RESUMO
We assessed the ability of gene transfer to reverse vancomycin resistance in class A (VanA) glycopeptide-resistant Enterococcus faecalis. Recombinant shuttle vectors containing a vanH promoter-vanA antisense gene cassette fully restored vancomycin susceptibility through a combined transcriptional activator binding domain decoy and inducible vanA antisense RNA effect.
Assuntos
Antibacterianos/farmacologia , Enterococcus faecalis/genética , Oligonucleotídeos Antissenso/farmacologia , Resistência a Vancomicina/genética , Vancomicina/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Carbono-Oxigênio Ligases/antagonistas & inibidores , Carbono-Oxigênio Ligases/genética , Interações Medicamentosas , Enterococcus faecalis/efeitos dos fármacos , Técnicas de Transferência de Genes , Vetores Genéticos , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Oligonucleotídeos Antissenso/genética , Transformação BacterianaRESUMO
The highly conserved central loop of domain V of 23S RNA (nucleotides 2042 to 2628; Escherichia coli numbering) is implicated in peptidyltransferase activity and represents one of the target sites for macrolide, lincosamide, and streptogramin B antibiotics. DNA encoding domain V (590 bp) of several species of Enterococcus was amplified by PCR. Twenty enterococcal isolates were tested, including Enterococcus faecium (six isolates), Enterococcus faecalis, Enterococcus avium, Enterococcus durans, Enterococcus gallinarum, Enterococcus casseliflavus (two isolates of each), and Enterococcus raffinosus, Enterococcus mundtii, Enterococcus malodoratus, and Enterococcus hirae (one isolate of each). For all isolates, species identification by biochemical testing was corroborated by 16S rRNA gene sequencing. The sequence of domain V of the 23S rRNA gene from E. faecium and E. faecalis differed from those of all other enterococci. The domain V sequences of E. durans and E. hirae were identical. This was also true for E. gallinarum and E. casseliflavus. E. avium differed from E. casseliflavus by 23 bases, from E. durans by 16 bases, and from E. malodoratus by 2 bases. E. avium differed from E. raffinosus by one base. Despite the fact that domain V is considered to be highly conserved, substantial differences were identified between several enterococcal species.
Assuntos
Enterococcus/genética , Genes de RNAr/genética , Variação Genética , Infecções por Bactérias Gram-Positivas/microbiologia , RNA Ribossômico 23S/genética , Animais , Enterococcus/isolamento & purificação , Genes Bacterianos , Humanos , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase , RNA Ribossômico 23S/química , Análise de Sequência de DNARESUMO
OBJECTIVE: To evaluate the HIV-1 RNA level as a predictor of survival time among individuals with advanced AIDS. METHODS: The serum HIV-1 RNA level, the CD4 cell count, and other clinical variables were evaluated at baseline, as predictors of survival time, among 56 retrospectively identified HIV-1 positive individuals with < or = 50 x 10(6) CD4 cells/l who attended the Beth Israel Deaconess Medical Center, Division of Infectious Diseases, between 1 July 1989 and 30 September 1993. RESULTS: During follow-up, 55 of these 56 patients died. The median survival time was 20.5 months. In univariate Cox proportional hazard modeling neither the baseline HIV-1 RNA level nor the CD4 cell count were predictive of survival time. However, in multivariate models longer survival time was associated with the use of trimethoprim-sulphamethoxazole at entry [hazard ratio (HR), 0.42; P = 0.007], whereas shorter survival time was associated with a history of an AIDS-defining illness other than Pneumocystis carinii pneumonia (HR, 2.87; P = 0.007). Correlative analysis revealed a modest correlation of the baseline CD4 cell count with survival time (Spearman p = 0.41; P = 0.002). However, no correlation was found between HIV RNA levels and survival time (P = 0.5). CONCLUSIONS: In this population with very advanced disease, the HIV-1 RNA level was a poor discriminator of survival time, being inferior to the CD4 cell count and to specific clinical variables such as the nature of the prior AIDS-defining illness and the type of Pneumocystis carinii pneumonia prophylaxis employed. Among individuals with advanced AIDS, these data emphasize the relative importance of the CD4 cell count and of specific clinical factors, over the HIV-1 RNA level in predicting survival time.
Assuntos
Síndrome da Imunodeficiência Adquirida/imunologia , Síndrome da Imunodeficiência Adquirida/virologia , Contagem de Linfócito CD4 , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/isolamento & purificação , RNA Viral/sangue , Infecções Oportunistas Relacionadas com a AIDS/mortalidade , Síndrome da Imunodeficiência Adquirida/mortalidade , Adulto , Feminino , Infecções por HIV/mortalidade , HIV-1/genética , Humanos , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Prognóstico , Análise de Regressão , Estudos Retrospectivos , Análise de Sobrevida , Fatores de Tempo , Carga ViralRESUMO
OBJECTIVE: To evaluate the phenotypic susceptibilities and genotypic resistance patterns to both didanosine and stavudine of baseline and follow-up HIV-1 isolate pairs, derived from antiretroviral naive subjects treated with this dual nucleoside combination. DESIGN AND METHODS: Phenotypic drug susceptibility testing was performed in peripheral blood mononuclear cells on 34 viral isolate pairs derived from patients participating in the BMS AI-460 trial. Sequencing of the complete reverse transcriptase of 36 study isolate pairs, baseline and follow-up, was performed using standard dideoxy techniques. RESULTS: The mean fold change in susceptibilities to didanosine was 1.6 (P= 0.278) and to stavudine 1.9 (P= 0.002, Wilcoxon's signed rank test). Mutations classically associated with zidovudine resistance were observed to emerge in 7 out of 36 isolates, T215Y/F (four), M41L +T215Y/F (two) and D67N (one). Other mutations observed included the A62V, V751, F77L, F116Y, Q151 M multinucleoside resistance complex (one), the Q151M mutation (two) and the rare V75T mutation (two). No mutations classically associated with didanosine exposure and resistance were observed. No relationship was evident between the emergence of zidovudine associated mutations and the level of phenotypic resistance to either stavudine or didanosine or between the emergence of zidovudine associated mutations and changes in plasma HIV RNA levels. CONCLUSION: These comprehensive data demonstrate modest (< twofold) mean reductions in didanosine and stavudine susceptibilities at follow-up. The emergence of zidovudine associated mutations in this retroviral-naive population treated with combination didanosine and stavudine therapy is notable. Furthermore, the emergence of these mutations and of the Q151 M multinucleoside resistance complex raise concerns for potential nucleoside analog cross-resistance. The potential mechanisms driving the selection of the zidovudine associated mutations in the setting of didanosine and stavudine therapy and the relevance of these findings to current three and four drug regimens merit further evaluation.