respect to resolution. In the fifth paper we simplified the methodology by developing and validating a multilocus typing (MLT) DNA microarray based on the MLST system. In more detail, MLST analysis of consecutive specimens from 2006 in Örebro County in Sweden, and comparison to specimens from 1999-2000, showed that the new variant C. trachomatis (nvCT) is monoclonal and likely has appeared in recent years. MLST analysis of LGV specimens from men who have sex with men (MSM) showed that the increase of LGV in Europe in the last decade indeed was a clonal outbreak, contrary to the USA where LGV might have been present all along. In the third paper, clinical symptoms could not be correlated with the MLST genotypes, suggesting, together with the combined results of all previous studies, that bacterial factors, if important, need to be understood in the context of host factors. MLST analysis of specimens from a high incidence C. trachomatis area in North Norway revealed interesting epidemiological details concerning unusual genetic variants, the nvCT and MSM, but found no significant difference in genetic diversity compared to two other geographic areas in Norway. Lastly, we developed a MLT array that provides high resolution while being rapid and cost-effective, which makes it an interesting alternative for C. trachomatis genotyping. In conclusion, the MLST system and the MLT array have proven to be useful tools and should now be applied in further investigations to improve our understanding of C. trachomatis epidemiology.

Background: Group B streptococcus (GBS) is regarded as one of the most important causes of maternal and neonatal morbidity and mortality in many parts of the world. GBS recto-vaginal colonization is important in the health of a mother and her neonate, especially in developing countries. Maternal vaginal colonization with GBS at the time of delivery can cause vertical transmission to the neonate. Multilocus sequence typing (MLST) is a technique used to characterize microbial isolates by means of sequencing internal fragments of housekeeping genes and has the advantage of reproducibility and has been shown to correlate with the other typing techniques and thus has emerged as the standard for delineating the clonal population of GBS. The study aimed to investigate the epidemiology of GBS colonization among pregnant women and their neonates, and to characterize the isolates by multilocus sequence typing technique at Dr George Mukhari Academic Hospital, Pretoria. Methodology: A total of 413 pregnant women who visited the antenatal clinic were recruited and screened. Participants were interviewed using a questionnaire to gather demographic and other relevant information such as history of current pregnancy, previous miscarriages and still births. Samples from maternal rectum and vagina as well as neonate ear and umbilical cord were taken for culture using colistin and nalidixic acid (CNA) blood agar and incubated for 24-48 hours. If negative after 48 hours, Todd-Hewitt broth was subcultured after 18-48 hours onto sheep blood agar. Multilocus sequence typing (MLST) was used to characterize seven group B streptococcus isolates collected at Dr George Mukhari academic hospital. Fragments of seven housekeeping genes were amplified by polymerase chain reaction (PCR) for each strain and sequenced. CLC bio software (Inqaba biotech, South Africa; Pretoria) was used to analyse sequenced loci and UPGMA dendrogram was constructed. Results: The colonization rate for GBS in pregnant women and their neonates was 30.9% and 0%, respectively. A higher proportion of GBS were isolated from the rectum (37.9%) as compared to the vagina (20.6%). Most socio-economic, demographic and obstetric factors analysed were not significantly associated with.GBS colonization. On 128 positive samples, the results of Todd-Hewitt enrichment broth and direct plating method using CNA were compared. A total of 45.3% of colonised were positive on direct selective agar (CNA); an additional 54.7% samples were recovered from Todd-Hewitt broth. Three genes (adhP, glnA and tkt) were sequenced successfully for six samples (1, 2. 4,6,12 and 65). The UPGMA tree with 1000 bootstrap showing the relationship between six samples was drawn.Conclusion: This study revealed that pregnant women of all ages are at risk of group B streptococcus colonization. Group B streptococcus was common among pregnant women at Dr George Mukhari Academic Hospital. No socio-economic risk factor was associated with group B streptococcus colonization. Results confirm that the combination of Todd-Hewitt broth and CNA agar plate is a time saving and sensitive method. The allelic profile, characteristics such as G+C (guanine+cytosine) content and dN/dS ratio were not analysed because of the smaller sample size used in this study, which shows that the MLST method was unsuccessful in this study. The UPGMA tree based on differences in consensus of the isolates showed that all group B streptococcus isolates are clustered and descend from a single node. / Life & Consumer Sciences / Life Sciences / M.Sc. (Life Sciences)

Το βακτήριο Wolbachia είναι ένα ενδοκυττάριο και μητρικά κληρονομούμενο συμβιωτικό βακτήριο. Ανήκει στην ομοταξία των Alphaproteobacteria και την τάξη των Rickettsiales. Αποτελεί ίσως τον πιο διαδεδομένο ενδοκυττάριο συμβιωτικό οργανισμό στον πλανήτη, καθώς έχει εντοπιστεί μέχρι στιγμής σε πληθώρα αρθροπόδων και νηματωδών της φιλαρίασης. Πρόσφατες μελέτες εκτιμούν ότι πάνω από το 40% των ειδών αρθροπόδων είναι μολυσμένα με το βακτήριο Wolbachia. Το συμβιωτικό αυτό βακτήριο επηρεάζει τις βιολογικές λειτουργίες και ιδιότητες των ξενιστών του και είναι υπεύθυνο για μια σειρά αναπαραγωγικών ανωμαλιών, όπως η κυτταροπλασματική ασυμβατότητα, η παρθενογένεση, η θανάτωση των αρσενικών εμβρύων και η θηλυκοποίηση. Τα μοναδικά αυτά βιολογικά χαρακτηριστικά του βακτηρίου Wolbachia προσελκύουν όλο και περισσότερο το ενδιαφέρον διαφόρων ερευνητών τόσο για το ρόλο του βακτηρίου σε εξελικτικές διαδικασίες (κυρίως ειδογένεση) όσο και για τη χρησιμοποίησή του σε περιβαλλοντικά φιλικές εφαρμογές καταπολέμησης οργανισμών που είναι επιβλαβείς στους τομείς του γεωργικού και δασικού περιβάλλοντος, και της υγείας. Τα είδη του γένους Glossina (Diptera: Glossinidae), γνωστά και ως μύγες τσε-τσε, αποτελούν ξενιστές του βακτηρίου Wolbachia. Η μύγα τσε-τσε είναι ο σημαντικότερος φορέας των παθογόνων τρυπανοσωμάτων στην τροπική Αφρική, τα οποία προκαλούν την ασθένεια του ύπνου (sleeping sickness) στον άνθρωπο και την αντίστοιχη τρυπανοσωμίαση, γνωστή ως nagana, στα ζώα. Η χρησιμοποίηση του βακτηρίου Wolbachia σε μεθόδους βιολογικής καταπολέμησης της μύγας τσε-τσε προαπαιτεί την πλήρη γνώση της γενετικής του ταυτότητας και των αλληλεπιδράσεων του με το ξενιστή. Προς την κατεύθυνση αυτή, και στα πλαίσια της παρούσας διατριβής, πραγματοποιήθηκε η ανίχνευση του συμβιωτικού βακτηρίου Wolbachia σε περισσότερα από 5300 άτομα από φυσικούς και εργαστηριακούς πληθυσμούς 11 διαφορετικών ειδών μύγας τσε-τσε από 13 Αφρικανικές χώρες. Τα αποτελέσματα έδειξαν τεράστια απόκλιση της παρουσίας του βακτηρίου τόσο μεταξύ ειδών όσο και μεταξύ πληθυσμών του ίδιου είδους. Επίσης, πραγματοποιήθηκε ο γενετικός χαρακτηρισμός των στελεχών Wolbachia από συνολικά 29 αντιπροσωπευτικά δείγματα διαφόρων πληθυσμών και ειδών μύγας τσε-τσε, ενώ σε αρκετά από αυτά παρατηρήθηκαν πολλαπλά στελέχη του βακτηρίου. Διαπιστώθηκε εντυπωσιακή γενετική ποικιλότητα στελεχών Wolbachia που απαντούν στα διάφορα είδη μύγας τσε-τσε καθώς και ασυμφωνία μεταξύ των φυλογενειών των στελεχών Wolbachia και των μυγών τσε-τσε ξενιστών της, γεγονός που σημαίνει οριζόντια μετακίνηση του συμβιωτικού βακτηρίου κατά την εξέλιξη. Επιπρόσθετα, εντοπίστηκαν για πρώτη φορά εκτεταμένα γεγονότα οριζόντιας μεταφοράς βακτηριακών γονιδίων στο γονιδίωμα τριών ειδών μύγας τσε-τσε: στο Glossina morsitans morsitans, Glossina pallidipes και Glossina austeni. Από εξελικτικής σκοπιάς, κρίσιμα ερωτήματα προκύπτουν από τα παραπάνω ευρήματα, και πιο συγκεκριμένα σχετικά με: την προέλευση-μηχανισμό αυτών των γεγονότων οριζόντιας μεταφοράς, τον χρονικό προσδιορισμό τους, τον πιθανό ρόλο τους σε διαδικασίες ειδογένεσης και την επιλεκτική εμφάνισή τους σε ορισμένα μόνο είδη Glossina π.χ. στo υποείδos Glossina morsitans centralis που είναι πολύ συγγενικό του Glossina morsitans morsitans δεν παρατηρήθηκε το φαινόμενο. Εξίσου σημαντική και επιβεβλημένη κρίνεται η διεξοδική διερεύνηση του ενδεχομένου τα βακτηριακά γονίδια που ενσωματώθηκαν στο ευκαρυωτικό γονιδίωμα της μύγας τσε-τσε να ευθύνονται για την έκφραση νέων λειτουργιών-ιδιοτήτων (ή να μεταβάλλουν τις ήδη υπάρχουσες), ιδίως μάλιστα εάν αυτές συνδέονται με την αποδοτικότητα μετάδοσης της νόσου της τρυπανοσωμίασης μέσω του φορέα της, δηλαδή της μύγας τσε-τσε. Τέλος, διαπιστώθηκε πιθανή αρνητική συσχέτιση της παρουσίας του βακτηρίου Wolbachia με τον παθογόνο ιό Salivary Gland hypertrophy Virus (SGHV), γεγονός που συζητείται στα πλαίσια βιολογικών εφαρμογών καταπολέμησης του εντόμου-φορέα και της τρυπανοσωμίασης. Παράλληλα, μεγάλο ενδιαφέρον παρουσιάζει η προοπτική χρησιμοποίησης του βακτηρίου Wolbachia για τη βιολογική καταπολέμηση εντόμων αγροτικής ή /και περιβαλλοντικής σημασίας, όπως είναι οι αφίδες και η καρπόκαψα καστανιάς. Το γεγονός αυτό προϋποθέτει την ανίχνευση και τη γενετική ταυτοποίηση του βακτηρίου σε φυσικούς πληθυσμούς εντόμων. Στα πλαίσια της παρούσας διατριβής πραγματοποιήθηκε ανίχνευση και χαρακτηρισμός του βακτηρίου Wolbachia σε 78 συνολικά άτομα από 22 είδη αφίδων, από 26 φυσικούς πληθυσμούς από την Ελλάδα. Από αυτούς τους 26 πληθυσμούς, μόλις οι 4 βρέθηκαν να είναι μολυσμένοι με το βακτήριο Wolbachia και συγκεκριμένα πληθυσμοί των ειδών: Aphis fabae, Aphis hederae, Metopolophium dirhodum και Baizongia pistaciae. Τα αποτελέσματα αυτά δείχνουν για πρώτη φορά ότι η παρουσία του βακτηρίου Wolbachia στις αφίδες είναι πιθανά πιο διαδεδομένη από ότι προέκυπτε από προηγούμενες μελέτες. Επίσης, μελετήθηκε η ανίχνευση και ο χαρακτηρισμός του βακτηρίου Wolbachia στα είδη Cydia splendana, Cydia fagiglandana και Pammene fasciana. Το βακτήριο Wolbachia ανιχνεύθηκε για πρώτη φορά στα συγκεκριμένα είδη και μάλιστα διαπιστώθηκε ότι η συχνότητα εμφάνισής του ποικίλει τόσο μεταξύ των δύο ειδών Cydia όσο και μεταξύ των πληθυσμών του κάθε είδους. Στο είδος Pammene fasciana, το βακτήριο ανιχνεύθηκε σε όλα τα άτομα που μελετήθηκαν. Τα αποτελέσματα της παρούσας διατριβής συζητούνται από τη σκοπιά τόσο της οικολογικής και εξελικτικής σημασίας τους όσο και της προοπτικής χρησιμοποίησης του συμβιωτικού βακτηρίου Wolbachia για τον πληθυσμιακό έλεγχο επιβλαβών εντόμων όπως οι μύγες τσε-τσε, οι αφίδες και η καρπόκαψα καστανιάς. / Wolbachia is an intracellular and maternally inherited symbiotic bacterium that belongs to the class of Alphaproteobacteria and the order of Rickettsiales. It is the most ubiquitous intracellular symbiotic organism of the planet, since it has been estimated that over 40% of insect species, in addition to filarial nematodes, crustaceans, and arachnids are infected with Wolbachia. In arthropods Wolbachia affects the biological functions and properties of its hosts and it is responsible for a number of reproductive abnormalities, such as cytoplasmic incompatibility (CI), thelytokous parthenogenesis, feminization of genetic males and male killing. These unique biological characteristics of Wolbachia are attracting the interest of various researchers for: (a) decyphering the role of Wolbachia in evolutionary processes (mainly speciation), and (b) for its use in environmentally friendly applications for the control of agricultural pests and disease vectors. The species of genus Glossina (Diptera: Glossinidae) known as tsetse flies, have been found to be infected with Wolbachia. Tsetse flies are the sole vectors of pathogenic trypanosomes in tropical Africa, causing the “sleeping sickness” in humans and the “nagana” in animals. The potential use of Wolbachia for the control of tsetse flies, prerequisite a thorough knowledge of its genetic identity and the interactions with the host. To further characterize the prevalence of Wolbachia in tsetse flies an extensive screen of more than 5300 specimens from natural and laboratory populations of 11 different Glossina species originating from 13 African countries was carried out. Our results indicated a huge divergence in the prevalence of Wolbachia, both among the species and among populations of the same species. Further characterization by MLST and wsp genotyping was carried out for the Wolbachia strains of 29 representative populations and species of tsetse flies. An impressive genetic diversity of Wolbachia strains in tsetse flies was revealed. Interestingly, disconcordance between the phylogeny of Wolbachia and that of the tsetse flies was observed, suggesting horizontal transmission of Wolbachia during the evolution. Moreover, extended horizontal gene transfer events were detected for first time in Glossina morsitans morsitans, Glossina pallidipes και Glossina austeni. These results raise critical questions concerning: (a) the origin/mechanism of these horizontal gene transfer events, (b) their temporal determination, (c) their potential role as agents of speciation and (d) their selective appearance in only some Glossina species e.g in the subspecies Glossina morsitans centralis which is closely related with Glossina morsitans morsitans the phenomenon was not observed. Equally important will be to examine if genes from the chromosomal insertions were potentially expressed and examine if these genes are associated with the vectorial capacity of tsetse flies for the trypanosoma transmission. Finally, a negative correlation between the presence of Wolbachia with the Salivary Gland Hypertrophy Virus (SGHV) was identified. This is further discussed in the context of biological applications for control of tsetse fly-vector and trypanosomiasis. Finally in this thesis, the detection and characterization of Wolbachia in 78 specimens of 22 aphids species, from 26 natural populations, from Greece was examined. Only 4 out of 26 populations were found to be infected with Wolbachia, and specifically the species: Aphis fabae, Aphis hederae, Metopolophium dirhodum και Baizongia pistaciae. These results indicated that the presence of Wolbachia in aphids is probably more prevalent than it was derived from previous studies. Also, detection and characterization of Wolbachia in the Cydia splendana, Cydia fagiglandana and Pammene fasciana was carried out. Wolbachia was detected for first time in these species, and it was found that the prevalence of Wolbachia varies between the two species of Cydia and among populations of each species, with the infection in Pammene fasciana being fixed. At the end the ecological and evolutionary importance of Wolbachia, together with the use of the bacterium for the population control of harmful insects like tsetse flies, aphids and moths is further discussed.

Background: Group B streptococcus (GBS) is regarded as one of the most important causes of maternal and neonatal morbidity and mortality in many parts of the world. GBS recto-vaginal colonization is important in the health of a mother and her neonate, especially in developing countries. Maternal vaginal colonization with GBS at the time of delivery can cause vertical transmission to the neonate. Multilocus sequence typing (MLST) is a technique used to characterize microbial isolates by means of sequencing internal fragments of housekeeping genes and has the advantage of reproducibility and has been shown to correlate with the other typing techniques and thus has emerged as the standard for delineating the clonal population of GBS. The study aimed to investigate the epidemiology of GBS colonization among pregnant women and their neonates, and to characterize the isolates by multilocus sequence typing technique at Dr George Mukhari Academic Hospital, Pretoria. Methodology: A total of 413 pregnant women who visited the antenatal clinic were recruited and screened. Participants were interviewed using a questionnaire to gather demographic and other relevant information such as history of current pregnancy, previous miscarriages and still births. Samples from maternal rectum and vagina as well as neonate ear and umbilical cord were taken for culture using colistin and nalidixic acid (CNA) blood agar and incubated for 24-48 hours. If negative after 48 hours, Todd-Hewitt broth was subcultured after 18-48 hours onto sheep blood agar. Multilocus sequence typing (MLST) was used to characterize seven group B streptococcus isolates collected at Dr George Mukhari academic hospital. Fragments of seven housekeeping genes were amplified by polymerase chain reaction (PCR) for each strain and sequenced. CLC bio software (Inqaba biotech, South Africa; Pretoria) was used to analyse sequenced loci and UPGMA dendrogram was constructed. Results: The colonization rate for GBS in pregnant women and their neonates was 30.9% and 0%, respectively. A higher proportion of GBS were isolated from the rectum (37.9%) as compared to the vagina (20.6%). Most socio-economic, demographic and obstetric factors analysed were not significantly associated with.GBS colonization. On 128 positive samples, the results of Todd-Hewitt enrichment broth and direct plating method using CNA were compared. A total of 45.3% of colonised were positive on direct selective agar (CNA); an additional 54.7% samples were recovered from Todd-Hewitt broth. Three genes (adhP, glnA and tkt) were sequenced successfully for six samples (1, 2. 4,6,12 and 65). The UPGMA tree with 1000 bootstrap showing the relationship between six samples was drawn.Conclusion: This study revealed that pregnant women of all ages are at risk of group B streptococcus colonization. Group B streptococcus was common among pregnant women at Dr George Mukhari Academic Hospital. No socio-economic risk factor was associated with group B streptococcus colonization. Results confirm that the combination of Todd-Hewitt broth and CNA agar plate is a time saving and sensitive method. The allelic profile, characteristics such as G+C (guanine+cytosine) content and dN/dS ratio were not analysed because of the smaller sample size used in this study, which shows that the MLST method was unsuccessful in this study. The UPGMA tree based on differences in consensus of the isolates showed that all group B streptococcus isolates are clustered and descend from a single node. / Life Sciences / M.Sc. (Life Sciences)