A multilocus sequence typing (MLST) scheme has been developed for Enterococcus faecium. Internal fragments from seven housekeeping genes of 123 epidemiologically unlinked isolates from humans and livestock and 16 human-derived isolates from several outbreaks in the United States, the United Kingdom, Australia, and The Netherlands were analyzed. A total of 62 sequence types were detected in vancomycin-sensitive E. faecium (VSEF) and vancomycin-resistant E. faecium (VREF) isolates. VSEF isolates were genetically more diverse than VREF isolates. Both VSEF and VREF isolates clustered in host-specific lineages that were similar to the host-specific clustering obtained by amplified fragment length polymorphism analysis. Outbreak isolates from hospitalized humans clustered in a subgroup that was defined by the presence of a unique allele from the housekeeping gene purK and the surface protein gene esp. The MLST results suggest that epidemic lineages of E. faecium emerged recently worldwide, while genetic variation in both VREF and VSEF was created by longer-term recombination. The results show that MLST of E. faecium provides an excellent tool for isolate characterization and long-term epidemiologic analysis.Vancomycin-resistant Enterococcus faecium (VREF) has recently emerged as an important threat in U.S. hospitals (5, 24). In Europe, VREF isolates are found relatively frequently in the community and farm animals, while prevalence in hospitals is generally low (14). The latter observation was explained by the use of the glycopeptide avoparcin as an antimicrobial growth promoter in animal feeding operations.Several molecular typing schemes have been developed to study the epidemiology of VREF. Of these, pulsed-field gel electrophoresis analysis of genomic restriction fragments has been considered the "gold standard" for the study of hospital outbreaks because of its high degree of isolate differentiation (15,17,20,23). However, due to this high degree of isolate differentiation, pulsed-field gel electrophoresis typing is less suitable for determining the degree of relatedness among epidemiologically unrelated isolates. Recently, amplified fragment length polymorphism (AFLP) analysis was applied as a new method for the typing of VREF (1, 33). AFLP analysis is a robust and fast typing technique with high intra-and interexperimental reproducibilities and appears to be discriminatory enough for the recognition of hospital outbreaks (1, 32, 33). In addition, AFLP analysis has allowed the detection of associations among different E. faecium genetic lineages and different human and animal hosts (33), suggesting the existence of host-specific VREF lineages. Whether this is also true for vancomycin-sensitive E. faecium (VSEF) is not known, since VSEF isolates were not included in that study. AFLP typing also disclosed two different human-associated lineages. One lineage comprised epidemic-related isolates recovered from hospitalized patients, while isolates of the other lineage were mainly from nonhospitalized persons. Interes...

No abstract

A multilocus sequence typing (MLST) scheme has been developed for Enterococcus faecium. Internal fragments from seven housekeeping genes of 123 epidemiologically unlinked isolates from humans and livestock and 16 human-derived isolates from several outbreaks in the United States, the United Kingdom, Australia, and The Netherlands were analyzed. A total of 62 sequence types were detected in vancomycin-sensitive E. faecium (VSEF) and vancomycin-resistant E. faecium (VREF) isolates. VSEF isolates were genetically more diverse than VREF isolates. Both VSEF and VREF isolates clustered in host-specific lineages that were similar to the host-specific clustering obtained by amplified fragment length polymorphism analysis. Outbreak isolates from hospitalized humans clustered in a subgroup that was defined by the presence of a unique allele from the housekeeping gene purK and the surface protein gene esp. The MLST results suggest that epidemic lineages of E. faecium emerged recently worldwide, while genetic variation in both VREF and VSEF was created by longer-term recombination. The results show that MLST of E. faecium provides an excellent tool for isolate characterization and long-term epidemiologic analysis.Vancomycin-resistant Enterococcus faecium (VREF) has recently emerged as an important threat in U.S. hospitals (5, 24). In Europe, VREF isolates are found relatively frequently in the community and farm animals, while prevalence in hospitals is generally low (14). The latter observation was explained by the use of the glycopeptide avoparcin as an antimicrobial growth promoter in animal feeding operations.Several molecular typing schemes have been developed to study the epidemiology of VREF. Of these, pulsed-field gel electrophoresis analysis of genomic restriction fragments has been considered the "gold standard" for the study of hospital outbreaks because of its high degree of isolate differentiation (15,17,20,23). However, due to this high degree of isolate differentiation, pulsed-field gel electrophoresis typing is less suitable for determining the degree of relatedness among epidemiologically unrelated isolates. Recently, amplified fragment length polymorphism (AFLP) analysis was applied as a new method for the typing of VREF (1, 33). AFLP analysis is a robust and fast typing technique with high intra-and interexperimental reproducibilities and appears to be discriminatory enough for the recognition of hospital outbreaks (1, 32, 33). In addition, AFLP analysis has allowed the detection of associations among different E. faecium genetic lineages and different human and animal hosts (33), suggesting the existence of host-specific VREF lineages. Whether this is also true for vancomycin-sensitive E. faecium (VSEF) is not known, since VSEF isolates were not included in that study. AFLP typing also disclosed two different human-associated lineages. One lineage comprised epidemic-related isolates recovered from hospitalized patients, while isolates of the other lineage were mainly from nonhospitalized persons. Interes...

No abstract

The virally encoded protease of human immunodeficiency virus is responsible for the processing of the gag and gag-pol polyprotein precursors to their mature polypeptides. Since correct processing of the viral polypeptides is essential for the production of infectious virus, HIV protease represents a potential target for therapeutic agents that may prove beneficial in the treatment of AIDS. In this study, full-length gag polyprotein has been synthesized in vitro to serve as a substrate for bacterially expressed HIV-1 protease. Expression of the protease in E. coli from the lac promoter was enhanced approximately five-fold by deletion of a potential hairpin loop upstream from the codon determining the amino terminus of mature protease. Extracts of induced cultures of E. coli harboring a protease-containing plasmid served as the source of protease activity. The gag polyprotein synthesized in vitro was cleaved by such lysates, producing fragments corresponding in size to p17 plus p24 and mature p24. Immunoprecipitations with monoclonal antibodies to p17 and p24 polypeptides suggest that initial cleavage of gag polyprotein occurs near the p24-p15 junction. The proteolysis was inhibited by pepstatin with an IC50 of 0.15 mM for cleavage at the p24-p15 junction and 0.02 mM for cleavage at the p17-p24 junction.

No abstract

SUMMARYStudies on the growth of Z.mobilis revealed that high concentrations of glucose (10-25%) can be efficiently and rapidly converted to ethanol in batch culture.By comparison with S. carlsbergensis, Z.mobilis had specific glucose uptake rates and specific ethanol productivies several times greater than the yeast. Z.mobilis also had ethanol yields of up to 97% of a theoretical value.

Blood donors reactive by enzyme-linked immunosorbent assay for antibody to the human immunodeficiency virus (HIV) who showed atypical patterns of viral core protein reactivity on Western blot were monitored for several months. Characterization of their antibodies was performed by 1) use of recombinant HIV proteins; 2) determination of cross-reactivity to HTLV-I, HTLV-II, and HTLV-IV: 3) assessment of immune status; and 4) identification of potentially interfering autoantibodies. Nineteen of 20 donors maintained the same HIV antibody reactivity throughout the follow-up period; the other donor became fully antibody-positive. Eighteen of 20 donors' sera showed clear reactivity with HIV recombinant core proteins. Ten of 19 donor samples demonstrated cross-reactivity to HTLV-IV; 3 of these 10 also cross-reacted with HTLV-I. The immune status of all donors was normal, although the medical histories and HLA antibody screens suggested possible autoimmune reactivity in 9 of 18 donors. During follow-up interviews, three donors reported possible risk factors for HIV infection that had not been acknowledged at the time of blood donation. We conclude that exclusion of donors with these atypical serologic test results is warranted while further studies to determine significance are being conducted.