Univ., Diss., 2010--Würzburg.

Thesis (M. Med. Sc.)--University of Hong Kong, 2005.

Thesis (M.S.)--Humboldt State University, 2006. / Includes bibliographical references (leaves 38-41). Also available via Humboldt Digital Scholar.

A previous survey of enterococci had identified five strains of Streptococcus faecalis (K55 and SB94) - two subspecies liquefaciens (K60 and K88) and one zymogenes (K87) - and two S. faecium strains (K46 and SB69) which were resistant to tetracycline and streptomycin but susceptible to gentamicin. All the S. faecalis strains and K46 were in addition resistant to erythromycin but only the S. faecium strains were penicillin and ampicillin resistant. The minimal inhibitory concentrations of a further six antibiotics were determined. These values confirmed that in S. faecalis strains, erythromycin resistance was accompanied by resistance to lincomycin and pristinamycin IA, a phenotype typical of macrolide - lincosamide - streptogramin B - type (MLS) antibiotics resistance. The erythromycin resistant K46 however, although resistant to lincomycin, was pristinamycin susceptible and so the basis of resistance is unknown. S. faecalis K60, K87 and SB94 were resistant to kanamycin and neomycin as was S. faecium K46 but all strains were susceptible to spectinomycin. The phenotypes were consistent with resistance mediated by enzymic modification of streptomycin with adenyltransferase (6) and of kanamycin and neomycin with phosphotransferase (3') (5")-III. Erythromycin and tetracycline resistances were expressed constitutively in all strains. Only one S. faecalis (K88) was found to be chloramphenicol resistant and as is typical of Gram-positive bacteria, resistance was inducible. The ability to produce bacteriocin was restricted to beta-haemolytic strain K87 and to strain SB94. Subsequent results indicated that strain K87 probably produced more than one bacteriocin, the activity of which was repressed in the parental strain but which, in derivatives, could be enhanced by the presence of streptomycin. Evidence for the location of resistance, haemolysin and bacteriocin genes was sought from study of the transfer characteristics and stability of markers and from examination of the plasmid content of parental strains and their derivatives. The well characterised S. faecalis subspecies zymogenes strain DS5 (Clewell et al., 1982b) was included for comparison in transfer and curing experiments. All the S. faecalis strains aggregated in response to a cell free filtrate of a plasmid free recipient strain JH2-1, indicating the presence of at least one conjugative plasmid although the low transfer frequencies of most resistance genes in broth matings suggested that response was not necessarily encoded by antibiotic resistance plasmids. Transfer of beta-haemolytic activity and all resistance markers was observed after broth matings but the range of transfer frequencies between strains was wide. Furthermore, the incidence of transfer could be variable particularly in the transfer of DS5 erythromycin resistance and all K87 antibiotic resistances which seemed to be dependent on the production of active donor bacteriocin. Matings of S. faecalis strains carried out on membrane filters were only marginally more efficient in terms of transfer frequencies but were superior with regard to reproducibility of transfer. No antibiotic resistance transfer from S. faecium donors was observed after broth matings and only SB69 tetracycline resistance transferred after filter mating at very low frequency. Several resistance determinants and those encoding β-haemolysin were found to be capable of retransfer indicative of association with genes specifying conjugative ability. Analysis of transconjugant phenotypes revealed that the tetracycline resistance gene of K55, the streptomycin resistance gene of K88 and β-haemolytic activities were always transferred alone but some resistance markers were usually co-transferred with other donor markers.

Extended-spectrum beta-lactamases (ESBL) mediate resistance to 3rd generation cephalosporins and aztreonam in Enterobacteriaceae and pose major clinical problems. Screened Enterobacteriaceae were collected from PHW Microbiology ABM Swansea laboratory and were demonstrated phenotypically to be ESBL- producers by BSAC methods. Isolates were identified using the BD Phoenix Automated system and Bruker Daltonics MALDI Biotyper. 138 isolates were genetically defined as ESBL-producers (103 Escherichia coli, 32 Klebsiella spp., 2 Enterobacter cloacae and 1 Citrobacter freundii) and 4 isolates (2 E. coli, 1 Enterobacter cloacae and 1 Morganella morganii) were genetically confirmed as AmpC-producers. PCR analysis revealed that the most prevalent ESBLs were CTX-M (n=133), predominantly Group 1 (n=128), of which 51% (66/128) contained the I526-CTX- M-15 link region, which is characteristic for epidemic E. coli strain A. PFGE confirmed that these isolates had a clonal relatedness to epidemic E. coli strain A. Allele-specific PCR revealed that all E. coli positive for I526-CTX-M-15 belonged to clone 025b-ST131 (found internationally), which has a high virulence potential and encompasses diverse PFGE patterns. With the molecular epidemiology established; the sensitivity and performance of phenotypic screening and confirmatory assays were analysed so that optimal strategies to handle difficult-to-identify ESBL resistance traits could be determined. In this study, the sensitivity of ESBL screening increased to 100% when ceftazidime was used alongside cefpodoxime. Isolates harbouring ESBL genes are often difficult to treat, as options are greatly limited. Susceptibility to various well-established antibiotics, along with temocillin and tigecycline, were investigated. Temocillin and tigecycline were effective against 98% and 89% of all isolates tested. The carbapenems were the most active antibiotics with 100% of isolates susceptible to imipenem and meropenem and 99% susceptible to ertapenem. Biofilm production in E. coli was also investigated. The pgaABCD gene locus was detected in all ESBL and AmpC-producing E. coli isolates (n=105); however, only 38% of these produced a phenotypic biofilm.

Orientador: Elerson Gaetti-Jardim Júnior / Coorientador: Alvimar Lima de Castro / Banca: Hélio Massaiochi Tanimoto / Banca: Luís Fernando Landucci / Banca: Ana Cláudia Okamoto / Banca: Marcelo Coelho Goiato / Resumo: Este estudo avaliou a ocorrência de leveduras do gênero Candida sp além dos principais microrganismos periodontopatogênicos e enterobactérias na saliva, em mucosa e no biofilme aderido à prótese total, correlacionando com aspectos clínicos e condição de higiene bucal de 90 indivíduos edêntulos e portadores de prótese total, por meio de métodos moleculares (PCR). Espécimes clínicos intrabucais foram coletados desses indivíduos após avaliação das condições sócio-econômicas e comportamentais. A microbiota bucal dos pacientes foi caracterizada por meio da obtenção de amostras de biofilme aderido às próteses totais, mucosa e saliva, as quais foram processadas, por meio de PCR. As inter-relações entre os diferentes microrganismos foram determinadas por meio dos testes de Qui-quadrado e Mann- Whitney. Verificaram-se diferenças na ocorrência de Prevotella intermedia e Enterobacteriaceae na saliva dos pacientes edêntulos, o mesmo ocorrendo com Enterobacteriaceae, Camphylobacter rectus e gênero Pseudomonas no biofilme aderido às próteses totais. As condições de higiene bucal e estado de conservação da prótese total precários favoreceram a ocorrência de leveduras do tipo Candida sp, em especial Candida albicans, em níveis estatisticamente significante nas amostras de mucosa e biofilme aderido à prótese total, tornando este dispositivo protético um potencial reservatório de leveduras e bactérias entéricas que podem ser de relevância na patogênese das infecções oportunistas / Abstract: The aim of this study was to evaluate the occurrence of major periodontopathogenic microorganisms and Enterobacteriaceae and biofilm adhered to the denture, mucosa and saliva in 90 edentulous subjects with complete dentures, using molecular methods (PCR). Clinical specimens were collected from these individuals after assessing the socio-economic circumstances and behavioral. The oral microbiota of patients was characterized by obtaining samples of the biofilm adhered to the dental prothesis and saliva, for detection of major pathogens using PCR. The possibility of inter-relationships between different microorganisms was determined using the chi-square test and Mann-Whitney test. There were differences in the occurrence of Prevotella intermedia and Enterobacteriaceae in the saliva of edentulous patients, likewise, Enterobacteriaceae, Pseudomonas, Camphylobacter rectus and the biofilm attached to denture patients. The conditions for oral hygiene and stat of preservation of prosthesis total precarious favored the occurrence of yeasts of the Candida sp, particularly Candida albicans, statistically significant levels in samples of mucosa and biofilm acceded to total prosthesis, prothetic device, making it a potential reservoir of enteric bacteria and yeasts that may be of relevance in the pathogenesis of opportunistic infections / Doutor

From Introduction: Bacterial cells all have a cytoplasmic membrane (see Figure 1) which regulates the movement of ions and molecules into and out of the bacterium. Enclosing this membrane is a cell wall of which there are two general types, which are differentiated by the Gram stain(02) as being either gram positive or gram negative (depending upon whether they hold the gram stain after washing with ethanol). The cell wall provides the cell with shape and rigidity and is composed, in the case of gram positive types, of peptidoglycan, and in the case of gram negative bacteria, of a peptidoglycan and an outer membrane (see Figure 2). The peptidoglycan layer, common to both cell wall types, consists of a backbone of alternating units of N-acetylglucosamine and N-acetylmuramic acid to which peptides are attached by amide links. This heteropolymer is a highly cross linked mosaic and this gives it strength and rigidity. In gram positive bacteria, this layer also contains two carbohydr ate antigens, a simple polysaccharide and a teichoic acid; these are usually the type specific or major group antigens of the bacterium. Many of the bacteria also produce exopolysaccharides (see Figure 3) either as discrete capsules (for example, the Enterobacteriaceae K antigens) or unattached slime layers (for example, the Enterobacteriaceae M antigens). The vast majority of these polysaccharides are heteroglycans(03) composed of contiguous oligosaccharide repeating units. Their monosaccharide components are largely neutral hexoses, 6-deoxy hexoses and also amino sugars. (03) Pentose units are rare. (03) The capsular polysaccharides usually have a high content of acidic constituents such as uronic acids, phosphate groups, or pyruvate ketals. (01) / KMBT_363 / Adobe Acrobat 9.53 Paper Capture Plug-in

This thesis presents a detailed chemical and spectroscopic determination of the capsular, polysaccharide K-antigen isolated from the Klebsiella bacterium, serotype K43 (culture #2482). The repeating unit of the capsular polysaccharide was found to be of the "3 + 2" repeating unit type. A uronic acid was found as part of a disaccharide side chain and the main chain of the polysaccharide was found to be composed of a neutral trisaccharide of mannose and galactose. The work forms part of an ongoing research interest in bacterial polysaccharides of this laboratory and now completes the structural elucidation of all the Klebsiella K-antigens, bar three antigens which were originally assigned to other laboratories. These data together with the respective serological characteristics of each serotype are available to the molecular biologist, and may result in the production of: vaccine(s) against Klebsiella infections, diagnostic products and novel carrier molecules enabling targeted drug delivery.

The work presented in this thesis forms part of a collaborative effort to determine the chemical structures of the surface antigens of bacteria which belong to the Enterobacteriaceae. These antigens are largely polysaccharides and occur as lipopolysaccharides and capsular polysaccharides which give rise to the somatic or 0 antigens and the capsular or K antigens, respectively. In recent years interest has mostly been focused on the extracellular polysaccharide antigens expressed by the genus Escherichia coli because of the effect they exert on normal immunological processes and their structural relatedness to the surface antigens of other more pathogenic bacteria. Therefore the molecular structures of the capsular polysaccharides (Kantigens)produced by E. coli 09:K35(AI04a) and 09:K38(A262a) have been determined by novel enzymic, chemical and spectroscopic procedures. These investigations show that the structures of these polysaccharides can be determined by a combination of chemical and spectroscopic procedures , or almost entirely by n.m.r. spectroscopy alone. The in vitro bacteriophage mediated depolymerisation of the native E. coli K35 polysaccharide demonstrates the value of this method for the isolation of oligosaccharides representing the repeating- unit and multiples thereof. Finally E. coli K37 and K38 capsular polysaccharides were used as model compounds for the evaluation of partial and selective reductive cleavage as methods of generating oligosaccharide for further structural analysis. The products of these reactions were analysed largely by a combination of mass spectrometric procedures.

The research presented in this thesis forms part of an on-going collaborative programme concerned with the determination of the chemical structures of the surface antigens of bacteria belonging to genera within the family Enterobacteriaceae. Bacteria of this family are opportunistic pathogens and are frequently responsible for serious infections in animals and man. Surface antigens produced by virulent strains are largely polysaccharides and occur as lipopolysaccharides (the O-antigens) and capsular polysaccharides (the K-antigens) respectively. The extracellular polysaccharide antigens expressed by strains of the species Escherichia coli are of considerable . interest due to their effect on immunological processes and the relationship which exists between their chemical structure and virulence. To date, some seventy-four K-antigens have been distinguished serologically within the species E. coli and structures have been determined for most of these. The K-antigens of E. coli are structurally diverse and exhibit serological cross-reactivity with other pathogenic bacteria. The structures of five previously unstudied E. coli K-antigens, viz. those produced by serotypes 020:K1 01 :H-, 08:K45:H9, 08:K50:H-, 0101 :K1 03:H-, and 08:K43:H11, are presented in this thesis. A variety of chemical techniques has been employed in the structural analysis, and these are discussed. Two-dimensional NMR spectroscopic techniques proved invaluable for the structural elucidation of these complex carbohydrates, and high-field NMR spectroscopy alone was used in the analysis of the K43 antigen. Structural analysis of the K1 03 antigen was facilitated by specific enzymatic degradation, using a bacteriophage-borne endoglycanase. The K45 antigen was found to contain the unusual sugar 3-acetamido-3,6-dideoxygalactopyranose, while the K50 and K103 antigens join a minority group of polysaccharides which contain pyruvate as their only acidic component.