RESUMO
Plasmid profiles were used to analyse 39 Acinetobacter baumannii isolates from 36 patients at three hospitals. The isolates were prevously classified by biotyping and rDNA fingerprinting. Ribotyping was useful to establish the lineage of isolates and to confirm genospecies identification. Thirty-seven isolates (94.9%) contained plasmids. The variable number of plasmids with different molecular weights in each isolate enabled the identification of 13 profiles without the need for endonuclease digestion. Fifteen A. baumannii biotype 2 isolates of similar ribotype and antibiotype contained identical plasmids over a two-month outbreak at one hospital. Plasmid typing discriminated these isolates from sporadic A. baumannii isolates of close ribotype obtained from different hospitals. A few isolates of different lineage, however, showed similar plasmid profile. Our results suggest that plasmid typing is a practical method to assist infection control of nosocomial A baumannii. A combination of plasmid typing and ribotyping is suggested to confirm genospecies classification and to identify strains against reference band profiles.
Assuntos
Acinetobacter/classificação , Acinetobacter/isolamento & purificação , Infecção Hospitalar/microbiologia , Plasmídeos/classificação , Plasmídeos/isolamento & purificação , Acinetobacter/efeitos dos fármacos , Técnicas Bacteriológicas , Humanos , Testes de Sensibilidade MicrobianaRESUMO
Fifty-one Pseudomonas aeruginosa isolates were differentiated into 21 types by ribotyping. Several enzyme combinations, including the best ones proposed in literature, were utilized and the highest discrimination was reached by individual digestion with PvuII, HindII, and EcoRI or BamHI. Clinical isolates from outbreaks were clonally related as identified by this molecular approach. Restriction rDNA profiles were composed of strong and weak bands. Using 6 micrograms DNA we were able to demonstrate that PvuII, HindIII, and BamHI weak bands were reproducible. These weak bands should be considered not only to accomplish the highest discrimination but also to correctly assign isolate clonality. Conversely, we found that EcoRI weak bands were not reproducible and, therefore, are not recommended for ribotype analysis. Finally, profiles differing in one single band actually represented isolates of different genotype, as confirmed by further analysis using other molecular methods. In this report on P. Aeruginosa ribotyping of clinical isolates, criteria for band pattern interpretation are established.
Assuntos
DNA Ribossômico/análise , Pseudomonas aeruginosa/genética , Técnicas de Tipagem Bacteriana/normas , Southern Blotting , Epidemiologia Molecular , Dados de Sequência Molecular , Óperon , Polimorfismo de Fragmento de Restrição , Reprodutibilidade dos Testes , Mapeamento por RestriçãoRESUMO
Ribotyping, exotoxin A genotyping (EAGP), and restriction fragment length polymorphism (RFLP) analysis of total DNA with SalI (SalI RFLP) were compared for intraspecies discrimination of 93 Pseudomonas aeruginosa isolates. Type-ability of all methods was 100% and the results of typing with each method remained unchanged during laboratory manipulation. Clonal groups defined with each molecular method were largely coincident and, in those cases where inconsistencies were detected, isolates were analyzed by transverse alternating field gel electrophoresis (TAFE) and arbitrarily primed polymerase chain reaction (AP-PCR). SalI RFLP analysis was highly discriminative so as to distinguish unrelated isolates of close lineage. However, it was not a good method to identify isolates of unrelated lineage because SalI RFLP appeared to be subjected to convergent evolution. The index of discrimination suggested by Hunter and Gaston was determined to assess the discriminatory power of the molecular methods utilized either alone or in several combinations. Combined use of ribotyping and SalI RFLP analysis reached the highest index of discrimination (0.982) and proved to be a very valuable tool for epidemiological differentiation of P. aeruginosa isolates.
Assuntos
Técnicas de Tipagem Bacteriana , DNA Ribossômico/análise , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Exotoxinas/genética , Pseudomonas aeruginosa/genética , Southern Blotting , Eletroforese em Gel de Ágar , Genótipo , Testes de Sensibilidade Microbiana , Filogenia , Polimorfismo de Fragmento de RestriçãoRESUMO
We have recently demonstrated that treatment with interleukin 1 beta (IL-1 beta) plus tumor necrosis factor alpha (TNF alpha) protects granulocytopenic hosts from Pseudomonas aeruginosa aerosol challenge. In this study we characterized the inflammatory response induced by P. aerugionsa in granulocytopenic mice treated with 2,000 U IL-1 beta plus 2,000 U TNF alpha. Treatment with the nonsteroidal anti-inflammatory agent piroxicam abolished both the protective effect of cytokine treatment and the increase in myeloperoxidase (MPO) pulmonary activity. Histopathological studies revealed that, after aerosol challenge with P. aeruginosa, treatment with these cytokines induced migration and extravasation of mononuclear cells of immature appearance into the lung parenchyma. These cells contained MPO in their cytoplasm and displayed phagocytic capacity. Resident alveolar macrophages exhibited signs of activation and appeared in reduced numbers in bronchoalveolar lavage fluid. We suggest that the inflammatory response promoted by low TNF alpha plus IL-1 beta doses may be one mechanism responsible for protection of granulocytopenic hosts from P. aeruginosa aerosol challenge.