Molecular evaluation of the mechanisms that determine antimicrobial resistance in Pseudomonas aeruginosa and Acinetobacter spp.

Author(s)

Eduardo Carneiro Clímaco

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto (PCARP/BC)
• Defense date: 2011-08-19

Examining board members

Ana Lucia da Costa Darini; Juliana Pfrimer Falcão; Nilton Erbet Lincopan Huenuman; Luisa Maria Sobreira Vieira Peixe; Fernando Bellissimo Rodrigues

• Advisor: Ana Lucia da Costa Darini

Abstract

The non-fermenting pathogenic bacteria Pseudomonas aeruginosa and Acinetobacter spp. are important causes of nosocomial infections. Theses species are often associated with a multidrug resistance (MDR) phenotype, due to intrinsic and acquired resistance genes. Some determinants of resistance, such as integrons, carbapenemases, overexpression of efflux systems and porins loss may be associated with the MDR phenotype. The aim of this study was to evaluate the association of non-MDR and MDR phenotypes in P. aeruginosa and Acinetobacter spp. to the presence of integrons and carbapenemases encoding genes, the overexpression of mexY, mexB, mexD and adeB genes and loss of the outer membrane protein, OprD. These resistance determinants were evaluated in 147 P. aeruginosa and 57 Acinetobacter spp., isolated from in-patients of University Hospital of UFJF. Isolates with different PFGE and non-susceptibility profiles were grouped according to MDR or non-MDR phenotypes. PCR and real-time RT-PCR were used to investigate the presence of class 1, 2 and 3 integrons and carbapenemase encoding genes and the expression of mexY, mexB, mexD and adeB efflux pumps and OprD porin, respectively. Class 1 integrons were one of the most common genetic elements present in MDR P. aeruginosa (44,2%), but the phenotype could not be attributed to these elements, since they showed empty (43/65) or only aminoglycoside gene cassettes (19/65). Class 2 integrons were the most common genetic elements in MDR Acinetobacter spp., and this association was statistically significant. SPM encoding gene was the only carbapenemase gene found in P. aeruginosa and, predominantly, in the PFGE cluster A. Expression of MexXY-OprM determined by real-time RT-PCR was the highest variable between MDR and non-MDR P. aeruginosa isolates (almost 10-fold). Reduction of 66.4% in OprD expression was observed in MDR P. aeruginosa, in comparison with non-MDR ones. It is concluded that the most important genetic determinants in the MDR phenotype of P. aeruginosa were SPM-1 production, followed by MexXY-OprM over expression and diminished production of OprD, while class 2 integrons was the most important genetic determinant of MDR phenotype in A. baumannii. (AU)

• FAPESP's process: 07/04580-0 - Molecular analysis of resistance mechanism in Pseudomonas aeruginosa and Acinetobacter spp.
• Grantee: Eduardo Carneiro Clímaco
• Support Opportunities: Scholarships in Brazil - Doctorate