RODRIGUEZ, M. T. T. Streptococcus e Enterococcus isolados de águas marinhas e de galerias pluviais na costa de Fortaleza: perfil de resistência a antibióticos. 2015. 143 f. Tese (Doutorado em Ciências Marinhas Tropicais) - Instituto de Ciências do Mar, Universidade Federal do Ceará, Fortaleza, 2015. / Submitted by Geovane Uchoa (geovane@ufc.br) on 2016-06-24T14:41:57Z No. of bitstreams: 1 2015_tese_mttrodriguez.pdf: 1869791 bytes, checksum: f622e9a6a99f6ac14b10ed77d612e052 (MD5) / Approved for entry into archive by Nadsa Cid (nadsa@ufc.br) on 2016-06-24T16:31:19Z (GMT) No. of bitstreams: 1 2015_tese_mttrodriguez.pdf: 1869791 bytes, checksum: f622e9a6a99f6ac14b10ed77d612e052 (MD5) / Made available in DSpace on 2016-06-24T16:31:19Z (GMT). No. of bitstreams: 1 2015_tese_mttrodriguez.pdf: 1869791 bytes, checksum: f622e9a6a99f6ac14b10ed77d612e052 (MD5) Previous issue date: 2015 / The term of faecal streptococci are represented by bacteria from Streptococcus and Enterococcus genera with different sanitary significance and survival characteristics. Enterococcus genera have been suggested as indicators of fecal contamination in marine waters since numerous studies related to water exposure with the presence of these bacteria and the occurrence of gastrointestinal diseases and acute febrile respiratory illness. A motive to concern in relation to the increase of the population of these bacteria in aquatic environments resistant to antimicrobials resulting mainly from the widespread use of these substances in human and veterinary therapy. The aim of this research was to quantify and identify contaminating bacteria of the genera Streptococcus e Enterococcus in water of two storm sewers (Riacho Maceió gallery-G1 and the Gallery in front to Meireles beach-G2) and points of adjacent sea to every gallery (P1 and P2) on the edge of the Fortaleza city, establishing antimicrobial resistance patterns. There were 5 continuous collections with a weekly periodicity in the four selected points. The values of the standard plate counts ranged from ≤10 in G2 gallery to 192 x 102 UFC /100 mL in G1 gallery and the sea water points from ≤10 in P2 to 94 x 102 UFC/ 100 mL in P1. Once carried out the biochemical tests, 192 strains (86.45%) were identified as belonging to the genus Enterococcus and 26 strains (13.54%) to the genus Streptococcus. Six Enterococcus species were identified and E. faecium was the higher frequency. It showed high resistance (˃ 50%) to ceftriaxone, clindamycin, cefotaxime and streptomycin. Antimicrobial resistance (36 profiles) was observed in 96.15% of the strains. Of the 156 strains tested in antibiotic susceptibility test, 137 (87.88%) showed multidrug resistance (29 multidrug resistance profiles). The highest IRA value (0.05) was presented by the gallery isolates. The MRA index ≥ 0.2 was found in 131/150 resistant strains (87.33%). Resistance associated with chromosomal genes or indicative of possible plasmid transfer was verified in isolated after plasmid cure. High genetic diversity from multidrug resistant analyzed isolates confirmed the necessity to polyphasic approach in order to identify environmental strains. / O termo estreptococos fecais abrange bactérias dos gêneros Streptococcus e Enterococcus com diferentes significado sanitário e características de sobrevivência. Enterococcus tem sido sugerido como indicador de contaminação fecal em águas marinhas já que numerosos estudos relacionam a exposição a águas com a presença dessas bactérias, a ocorrência de doenças e a infecções em humanos. O aumento dessas bactérias em ambientes aquáticos resistentes a antimicrobianos, resultado do amplo emprego dessas substâncias na terapêutica humana e veterinária, é motivo de preocupação. O objetivo desta pesquisa foi quantificar e identificar bactérias contaminantes dos gêneros Streptococcus e Enterococcus em duas galerias pluviais (Galeria Riacho Maceió-G1 e Galeria frente à praia do Meireles-G2) e pontos de mar adjacentes a cada galeria (P1 e P2) na orla da cidade de Fortaleza estabelecendo padrões de resistência a antimicrobianos. Foram realizadas cinco coletas contínuas com periodicidade semanal nos quatro pontos selecionados. Os valores das Contagens Padrão em Placas variaram de ≤10 na galeria G2 a 192 x 102 UFC /100 mL na galeria G1 e para os pontos de água de mar de 1x 102 em P2 a 94 x 102 UFC/ 100 mL em P1. Uma vez realizadas as provas bioquímicas, 192 estirpes (86,45%) foram identificadas como pertencentes ao gênero Enterococcus e 26 (13,54%) ao gênero Streptococcus. Seis espécies de Enterococcus foram identificadas, sendo E. faecium a espécie de maior frequência. Foi observada alta resistência (˃ 50%) a ceftriaxone, clindamicina, cefotaxime e estreptomicina. A resistência antimicrobiana (36 perfis) foi observada em 96,15% das cepas testadas. De 156 cepas testadas no antibiograma, 137 (87,88%) apresentaram multirresistência (29 perfis). O maior valor de IRA (0,05) foi apresentado pelos isolados de galeria. O índice MRA ≥ 0,2 foi encontrado em 131/150 cepas resistentes (87,33%). Resistência associada a genes cromossômicos ou indicativa de possível transferência plasmidial foi verificada nos isolados após cura plasmidial. Alta diversidade genética dos isolados multirresistentes analisados confirmou a necessidade da abordagem polifásica na identificação de espécies ambientais.

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Streptococcus pneumoniae (pneumococcus) is one of the leading causes of mortality in Africa. It asymptomatically colonizes the human nasopharynx. The invasive pneumococcal disease occurs when isolates spread to normally sterile sites such as lungs, blood, and the central nervous system. Colonization, though, does not necessarily lead to infection. Some isolates remain in the upper respiratory tract only, without causing any pathogenic symptoms. This thesis hypothesized that invasive and non-invasive isolates differ genetically. We tested this hypothesis by applying a pan-genome approach using whole-genome sequencing short reads of 1477 samples from Malawi, including those obtained from the nasopharynx of carriers (825 samples) and from the blood and cerebrospinal fluid of patients (652 samples). In-silico serotyping identified 56 serotypes in the cohort and statistical analysis showed that despite the vaccination, the prevalence of serotypes 1 and 12F increased amongst patients. Genomes were assembled, and a reference pan-genome for all strains was built. Short reads were aligned to the core genome, and core variants were called. The population structure was determined based on the distribution of variants in the pan-genome. Finally, genes with a significant presence in the invasive isolates were identified. Functional enrichment analysis of potential virulence genes was carried out to address how specific genes may contribute to the pathogenesis. The findings highlighted the features of the pneumococcus pan-genome in Malawi. The core- and accessory-genome were characterized based on the functional analysis of genes. The core components included: Ribosomal subunits. Subunits of F-type ATP synthase. Enzymes that catalyze the attachment of amino acids to tRNA molecules, DNA replication, DNA repair, and homologous recombination. 10.13% of the core and soft-core genes were uncharacterized. In the accessory genome, the study detected the presence of genes from Regions of Diversity (RDs), including Subunits of V-type ATPases and Sodium/solute symporter from RD8a. Enzymes from RD3 catalyzing the capsule synthesis. Subunits of PsrP secY2A2 pathogenicity island from RD10. Genes from RD6 and RD7 involved in transposing mobile genetic elements. Genes from RD2 RD8b, and RD12 participating in communication and competition. Genes from RD4 that assemble pilins into pili and anchor pili to the cell wall. 53.58% of accessory genes were uncharacterized. Most serotypes showed a similar prevalence in carriage and disease groups. However, the significant abundance of serotypes 1, 5, and 12F among patients compared to the carriage group suggested they are highly invasive with a short colonization period. These serotypes exhibited a remarkable genetic distinction from others. Their divergence included the absence and presence of several genes in their genome structure. The lack of genes from a genomic island known as RD8a was the most pronounced difference between serotypes 1, 5, and 12F compared to significantly prevalent serotypes in the nasopharynx. Genes in RD8a are involved in binding to epithelial cells and doing aerobics respiration to synthesize ATP through oxidative phosphorylation. The absence of RD8a from serotypes 1, 5, and 12F may be associated with their short duration in the nasopharynx where they need to bind to epithelial cells and access free oxygen molecules required for aerobic respiration. Given this, the amount of ATP is likely to decline in serotypes 1, 5, and 12F, causing them to harbour more phosphotransferase systems to transport carbohydrates since these transporters use phosphoenolpyruvate as the energy source instead of ATP. In conclusion, serotypes 1, 5, and 12F, the most prevalent and invasive pneumococcal strains in Malawi, showed a considerable genetic distinction from other strains that may be associated with their short colonization period and quickness to infect the blood and cerebrospinal fluid.

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Streptomycin resistance in both gram-positive and gram-negative bacteria is usually caused by a single mutation in the rpsL gene. The rpsL gene encodes the S12 protein of the ribosomal complex. The rpsL genes of various bacteria have consensus regions in their sequences. Primers were designed from these consensus pockets and a fragment of the rpsL gene was sequenced from S. gordonii using PCR based methodologies. Using the Multiplex Restriction Sequence PCR(mRS PCR), which used the known primer at one end and a restriction site primer on the other, a gene walk was conducted. In streptomycin resistant strains of S. gordonii, namely GP204, SP204 and SP635, the AAA coding for Lys56 was mutated to ACA, coding for Thr56. The lysine to threonine transition, causing resistance to streptomycin was identical to that expected from the literature. The streptomycin resistance gene of S. pyogenes was mapped using similar techniques. Streptomycin resistant strains S43 ATCC, 543/192/4 and S43/192/30R were studied. In streptomycin resistant S43 ATCC and S43/192/30R strains, the lysine 56 changed to isoleucine and threonine respectively. Surprisingly, the 192/4 had two mutations, in each of the two hotspots in the rpsL gene where mutations due to streptomycin resistance occur. It had the amino acid 56, lysine, mutated to arginine and lysine 101 changed to asparagine. To check if this mutation was stable in the host animal, S43/192/4 P8 (S43/192/4 passaged eight times in mice) was sequenced and the sequence was identical to the streptomycin resistant 192/4. Hence, the lys101 mutation was stable and unlike the ancillary mutations in E.coli and S. typhimurium, which are compensated by new mutations. The pathogenesis of S. pyogenes depends in part on the ability of the pathogen to adhere to the epithelial cells of the throat and the quantity of M protein. Pathogenesis studies done on mice revealed the avirulence of S43/192/4smR strain. To elucidate the reason for this avirulence, the adherence properties and the production of M protein of the two strains S43/192/4smR and S43/192/30R were tested. Qualitative immunoblot analysis of the M protein of 192/4 and 30R revealed no significant difference. Competition ELISA was conducted to quantitate the M protein, and this also did not show any significant difference in the M protein levels. The adherence of 30R and 192/4 was measured on human pharyngeal epithelial cell line. The adherence properties of S43/192/4 SmR, was no different from other strains in this experiment. Electron microscopy, using immunogold to highlight the M protein on the cell surface showed no differences. / Graduation date: 2000

Thesis (M.S.)--University of Louisville, 1998. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (M.S.)--University of Louisville, 1998. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (M.S.)--University of Louisville, 1996. / School of Dentistry, Program in Oral Biology. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (M.S.)--University of Louisville, 1996. / School of Dentistry, Program in Oral Biology. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.