Thesis (M. Med. Sc.)--University of Hong Kong, 2000. / Includes bibliographical references (leaves 31-37).

Thesis (M.Med.Sc.)--University of Hong Kong, 2000. / Includes bibliographical references (leaves 31-37). Also available in print.

Thesis (Ph.D.)--Tufts University, 2004. / Adviser: Krishna Kumar. Submitted to the Dept. of Chemistry. Includes bibliographical references (leaves 190-197). Access restricted to members of the Tufts University community. Also available via the World Wide Web;

Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2002. / Includes bibliographical references (leaves 71-76). Also available in electronic version. Access restricted to campus users.

Thesis (Ph. D.)--Illinois State University, 2002. / Title from title page screen, viewed January 31, 2006. Dissertation Committee: Brian J. Wilkinson (chair), Radheshyam K. Jayaswal, Alan J. Katz, Wade A. Nichols, Anthony J. Otsuka. Includes bibliographical references (leaves 144-149) and abstract. Also available in print.

Thesis (Ph. D.)--Illinois State University, 1999. / Title from title page screen, viewed July 20, 2006. Dissertation Committee: Brian J. Wilkinson (chair), Radheshyam K. Jayaswal, Alan J. Katz, Anthony J. Otsuka, David L. Williams. Includes bibliographical references and abstract. Also available in print.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Autolysins are enzymes that cleave specific structural components within the bacterial cell wall. They contribute to numerous cellular activities such as cell growth, cell division, peptidoglycan recycling and turnover. In this study, twelve Enterococcal isolates (previously from the genus Streptococcus) were examined for susceptibility to the antibiotics Penicillin G and Vancomycin, using a Disk Diffusion and a Microtitre plate assay. In both methods, all twelve strains were resistant to Vancomycin. Six of these strains were susceptible to Penicillin G. The minimum inhibitory concentration (MIC) values were twice that of the disk diffusion assay values. In the presence of antibiotic, the growth rates for the six strains were halved. Autolysins were extracted from the respective cell cultures using a 4% SDS precipitation method. The protein concentrations were calculated and estimated to be within the range of 5.47- to 6.35 μg/μl. Profiles of the SDS precipitate were analyzed on SDS-PAGE. Autolytic proteins were identified and partially analyzed by renaturing SDS-PAGE (zymograms) using gels containing cell wall substrate. Seven lytic bands of molecular weights 25, 30, 50, 63, 75 95 and 145 kDa (designated Autolysin A to G, respectively) were selected for further analysis. The temporal distribution of the enzymes ranged from the mid exponential phase to the early death phase. The seven proteins were blotted onto polyvinylidene difluoride (PVDF) membranes and excised for N-terminal sequencing. Blast analysis of the respective N-terminal sequences showed autolysins A, C, D, E and F to have 100% similarity to the muramidase, amidase and peptidase from S. cremoris, S. suis, S. pneumonia, S. pyogenes and E. faecium, respectively. Biochemical characterization confirmed autolysins A, B, E and F to exhibit muramidase activity, and autolysin C and G to exhibit peptidase activity. Autolysin D displayed 100% similarity to the protein LytA, a peptidoglycan hydrolase that is known to exhibit amidase activity. Blast analysis could not determine any significant similarities for autolysins B and G to previously identified autolysins, thus indicating they may perhaps be novel autolysins. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2007.

Submitted by Sandra Pereira (srpereira@ufrrj.br) on 2016-07-29T13:36:35Z No. of bitstreams: 1 2011 - Bruno Rocha Pribul.pdf: 1463809 bytes, checksum: ba7b3b0f00c0b4469b45fe02ec9f8a7f (MD5) / Made available in DSpace on 2016-07-29T13:36:35Z (GMT). No. of bitstreams: 1 2011 - Bruno Rocha Pribul.pdf: 1463809 bytes, checksum: ba7b3b0f00c0b4469b45fe02ec9f8a7f (MD5) Previous issue date: 2011-02-24 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico - CNPq / PRIBUL, Bruno Rocha. Evaluation of Vancomycin Resistance and Susceptibility Profile Bacteriocins of staphylococci isolated from bovine mastitis in Southern State of Rio de Janeiro. 41p Thesis (Master of Veterinary Science). Instituto de Veterin?ria, Departamento de Parasitologia Animal, Universidade Federal Rural do Rio de Janeiro, Serop?dica, RJ, 2011. Bacteria of the genus Staphylococcus are among the most implicated in the etiology of mastitis. An additional difficulty in controlling bacterial infections by this genus is represented by its frequent resistance to antibiotics. The glycopeptides (eg vancomycin) appear as an alternative therapy because of the increasing strains of staphylococci resistant to beta-lactams, which are the drugs of choice in these infections. Thus, glycopeptide resistance represents a threat to the future of antimicrobial therapy in humans and animals. This study aimed to evaluate the profile of vancomycin resistance pheno-genotypically in Staphylococcus spp. milk samples from cows with mastitis. All 150 isolates (50 isolates of Staphylococcus aureus, 50 isolates of Staphylococcus intermedius and 50 isolates of Coagulase-negative Staphylococcus spp. (CNS) were evaluated using the tests of disk diffusion, agar microdilution and detection of vanA e vanB genes. The antimicrobial susceptibility test showed high resistance to beta-lactam antibiotics for all groups. In the disk diffusion test, 24% of coagulase-positive isolates showed resistance to teicoplanin (24/100) and 23% to vancomycin (23/100). In the agar microdilution test for detection of CIM, 16% of the isolates were resistant to vancomycin (16/100). Among the resistant isolates in the agar microdilution testing, 56.25% of the isolates (9 / 16) had MIC values ranging from 4-8?g/ml. Staphylococcus coagulase-negative showed no resistance to the evaluated glycopeptides. Genes for resistance to vancomycin were detected in 37.5% of vancomycin-resistant isolates (6 / 16). To seek a correlation between the presence of glycopeptide resistance and the ability of producing biofilm, tests were performed to characterize growth in Congo red agar and also detection of the biofilm adherence test in microplates. It were detected 66.66% and 90, 00%, respectively, of isolates producing biofilm. The production of "slime" presented no statistical correlation with resistance to glycopeptides. The sensitivity profile of Staphylococcus spp. was assessed against the bacteriocins produced by Lactobacillus paracasei, Lactobacillus acidophilus and Lactobacillus fermentum. Lactobacillus paracasei bacteriocins presented the highest inhibition capacity. / PRIBUL, Bruno Rocha. Avalia??o da Resist?ncia a Vancomicina e do Perfil de Suscetibilidade a Bacteriocinas de Staphylococcus spp Isolados de Mastite Bovina da Regi?o Sul do Estado do Rio de Janeiro. 41p Disserta??o (Mestrado em Ci?ncias Veterin?rias). Instituto de Veterin?ria, Departamento de Parasitologia Animal, Universidade Federal Rural do Rio de Janeiro, Serop?dica, RJ, 2011. As bact?rias do g?nero Staphylococcus est?o entre as mais implicadas na etiologia das mastites. Uma dificuldade adicional no controle das infec??es bacterianas por este g?nero ? representada por sua freq?ente resist?ncia aos antimicrobianos. Os glicopept?deos, como a vancomicina, aparecem como uma alternativa terap?utica devido ao crescente aumento de cepas estafiloc?cicas resistentes aos betalact?micos, que s?o os f?rmacos de elei??o nestas infec??es. Desse modo, a resist?ncia aos glicopept?deos representa uma amea?a ao futuro da terapia antimicrobiana em humanos e animais. O presente trabalho buscou avaliar o perfil de resist?ncia ? vancomicina em isolados de Staphylococcus spp. oriundos de amostras de leite de vacas com mastite. Todos os 150 isolados estudados (50 Staphylococcus aureus, 50 Staphylococcus intermedius e 50 Staphylococcus spp. coagulase negativos), foram avaliados atrav?s dos testes de difus?o em disco simples, microdilui??o em ?gar e detec??o dos genes vanA e B. O teste de suscetibilidade antimicrobiana revelou elevada resist?ncia aos beta-lact?micos para todos os grupos avaliados. No ensaio de difus?o em disco, os isolados coagulasepositivos apresentaram um perfil de resist?ncia de 24% a teicoplanina (24/100) e 23% a vancomicina (23/100). No teste de microdilui??o em ?gar para detec??o da CIM, 16% dos isolados apresentaram resist?ncia ? vancomicina (16/100). Entre os isolados resistentes no teste de microdilui??o em ?gar, 56,25% dos isolados (9/16) apresentaram valores de CIM que variaram entre 4-8?g/ml. Os Staphylococcus coagulase-negativos n?o apresentaram resist?ncia aos glicopeptideos. Os genes de resist?ncia ? vancomicina foram detectados em 37,5% dos isolados vancomicina-resistentes (6/16). Para se buscar uma correla??o entre a presen?a de resist?ncia aos glicopeptideos e a capacidade de forma??o de biofilme foram realizados os testes de caracteriza??o de crescimento em ?gar vermelho congo e detec??o de biofilme pelo teste de ader?ncia em microplacas, onde foram detectados 66,66% e 90,00% respectivamente, de isolados produtores de biofilme. A produ??o de ?slime? n?o apresentou correla??o estat?stica com a resist?ncia aos glicopept?deos. O perfil de sensibilidade dos isolados de Staphylococcus spp. foi avaliado frente as bacteriocinas produzidas pelos Lactobacillus paracasei, Lactobacillus acidophillus e Lactobacillus fermentum. As bacteriocinas que apresentaram maior capacidade de inibi??o frente aos Saphylococcus spp. testados foram as produzidas pelos Lactobacillus paracasei

Three Streptococcal strains: S. milleri P213, S. milleri P35 and S. milleri B200 and three enterococcal strains: E. faecalis 123, E. faecalis 126 and E. faecium were used to test for vancomycin resistance. Two strains were used as reference strains that were already characterized as vancomycin resistant. E. faecium BM4147 was used as a VanA control and E. faecalis ATCC was used as a VanB control. Susceptibility of each strain to this antibiotic was tested by disk-diffusion assay and the MIC values for the strains were found to be between 5 - 10 ug/ml and for the VanA control, the MIC was > 64 ug/ml and for the VanB control was 32 ug/ml. These MIC values indicate that S. milleri P213, S. milleri P35, S. milleri B200, E. faecalis 123, E. faecalis 126, and E. faecium are all of the VanC phenotype. All strains were tested for lysis by means of addition of vancomycin (10 ug/ml) to the bacterial cultures. Lytic curves were constructed and the VanB control was found to be most autolytic upon addition of vancomycin and E. faecalis 123 was the least autolytic. However, under normal conditions in phosphate buffer, lytic curves showed that S. milleri P213 was the most autolytic and the VanA control, the least autolytic. PCR assays were performed to detect specific antibiotic resistant genes. Primers were selected from Dukta-Malen et al., 1995. The VanA primer yielded amplification of 732 bp for only the VanA control DNA and the VanB primer set yielded products for the VanB control DNA. S. milleri P213, P35, B200 and E. faecalis 123 and 126, and E. faecium DNA were amplified with the VanC primers. This supports the results obtained in MIC that these strains are possibly VanC resistant strains. Amplified VanA control and that of E. faecalis 126 were thereafter sequenced. VanA control amplicon was correctly amplified since it showed homology to E. faecium BM4147 as well as the VanB amplicons which was found to be homologous to the transposon Tn1549 found on the well-characterized E. faecalis strain which is known to harbour the VanB vancomycin-resistant genes. Whilst E. faecalis 126 which represented the VanC phenotype showed 96% homology to E. gallinarum BM4147 which is a well-characterized glycopeptide-resistant enterococci belonging to the VanC phenotype. Southern blots were performed using specific primers as a probe to verify whether the gene sequences for the specific genotype were present in these strains and results confirmed those found in the PCR assays and in DNA sequencing. The peptidoglycan precursors of each strain were arrested in vancomycin (20 ug/ml) to block transpeptidation and transglycosylation steps of peptidoglycan synthesis and bacitracin (100 ug/ml) was used to amplify precursors at the transglycosylation step. Precursors were extracted and analysed by reverse-phase HPLC. UDP-MurNAc-tetrapeptides cell wall precursors, which are found abundantly in vancomycin-resistant strains, were found in large proportions in all strains, except in E. faecalis 123 when arrested with vancomycin. This precursor has a noticeably decreased affinity for vancomycin, hence contributing to its resistance. The precursor accumulated when arrested with bacitracin, was, UDPMurNAc-tetrapeptide in all strains except in E. faecalis 126. UDP-MurNAc-pentapeptides were also found in moderate amounts in most strains. The molecular masses of the peptidoglycan precursors obtained from mass spectrometry correctly identified them. This confirmed that the bacterial strains investigated were in fact resistant to the antibiotic vancomycin and this study shows that results obtained from conventional phenotypical screening methods reliably correlated with the genotypes classified using more advanced techniques such as PCR, southern blot/hybridisation and DNA sequencing. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2005.