HB - Patologia Clínica
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Browsing HB - Patologia Clínica by Author "Azeredo, J"
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- Candida clinical species identification: molecular and biochemical methodsPublication . Costa, AR; Silva, F; Henriques, M; Azeredo, J; Oliveira, R; Faustino, AIn the last decade, the number and diversity of nosocomial Candida infections has increased significantly, resulting in an emergent need for rapid and accurate methods for Candida identification. Therefore, the aim of this study was to evaluate the performance of three biochemical systems (Auxacolor, ID32C, and Vitek 2 YST) for the identification of Candida species, comparing them with molecular identification (polymerase chain reaction and gel agarose electrophoresis). These methods were used to assess Candida spp. (229 clinical isolates) prevalence and distribution among clinical specimens. The biochemical methods with higher percentages of correct identification were Vitek 2 YST (79.6%) and Auxacolor (78.6%). However, overall the biochemical methods assayed differed from the molecular identification. Thus, due to their rapid and precise identification, molecular methods are promising techniques for Candida species identification in clinical laboratories. Candida albicans and Non Candida albicans Candida species had a similar prevalence (50.4 and 49.6%, respectively), corroborating the epidemiological shift observed for these pathogens in the recent years.
- Characterization and genome sequencing of a Citrobacter freundii phage CfP1 harboring a lysin active against multidrug-resistant isolatesPublication . Oliveira, H; Pinto, G; Oliveira, A; Oliveira, C; Faustino, MA; Briers, Y; Domingues, L; Azeredo, JCitrobacter spp., although frequently ignored, is emerging as an important nosocomial bacterium able to cause various superficial and systemic life-threatening infections. Considered to be hard-to-treat bacterium due to its pattern of high antibiotic resistance, it is important to develop effective measures for early and efficient therapy. In this study, the first myovirus (vB_CfrM_CfP1) lytic for Citrobacter freundii was microbiologically and genomically characterized. Its morphology, activity spectrum, burst size, and biophysical stability spectrum were determined. CfP1 specifically infects C. freundii, has broad host range (>85 %; 21 strains tested), a burst size of 45 PFU/cell, and is very stable under different temperatures (-20 to 50 °C) and pH (3 to 11) values. CfP1 demonstrated to be highly virulent against multidrug-resistant clinical isolates up to 12 antibiotics, including penicillins, cephalosporins, carbapenems, and fluroquinoles. Genomically, CfP1 has a dsDNA molecule with 180,219 bp with average GC content of 43.1 % and codes for 273 CDSs. The genome architecture is organized into function-specific gene clusters typical for tailed phages, sharing 46 to 94 % nucleotide identity to other Citrobacter phages. The lysin gene encoding a predicted D-Ala-D-Ala carboxypeptidase was also cloned and expressed in Escherichia coli and its activity evaluated in terms of pH, ionic strength, and temperature. The lysine optimum activity was reached at 20 mM HEPES, pH 7 at 37 °C, and was able to significantly reduce all C. freundii (>2 logs) as well as Citrobacter koseri (>4 logs) strains tested. Interestingly, the antimicrobial activity of this enzyme was performed without the need of pretreatment with outer membrane-destabilizing agents. These results indicate that CfP1 lysin is a good candidate to control problematic Citrobacter infections, for which current antibiotics are no longer effective.
- Efficacy of a broad host range lytic bacteriophage against E. coli adhered to urotheliumPublication . Sillankorva, S; Oliveira, D; Moura, A; Henriques, M; Faustino, A; Nicolau, A; Azeredo, JPersistent urinary tract infections (UTI) are often caused by E. coli adhered to urothelium. This type of cells is generally recognized as very tolerant to antibiotics which renders difficult the treatment of chronic UTI. This study investigates the use of lytic bacteriophages as alternative antimicrobial agents, particularly the interaction of phages with E. coli adhered to urothelium and specifically determines their efficiency against this type of cells. The bacterial adhesion to urothelium was performed varying the bacterial cell concentrations and the period and conditions (static, shaken) of adhesion. Three collection bacteriophages (T1, T4, and phiX174 like phages) were tested against clinical E. coli isolates and only one was selected for further infection experiments. Based on the lytic spectrum against clinical isolates and its ability to infect the highest number of antibiotic resistant strains, the T1-like bacteriophage was selected. This bacteriophage caused nearly a 45% reduction of the bacterial population after 2 h of treatment. This study provides evidence that bacteriophages are effective in controlling suspended and adhered cells and therefore can be a viable alternative to antibiotics to control urothelium- adhered bacteria.
- Use of newly isolated phages for control of Pseudomonas aeruginosa PAO1 and ATCC 10145 biofilmsPublication . Pires, D; Sillankorva, S; Faustino, A; Azeredo, JPseudomonas aeruginosa is a relevant opportunistic pathogen involved in nosocomial infections that frequently shows low antibiotic susceptibility. One of its virulence factors is associated with the ability to adhere to surfaces and form virulent biofilms. This work describes the isolation and characterization of lytic phages capable of infecting antibiotic-resistant P. aeruginosa strains. In addition, characterization of P. aeruginosa biofilms and the potential of newly isolated phages for planktonic and biofilm control was accessed. According to the results, the isolated phages showed different spectra of activity and efficiency of lysis. Four broad lytic phages were selected for infection of planktonic cells; however, despite their broad range of activity, two of the selected phages failed to efficiently control planktonic cultures. Therefore, only two phages (phiIBB-PAA2 and phiIBB-PAP21), highly capable of causing strong biomass reduction of planktonic cells, were tested against 24 h biofilms using a m.o.i. of 1. Both phages reduced approximately 1-2 log the biofilm population after 2 h of infection and reduction was further enhanced after 6 h of biofilm infection. However, biofilm cells of P. aeruginosa PAO1 acquired resistance to phiIBB-PAP21; consequently, an increase in the number of cells after 24 h of treatment was observed. Conversely, phage phiIB-PAA2 for P. aeruginosa ATCC10145 continued to destroy biofilm cells, even after 24 h of infection. In these biofilms, phages caused a 3 log reduction in the number of viable counts of biofilm cells.