Aplicação de porfirinas como fotossensibilizadores para a inativação fotodinâmica de microrganismos da cavidade oral

A cavidade oral é um habitat favorável ao desenvolvimento de microrganismos, alguns dos quais podem causar doenças, sendo Enterococcus faecalis uma bactéria frequentemente encontrada em biofilmes instalados em diferentes nichos da cavidade oral. Este trabalho teve como objetivo testar a aplicabilidade da inativação fotodinâmica (PDI), usando porfirinas como fotossensibilizadores, como estratégia de controlo de biofilmes da cavidade oral, tomando E. faecalis como microrganismo modelo. Como fotossensibilizadores, foram testadas as porfirinas catiónicas Tetra-Py+-Me, Tri-Py+-Me-PF, PCat 2, PCat 3, PCat 4 e o corante azul de toluidina O (TBO), incluído como fotossensibilizador de referência. Os biofilmes de E. faecalis foram irradiados com luz branca (270 J.cm-2) a uma intensidade de 150 mW.cm-2, na presença de até 50 µM de porfirina ou até 20 µM de TBO. A cinética de inativação foi caracterizada pela variação da concentração de células viáveis ao longo da experiência. Foi também testada a inativação de células na forma livre, em condições equivalentes. Os biofilmes de E. faecalis mostraram-se muito resistentes à PDI com qualquer dos PS testados, não tendo sido conseguidos fatores de inativação superiores a 2 log com a concentração máxima de PS (50 µM) e a dose máxima de luz (270 J.cm-2). Na forma livre as células foram inativadas até ao limite de quantificação com concentrações de PS de 0,5 µM e doses de luz até 108 J.cm-2, com uma intensidade de 10 mW.cm-2. No entanto, a eficiência de ligação dos PS às células livres não foi maior do que aos biofilmes. Embora os fatores de inativação obtidos não permitam ainda considerar que a PDI com os compostos testados seja uma abordagem antimicrobiana eficiente contra biofilmes de E. faecalis, o facto de se confirmar uma relação entre as propriedades químicas e físicas do PS e a sua eficiência, bem como os resultados muito promissores obtidos com uma das famílias de porfirinas testadas apenas em células livres, justifica a prossecução do desenvolvimento de novos PS para o controle de biofilmes bacterianos na cavidade oral.

Antimicrobial resistance and species composition of Enterococcus spp. isolated from waters and sands of marine recreational beaches in Southeastern Brazil

Density, species composition and antimicrobial resistance in bacteria of the Enterococcus genus were evaluated in seawater and sands from 2 marine recreational beaches with different levels of pollution. The 2 beaches showed predominance of Enterococcus faecalis and Enterococcus faecium, in the water and the sand. Dry sand presented higher densities of Enterococcus sp. and higher frequency of resistant strains than wet sand and seawater. The beach with a higher degree of pollution presented higher percentages of resistant strains (66.7% and 61.5%, in sand and in water, respectively) and resistance to a larger number of antimicrobials compared with the less polluted beach, Ilha Porchat (35.7% and 31.25% of resistant strains in sand and water, respectively). in water samples, the highest frequencies of resistance were obtained against streptomycin (38.5%) and erythromycin (25%), whilst in sand, the highest frequencies were observed in relation to erythromycin and tetracycline (38.1% and 14.3%, respectively). These results show that water and sands from beaches with high indexes of faecal contamination of human origin may be potential sources of contamination by pathogens and contribute to the dissemination of bacterial resistance. (C) 2007 Elsevier Ltd. All rights reserved.

Synthesis and antimicrobial activity of some 2- Piperidinomethylamino-4-(7-H/substituted coumarin-3-yl)- 6-chlorosubstitutedphenyl pyrimidines

Purpose: To prepare and evaluate some 2-piperidinomethylamino-4-(7-H/substitutedcoumarin-3-yl)-6- chlorosubstitutedphenyl pyrimidines as antimicrobial agents. Methods: Some 2-piperidinomethylamino-4-(7-H/substitutedcoumarin-3-yl)-6-chlorosubstitutedphenyl pyrimidines were prepared by reacting 2-amino-4-(7-H/substitutedcoumarin-3-yl)-6- (chlorosubstitutedphenyl) pyrimidines with piperidine and formaldehyde. The chemical structures of the synthesized compounds were elucidated by Fourier transform infrared (FTIR), 1H-nuclear magnetic resonance (1H-NMR), mass spectrometry and elemental analysis. These compounds were investigated for their antimicrobial activity against ten bacteria and five fungi by serial plate dilution method using standard drugs, namely, ofloxacin and ketoconazole, respectively, and their minimum inhibitory concentrations (MICs) were also determined. Results: A total of eighteen new compounds (1a-18a) were synthesized. Compound 6a (MIC = 50 μg/mL; p < 0.05 or less) displayed the highest activity against S. aureus , E. faecalis , Staphylococcus epidermidis , B. subtilis , and B. cereus . Compound 6a further showed good activity (MIC = 25 μg/mL; p < 0.05 or less) against E. coli ; P. aeruginosa K. pneumonia , B. bronchiseptica , and P. vulgaris . Compounds 6a (MIC = 25 μg/mL; p < 0.0001) and 17a (MIC = 25 μg/mL; p < 0.0001) displayed very good activity against C. albicans , A. niger , A. flavus , M. purpureous , and P. citrinum , respectively. Analysis of structure-activity relationship revealed that the presence of bromo group at 7-postion of the coumarin moiety along with the 4-chlorophenyl group at position-6 of the pyrimidine ring is critical for antimicrobial activity against Gram-positive bacteria, Gram negative bacteria and fungi. Conclusion: The synthesized 2-piperidino derivatives are better antifungal and antibacterial agents than the earlier reported 2-morpholino derivatives, but require further investigations against other microbial strains to ascertain their broad spectrum antimicrobial activity.

Convergent evolution of PICIs-mediated phage interference

Staphylococcal pathogenicity islands (SaPIs), the prototype members of the family of phage inducible chromosomal islands (PICIs), are extremely mobile phage satellites, which are transferred between bacterial hosts after their induction by a helper phage. The intimate relationship between SaPIs and their helper phages is one of the most studied examples of virus satellite interactions in prokaryotic cells. SaPIs encode and disseminate virulence and fitness factors, representing a driving force for bacterial adaptation and pathogenesis. Many SaPIs encode a conserved morphogenetic operon, including a core set of genes whose function allows them to parasitize and exploit the phage life cycle. One of the central mechanisms of this molecular piracy is the specific packaging of the SaPI genomes into reduced sized capsid structures derived from phage proteins. Pac phages were classically thought to be the only phages involved in the mobilisation of phage-mediated virulence genes, including the transfer of SaPIs within related and non-related bacteria. This study presents the involvement of S. aureus cos phages in the intra- and intergeneric transfer of cos SaPIs for the first time. A novel example of molecular parasitism is shown, by which this newly characterised group of cos SaPIs uses two distinct and complementary mechanisms to take over the helper phage packaging machinery for their own reproduction. SaPIbov5, the prototype of the cos SaPIs, does not encode the characteristic morphogenetic operon found in pac SaPIs. However, cos SaPIs features both pac and cos phage cleavage sequences in their genome, ensuring SaPI packaging in small- and full-sized phage particles, depending on the helper phage. Moreover, cos-site packaging in S. aureus was shown to require the activity of a phage HNH nuclease. The HNH protein functions together with the large terminase subunit, triggering cleavage and melting of the cos-site sequence. In addition, a novel piracy strategy, severely interfering with the helper phage reproduction, was identified in cos SaPIs and characterised. This mechanism of piracy depends on the cos SaPI-encoded ccm gene, which encodes a capsid protein involved in the formation of small phage particles, modifying the assembling process via a scaffolding mechanism. This strategy resembles the ones described for pac SaPIs and represents a remarkable example of convergent evolution. A further convergent mechanism of capsid size-reduction was identified and characterised for the Enterococcus faecalis EfCIV583 pathogenicity island, another member of the PICI family. In this case, the self-encoded CpmE conducts this molecular piracy through a putative scaffolding function. Similar to cos SaPIs, EfCIV583 carries the helper phage cleavage sequence in its genome enabling its mobilisation by the phage terminase complex. The results presented in this thesis show how two examples of non-related members of the PICI family follow the same evolutionary convergent strategy to interfere with their helper phage. These findings could indicate that the described strategies might be widespread among PICIs and implicate a significant impact of PICIs mediated-virulence gene transfer in bacterial evolution and the emergence of pathogenic bacteria.