RESUMO
We studied 40 E. coli strains from meat and hamburger and 64 strains from stools of people: 14/64 from traveler's diarrhea and 50/64 from infants with and without diarrhea. We want to know if they could be producing of cytotoxin and enterotoxin in Vero culture cells as well as phenotypic and genotypic relationships. The serotypes that we isolated were different than O:157H:7. We found 3 cytotoxic strains, 77 heat labile enterotoxic strains on Vero culture cells, and 19 E. coli strains with cytotoxin and LT enterotoxin. One strain isolated from infant with diarrhea was negative sorbitol but cytotoxic effect and it was O:55 serotype. The colony blot and cytotonic were found in 90 (86.5%) E. coli strains. The sensitivity of colony blot was 93.75%.
Assuntos
Citotoxinas/isolamento & purificação , Enterotoxinas/isolamento & purificação , Proteínas de Escherichia coli , Escherichia coli/química , Adulto , Animais , Toxinas Bacterianas/genética , Pré-Escolar , Chlorocebus aethiops , Meios de Cultura , Diarreia/epidemiologia , Diarreia/microbiologia , Diarreia Infantil/microbiologia , Surtos de Doenças , Enterotoxinas/genética , Escherichia coli/classificação , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Escherichia coli/metabolismo , Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/microbiologia , Fezes/microbiologia , Feminino , Microbiologia de Alimentos , Humanos , Lactente , Masculino , México/epidemiologia , Hibridização de Ácido Nucleico , Sensibilidade e Especificidade , Sorotipagem , Sorbitol/metabolismo , Células VeroRESUMO
PCR was made with ctx2 (CGG GCA GAT TCT AGA CCT CCT G) y ctx3 (CGA TGA TCT TGG AGC ATT CCC AC) primers for subunit A of cholera toxin, 30 cycles of temperature on samples of 50 g of oysters added in 450 ml of peptone alcaline water that were inoculated with 15 x 10(6), 0.75 x 10(6) and 0.15 x 10(6) CFU/ml of toxigenic 6707 V. cholerae O1 reference strain. The samples were tested by three microbiological methods: INDRE's method uses 1 x 10(-1) dilution of sample, two fold pass to peptone alcaline water pH 9 incubated 18 h and 6 h at 37 degrees C, the Food and Drugs Administration (FDA) method uses 10(-1) to 10(-6) dilutions of sample, 6 h incubation and reincubation for 18 h at 37 and 42 degrees C and the Mexican laboratories (LMD) with 10(-4) to 10(-3) dilutions, the samples were incubated for 6 h and then reincubated for 18 h at two temperatures 37 and 42 degrees C. The PCR by INDRE's method was positive with 3 x 10(2) CFU/ml/g oyster. In the FDA's method the PCR detected DNA in 10(-4) dilution with 3 x 10(1) CFU/ml/g oyster and in LMD's method the PCR was positive in 10(-3) with 3 CFU/ml/g oyster. The results of the PCR were obtained between 5-6 h, and later V. cholerae O1 was isolated by three microbiological methods. The PCR reproducibility was better on DNA sample diluted 1:4 and 10 microliters of sample increased from 1:1000 to 1:10000 the sensitivity of PCR.
Assuntos
Toxina da Cólera/genética , DNA Bacteriano/isolamento & purificação , Ostreidae/microbiologia , Reação em Cadeia da Polimerase , Vibrio cholerae/isolamento & purificação , Animais , Técnicas Bacteriológicas , Sequência de Bases , DNA Bacteriano/genética , Dados de Sequência Molecular , Vibrio cholerae/genética , Vibrio cholerae/crescimento & desenvolvimentoRESUMO
We made 52180 tests for isolation and identification of toxigenic V. cholerae O1 from rectal swabs and reference strains. We isolated 17.6% V. cholerae O1 strains in 1991, 43.5% in 1992 and 38.9% in 1993. The main serovar in 1991 was Inaba, whereas in 1993 a similar percentage was serovar Ogawa. The phenotype of V. cholerae strains was determined by hemolysis test, Voges-Proskauer test, polymyxin B resistance and phages 4 and 5 resistance. All of the mexican strains were El Tor. There were 2.9-0.75% hemolytic strains from 1991 to 1993, but they were negative when the test was made in tube with human erythrocytes. The resistotypes were performed in 24526 selected strains by Kirby-Bauer method and MIC tests. All of the strains were sensitive, except more than 100 strains isolated in Veracruz that were resistant to tetracycline and doxycycline. Detection of cholera toxin was made by ELISA and on culture of Vero and CHO cells. All the V. cholerae O1 strains were toxigenic. The genotype was determined by PCR and ribotyping. The PCR amplified one 564 pb fragment on V. cholerae O1. The ribotypes of mexican strains were 5 and 6a.
Assuntos
Vibrio cholerae/classificação , Técnicas de Tipagem Bacteriana , Cólera/epidemiologia , Cólera/microbiologia , Toxina da Cólera/análise , Toxina da Cólera/genética , Surtos de Doenças , Resistência Microbiana a Medicamentos , Fezes/microbiologia , Hemólise , Humanos , México/epidemiologia , Fenótipo , Reação em Cadeia da Polimerase , Sorotipagem , Esgotos , Vibrio cholerae/efeitos dos fármacos , Vibrio cholerae/genética , Vibrio cholerae/isolamento & purificaçãoRESUMO
We studied 40 Vibrio cholerae strains: 16 from stool, 16 from sewage and 8 from food. The serotypes were Inaba in 21 strains, 8 Ogawa strains and 11 V. cholerae non-O1. PCR was made with ctx2 and ctx3 primers with 25 cycles of temperature: 1 min at 94 degrees C, 1 min at 60 degrees C and 1 min at 72 degrees C. 24 V. cholerae strains were positive: 18/24 Inaba y 6/24 Ogawa. PCR was negative for 16 strains: 3 Inaba serotype, 2 Ogawa y 11 V. cholerae non-O1. In Vero culture cells 18 strains were cytotonic, 21 cytotoxic and 1 strain was negative. ELISA was positive for 11 strains with PCR positive. The PCR sensitivity was 95.83% compared with culture cells. V. cholerae O1 produced cytotoxic effect on Vero culture cells, maybe related to ACE factor. Colony blot was made with a specific probe labeled with digoxigenin and it could detect 4 Vibrio cholerae toxigenic strains with PCR negative. All V. cholerae Non O1 strains were PCR negative.