ABSTRACT
PFGE (pulsed field gel eletrophoresis) é a sigla usada para indicar qualquer técnica de eletroforese apropriada para separar grandes fragmentos de DNA, por meio da reorientação do DNA em gel pela ação de campos elétricos alternados. Esta técnica é reconhecida como padrão ouro para identificação de linhagens bacterianas, fúngicas e de protozoários. O principal objetivo deste estudo de revisão é o da elucidação dos fundamentos da técnica de PFGE ou eletroforese de campo pulsante aplicada em estudo com bactéria. Sua resolução depende de uma série de fatores como: voltagem, concentração de agarose, temperatura, solução tamponante, tempo de pulso e de corrida eletroforética. A compreensão dos mecanismos moleculares envolvidos nesse tipo de eletroforese é fundamental para a otimização e obtenção dos resultados apropriados.
Subject(s)
DNA, Bacterial , Electrophoresis, Gel, Pulsed-Field , Molecular Epidemiology , Bacteriological Techniques , GenotypeABSTRACT
An outbreak of bacteraemia in a haemodialysis unit where 65 episodes of infection involved 35 outpatients is reported. Burkholderia cepacia complex was the agent most frequently recovered from blood. Thirty-three environmental and clinical isolates of B. cepacia complex were characterized by whole-cell protein electrophoresis and recA-RFLP profile. Fourteen isolates were genomovar I and 16 isolates were not classifiable by their recA-RFLP pattern. Ribotyping, random amplification of polymorphic DNA (RAPD) and integron profile were used to explore the clonality of the isolates, and revealed multiple strain genotypes. Four ribotypes and RAPD types and three integron patterns were identified. The water supply was identified as the source of the outbreak, and inappropriate cleaning and a leak in the reverse osmosis tubing connection were the probable causes of contamination. B. cepacia complex was still recovered from blood of patients even after apparently adequate measures were taken and water quality standards were met, suggesting that higher standards for water quality should be adopted in haemodialysis units. The genomovars recovered here were distinct from those commonly reported for cystic fibrosis isolates.
Subject(s)
Burkholderia Infections/epidemiology , Burkholderia cepacia/genetics , Cross Infection/epidemiology , Disease Outbreaks , Hemodialysis Units, Hospital , Burkholderia cepacia/classification , Humans , Integrons , Random Amplified Polymorphic DNA Technique , Ribotyping , Water MicrobiologyABSTRACT
OBJECTIVE: To investigate an apparent outbreak involving simultaneous isolation of Pseudomonas aeruginosa and Serratia marcescens from bronchoalveolar lavage (BAL) samples. DESIGN: Retrospective and prospective cohort studies using chart review, environmental sampling, and ribotyping of all available isolates. Cleaning and disinfection procedures for the bronchoscopes were also evaluated. SETTING: A 380-bed private hospital in São Paulo, Brazil PATIENTS: Forty-one patients who underwent bronchoscopic procedures between December 1994 and October 1996 and from whom P. aeruginosa and S. marcescens were concomitantly isolated. Bronchoscopes and related items were microbiologically assessed. RESULTS: P. aeruginosa and S. marcescens were simultaneously isolated from BAL samples 12.6% of the time (41 of 324) during the epidemic period versus 1.8% of the time (1 of 54) in the pre-epidemic period (P = .035). Ribotyping revealed two strains of P. aeruginosa and one of S. marcescens that were isolated from BAL samples of patients with no signs of respiratory tract infection, suggesting a pseudo-outbreak. Evaluation of bronchoscope disinfection revealed that inappropriate methods were being used. Implementation of simple control measures resulted in a significant decrease in simultaneous isolation of these species. CONCLUSION: Prevention of pseudo-outbreaks requires meticulous use of preventive measures for infection-prone medical procedures.
Subject(s)
Bronchoscopes/microbiology , Bronchoscopy/adverse effects , Cross Infection/epidemiology , Cross Infection/etiology , Disease Outbreaks , Equipment Contamination , Pseudomonas Infections/transmission , Pseudomonas aeruginosa/isolation & purification , Serratia Infections/transmission , Serratia marcescens/isolation & purification , Brazil/epidemiology , Bronchoalveolar Lavage , Cohort Studies , Disinfection/methods , Equipment Reuse , Hospitals, Private , Humans , Prospective Studies , Pseudomonas aeruginosa/pathogenicity , Retrospective Studies , Ribotyping , Serratia marcescens/pathogenicityABSTRACT
Eighty-five Pseudomonas aeruginosa isolates resistant to various antibiotics were selected from the intensive care unit of a Brazilian hospital and analyzed for integron content by PCR. Fifty-four of them had class-1-related integrons, some of which were identical. Integron-positive isolates were statistically more resistant to aminoglicoside, quinolone and beta-lactam compounds. Ribotyping of integron-positive isolates demonstrated the presence of identical integrons among genetically unrelated strains in the hospital environment, confirming its role in the spread of gene cassettes in bacterial populations of clinical importance.
Subject(s)
Drug Resistance, Bacterial/genetics , Integrons , Intensive Care Units , Pseudomonas aeruginosa/drug effects , Anti-Bacterial Agents/pharmacology , Brazil/epidemiology , Humans , Microbial Sensitivity Tests , Pseudomonas Infections/microbiology , Pseudomonas Infections/transmission , Pseudomonas aeruginosa/genetics , Ribotyping , Sequence Analysis, DNAABSTRACT
A tipagem molecular do genoma bacteriano, na maioria das vezes, envolve a análise de fragmentos de restriçäo do DNA cromossômico. Desde que a ribotipagem foi descrita, em 1986, tem sido amplamente utilizada para analisar relaçöes taxonômicas e/ou epidemiológicas entre os diferentes grupos de organismos. A ribotipagem usa o padräo de restriçäo do opéron de RNA ribossômico (rrn) como ferramenta epidemiológica e tem fornecido ótimos resultados para a detecçäo de polimorfismo do comprimento dos fragmentos de restriçäo (RFLPs). O número de opérons rrn da bactéria está diretamente relacionado ao potencial discriminatório da técnica, fornecendo um maior ou menor número de bandas.
Subject(s)
Humans , Bacterial Infections , Bacterial Typing Techniques , Molecular Epidemiology , DNA, Bacterial , Electrophoresis, Agar Gel , Polymerase Chain Reaction , SerotypingABSTRACT
A freqüência de transposiçäo do Tn1 do plasmídio pTH10 para o cromossomo da célula hospedeira foi maior em Proteus mirabilis que em Escherichia coli. Contudo pode-se supor que os valores obtidos em P. mirabilis foram subestimados desde que o plamídio doador do transposoma é instavél na linhagem PG1342. Experimentos de hibridaçäo sugerem que existem mais do que uma inserçäo por cromossomo de P. mirabilis, mas análises adicionais säo necessárias para a localizaçäo dessas inserçöes. Se o transposon T n1 se insere em um ponto quente do cromossomo da célula hospedeira, esse alvo näo causou auxotrofia em nenhum dos clones testados de PG1342::Tn1. Transposiçöes entre plasmídios albergados por P. mirabilis parecem näo ocorrer em níveis maiores do que para o cromossomo, confrome relatados para E. coli. Outrossim, as freqüências de inserçäo do Tn1 variam de acordo com o DNA alvo. A seleçäo de um sítio de inserçäo ocorre diferentemente nos dois gêneros, sugerindo que fatores dos hospedeiros têm uma participaçäo importante nessa seleçäo. Por outro lado, Tn1 mostrou inserçäo preferencial em outrom transposon (Tn402), independentemente do hospedeiro. Neste caso a inserçäo do Tn1 näo é estável, levando à excisäo do transposon ampicilina
