- Volume 50, Issue 6, 2001

This report describes a method for the isolation of nucleic acid from a suspension of matured virus. Nucleic acid (DNA) was isolated from a prototype strain of adenovirus type 7 and a clinical isolate of adenovirus type 7. Instead of the usual method of ultracentifugation, a filtration method was applied to concentrate the virus rapidly and nucleic acid was then isolated by a standard phenol/chloroform/isoamyl-alcohol extraction procedure. The DNA was found to be sufficiently purified to generate a reproducible restriction endonuclease digestion pattern. The clinical isolate of adenovirus type 7 revealed loss of restriction site for the endonuclease HindIII when compared with the prototype strain.

Rabbits and mice immunised with chemically extracted O-antigens (O-Ags) of Vibrio cholerae O139 (O-AgB and O-AgD) developed antibodies (Abs) which appeared to be highly specific in ELISA for the relevant antigens and V. cholerae O139 strains without absorption, in contrast to the Abs against the heated O-Ag (O-AgH). An ELISA test based on the use of these Abs was shown to detect V. cholerae O139 strains down to concentrations of (9.4 × 104)–(7.5 × 105) vibrios/ml and demonstrated no cross-reaction with other vibrios including representatives of serogroup O22. Native and proteinase K-treated O-AgB, O-AgD, O-AgH, as well as whole-cell lysates of V. cholerae O139 strains of different origin were used in immunoblotting with these Abs. Clear differences in the patterns of zones of specific reaction between chemically extracted and heated O-Ags and between lipopolysaccharide profiles of the V. cholerae O139 strains of different origin were observed. Serogroup-specific protein bands in the native O-AgB and O-AgD preparations were defined. The approach described for obtaining serogroup-specific Abs against vibrios and other bacteria seems to be promising for the development of specific diagnostic tests and further investigation of bacterial antigenic structure.

New agents are urgently needed to meet the threat of multiple drug-resistant tuberculosis and to manage infection with the naturally resistant non-tuberculosis mycobacteria. Earlier fluoroquinolones have been shown to have promising in-vitro activity, although mouse infection and clinical studies suggested that they lack sufficient bactericidal activity. Methods were evaluated to measure the bactericidal activity of fluoroquinolones and to compare the new agent moxifloxacin with other fluoroquinolones with M. fortuitum as a model system. The optimum bactericidal concentrations (OBC) for the fluoroquinolones were: moxifloxacin, 0.55mumg/L; ciprofloxacin and sparfloxacin, 25mumg/L and ofloxacin, 85mumg/L. The bactericidal indices (BI) for moxifloxacin, ciprofloxacin, sparfloxacin and ofloxacin were 1.8, 0.5, 0.2 and 0.2, respectively. Similar ranking was obtained when the time taken to produce one log10 reduction in viable count was calculated. These data indicate that moxifloxacin was the most bactericidal of the fluoroquinolones tested. Such methods provide a simple in-vitro measure that correlates with in-vivo models.

Legionella pneumophila produces several extracellular proteins, but their role in the pathogenesis of Legionnaires’ disease is unclear. This study examined the effects of the L. pneumophila major secretory protein (Msp), a zinc metalloprotease, on the oxidative burst and chemotaxis of human phagocytes. Polymorphonuclear leucocytes (PMNLs) and adherent monocytes treated with sublethal amounts of Msp protease were stimulated with formyl-leucyl-methionyl-phenylalanine (fMLP) and opsonised zymosan particles (ZAP). A dose-dependent inhibition in superoxide anion production in response to both stimuli was seen, and complete inhibition was achieved in PMNLs and monocytes treated with Msp at concentrations of 1500 and 10005muU/ml, respectively. ZAP-induced chemiluminescence by PMNLs and mononuclear cells and fMLP-induced PMNL nitroblue tetrazolium dye reduction were both significantly inhibited. The chemotactic response of PMNLs to fMLP was inhibited in a dose-dependent manner and substantial inhibition (11% of control) was achieved with Msp 12005muU/ml. These results suggest that the L. pneumophila Msp protease alters human phagocyte functional responses significantly and may contribute to the pathogenesis of Legionnaires’ disease.

Immunoglobulin A (IgA) is important in protective immunity against infection by Vibrio cholerae. In this study, the immune response to and protective role of a 31-kDa antigen of V. cholerae O139, reacting with IgA antibodies present in the sera of cholera patients and common to V. cholerae strains O139 and O1 was evaluated in BALB/c mice. From the various antigens of V. cholerae O139 and V. cholerae O1 which reacted with IgA antibodies in sera of a cholera patient, a 31-kDa common antigen was selected and purified by DEAE-Sepharose CL 6B column chromatography. Oral administration of live V. cholerae O139 in BALB/c mice elicited an IgA response to the 31-kDa antigen in serum and intestinal fluid, and a proliferation of the splenic lymphocytes on stimulation with the same antigen. The cytokine profile of these splenic lymphocytes revealed a shift from a mixed Th1 and Th2 response – interleukin-10 (IL-10) and interferon-γ – in the first week after infection to a Th2 type of response – IL-10 – in the third week. In passive protection studies, hyperimmune serum to the 31-kDa antigen was able to protect infant mice against challenge with O139 and O1 strains. These results demonstrate the ability of the 31-kDa antigen of V. cholerae O139 to induce humoral and cellular immune responses in mice, and its immunoprotective nature.

A total of 128 MRSA isolates from a burns unit in 1992 and 1997 was studied by resistotyping, plasmid analysis and pulsed-field gel electrophoresis (PFGE) of SmaI-digested chromosomal DNA to ascertain whether a clone of MRSA had persisted in the unit or whether different clones had been introduced at different times. All the MRSA isolates produced β-lactamase and had high MICs to methicillin (>2565mumg/L). All were resistant to tetracycline, kanamycin, cadmium acetate and mercuric chloride. Most were resistant to gentamicin, neomycin, erythromycin, chloramphenicol, trimethoprim, ciprofloxacin, propamidine isethionate and ethidium bromide, and were susceptible to minocycline, vancomycin and teicoplanin. None of the 1992 isolates was resistant to mupirocin, but 56% and 19% of the 1997 isolates expressed high- and low-level mupirocin resistance, respectively. Many of the 1997 isolates had acquired a 38-kb plasmid encoding high-level mupirocin resistance. The 1992 isolates had two main PFGE patterns; 82% of them belonged to PFGE pattern 1. The 1997 isolates had PFGE pattern 1, the same as the majority of the 1992 isolates. All MRSA isolates from both years carried the mecA gene in the same SmaI fragment. These findings demonstrated that a clone of MRSA that was prevalent in the burns unit in 1992 had persisted and became the predominant clone in 1997.