RESUMO
Identification of presumptive foodborne pathogens grown on selective media may take one to several days and requires a different battery of biochemical tests for each microorganism. A molecular identification method was developed in which universal primers were used to amplify the 16S to 23S rDNA intergenic spacer of target microorganisms, and PCR products were hybridized to a panel of species-specific oligonucleotides that were immobilized on a nylon membrane. The seven target microorganisms were Bacillus cereus, Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella, Staphylococcus aureus, and Vibrio parahaemolyticus. After testing a large collection of target bacteria (29 to 51 strains) and nontarget bacteria (> 500 strains), the performances (sensitivity and specificity) of the oligonucleotide array were as follows: B. cereus (100 and 77%), E. coli (100 and 100%), L. monocytogenes (100 and 90%), P. aeruginosa (100 and 100%), Salmonella (100 and 100%), S. aureus (100 and 100%), and V. parahaemolyticus (100 and 94.2%). Other species in the B. cereus group cross-hybridized to the probes used for identification of B. cereus, and positive results should be confirmed by additional morphological observation of colonies. Listeria innocua cross-reacted with probes used to identify L. monocytogenes, but a simple hemolysis test was used to differentiate the two species. Some strains of Vibrio harveyi and Vibrio mimicus cross-hybridized with probes used for identification of V. parahaemolyticus and caused false-positive reactions. The advantage of the array is that a common protocol was used to identify the seven target microorganisms and multiple different microorganisms could be simultaneously identified on a single array.
Assuntos
Meios de Cultura/química , Contaminação de Alimentos/análise , Microbiologia de Alimentos , Análise de Sequência com Séries de Oligonucleotídeos , Pseudomonas aeruginosa/isolamento & purificação , Bacillus cereus/genética , Bacillus cereus/isolamento & purificação , Contagem de Colônia Microbiana , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Reações Falso-Positivas , Listeria monocytogenes/genética , Listeria monocytogenes/isolamento & purificação , Reação em Cadeia da Polimerase , Pseudomonas aeruginosa/genética , Salmonella/genética , Salmonella/isolamento & purificação , Sensibilidade e Especificidade , Especificidade da Espécie , Staphylococcus aureus/genética , Staphylococcus aureus/isolamento & purificação , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/isolamento & purificaçãoRESUMO
Eight isolates (no. 1 to 8) of Pseudomonas aeruginosa isolated from 8 burn patients were typed by pulsed-field gel electrophoresis (PFGE), arbitrarily primed polymerase chain reaction (PCR) (AP-PCR), biotyping, antimicrobial susceptibility testing, and a newly developed technique-impedance method. By both PFGE and AP-PCR, isolates 1 and 2 were designated type A, while isolates 3 to 8 were designated type B. However, isolates 3 to 8 could be further divided into three distinct subtypes (B, C, and D) by the impedance method. Four antibiograms were obtained by testing the 8 isolates against six antimicrobial agents and designation of antibiogram to each of the 8 isolates was in accordance with those obtained by the impedance method. The results of biotyping did not agree with any of the above typing methods. In conclusion, the impedance technique had a high discriminatory ability to differentiate genetically related clones into subtypes. The method is simple, reproducible, and has a high typeability.
