RESUMO
Five chromosomal genes, prfA, plcA, hlyA, mpl and plcB, are implicated in the virulence of Listeria monocytogenes and some of these genes have been used for the identification of bacteria by polymerase chain reaction (PCR). Using 6 strains of L. monocytogenes and 3 L. innocua strains, the relationship was examined between the presence of five virulence-associated genes and actual virulence to mice in terms of 50% lethal dose (LD50), bacterial viability in the organ of infected mice and the intracellular growth in cultured macrophages. None of the five genes could be amplified by PCR in all the L. innocua strains and they were actually avirulent to mice. All L. monocytogenes strains were shown to be virulent and to have intact virulence-associated genes except for the strain ATCC15313. This particular strain was revealed to be avirulent and defective in hlyA and plcA in PCR amplification. It was suggested that PCR detection of genes prfA, mpl, or plcB may not be sufficient to detect virulent strains of L. monocytogenes. It appeared that the ability to produce listeriolysin O (LLO), which is encoded by hlyA, was critical for the expression of virulence regardless of the amount of LLO produced.
Assuntos
Proteínas de Bactérias/fisiologia , Toxinas Bacterianas , Genes Bacterianos , Listeria/genética , Listeriose/microbiologia , Animais , Proteínas de Bactérias/genética , Sequência de Bases , DNA Bacteriano/genética , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/fisiologia , Proteínas Hemolisinas , Dose Letal Mediana , Listeria/patogenicidade , Listeria monocytogenes/genética , Listeria monocytogenes/patogenicidade , Macrófagos Peritoneais/microbiologia , Metaloendopeptidases/genética , Metaloendopeptidases/fisiologia , Camundongos , Dados de Sequência Molecular , Fatores de Terminação de Peptídeos , Fosfatidilinositol Diacilglicerol-Liase , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/fisiologia , Reação em Cadeia da Polimerase , Especificidade da Espécie , Transativadores/genética , Transativadores/fisiologia , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/fisiologia , Virulência/genéticaRESUMO
The inhibitory effect of milk in the PCR detection of Listeria monocytogenes could be overcome by washing the contaminated milk sample with phosphate-buffered saline and concentrating the bacteria to 1/10 of the original volume. In order to avoid a possible failure in the detection of virulent L. monocytogenes, a one-step procedure which enabled demonstration of three virulence-associated genes, prfA, hlyA, and plcB, simultaneously in a single PCR mixture was developed.
