- Volume 38, Issue 1, 1993

Biotypes, ribosomal RNA gene restriction patterns (ribopatterns), whole-cell protein patterns and plasmid profiles of paired Helicobacter pylori isolates from 17 patients were examined. Each pair comprised a pre- and a post-treatment isolate; nine of the 17 post-treatment isolates were obtained after treatment with tripotassium dicitrate bismuthate (DeNol) and metronidazole. All strains of H. pylori had identical biotypes, but exhibited diversity between pairs in their molecular fingerprints. Each of the 17 strain pairs had unique ribopatterns; the pre- and post-treatment isolates in most pairs (16 of 17) were similar or identical, irrespective of metronidazole susceptibility. DNA subtype variants were detected in three patient sets. Although nine post-treatment isolates had acquired resistance to metronidazole, most (six of nine) resembled the pre-treatment isolates in their ribopattern, protein and plasmid profiles. No significant correlation was observed between metronidazole resistance and plasmid content in these H. pylori isolates. Emergence of post-treatment metronidazole-resistant isolates of H. pylori isolates. Emergence of post-treatment metronidazole-resistant isolates of H. pylori was associated only rarely with colonisation by a novel strain or acquisition of a plasmid and, in most patients, probably resulted from spontaneous emergence of resistance in the original infecting strain.

Peritoneal cells from rats infected intraperitoneally with Escherichia coli and Bacteroides fragilis, alone or in combination were examined in vitro. Cells were harvested 6 h after implantation of fibrin clots infected with E. coli or B. fragilis, separately or containing both species, and assayed for their bactericidal capacities, chemiluminescence and production of cidal metabolites. Peritoneal cell populations from rats with implants of any of the infected clots showed similar distribution of different subpopulations. Bactericidal activity of peritoneal cells did not differ with the bacterial species used. Chemiluminescence values of peritoneal cells from rats with mono-infected B. fragilis or mixed-infected implanted clots, after stimulation with either particles or chemical stimuli, were significantly higher than those of rats with mono-infected E. coli or sterile clots. The same tendency was seen with regard to the production of cidal metabolites such as hydrogen peroxide and superoxide anions although no significant differences were found.

The increase in the number of cases of meningococcal disease reported to the Ministry of Health in Athens since 1989 prompted the present study to determine if isolates from patients or carriers expressed the same phenotypic characters as those in other parts of Europe. None of the isolates from patients (31) or carriers (547) expressed the antigenic combinations associated with outbreaks in northern Europe, i.e., B:15:P1.16 or B:4:P1.15. The majority of the Greek isolates did not react with any of the six monoclonal serotype reagents tested; however, most reacted with one or more of the 11 monoclonal subtype antibodies. The results suggest that additional serotype reagents are needed for epidemiological studies in southeastern Europe and that vaccines based on serotype antigens developed against outbreak strains in northern Europe would not be effective in Greece.

Dot-blot analysis of whole-cell suspensions of meningococci showed that 81% of B:15:P1.16 strains from patients reacted with a monoclonal antibody (MAb) against subtype P1.7. The remaining strains, which did not react on dot-blots or in ELISA, demonstrated the P1.7 subtype epitope on immunoblots after denaturation of the cells with sodium dodecyl sulphate. The monomeric class 1 proteins of the two P1.16 subtype variants had slightly different mol. wts, but bound the P1.7 antibody equally well. These results were explained by a deletion of three codons in the gene encoding the first variable region of the P1.16 class 1 protein. The deletion accounted for the non-exposure of the P1.7 epitope on native cells. Other patient strains, with subtypes P1.3, P1.9 or without any known subtype, also showed a binding site for the P1.7 MAb, which became available only after denaturation. Demonstration of inaccessible epitopes may have consequences for subtype designations and vaccine development.

The epidemiology of pulmonary colonisation by Pseudomonas aeruginosa was studied in 21 patients with cystic fibrosis (CF) by field inversion gel electrophoresis. DraI-DNA restriction patterns were analysed for 187 P. aeruginosa isolates from these patients. The results revealed that the strains present in individual patients varied during the course of chronic colonisation; the emergence of new strains often was associated with periods of antibiotic therapy. Patients often were colonised by more than one strain (two or three strains were present in 54% of the patients) and the strains obtained from unrelated patients were highly heterogeneous, in contrast to those isolated from a pair of twins. These results demonstrate the heterogeneity and variability of P. aeruginosa isolates in the pulmonary flora of chronically infected CF patients.

The microparticle capture enzyme immuno-assay (MEIA) is an automated system for measuring specific antibody by interaction with antigen-coated particles. An MEIA method for detecting toxoplasma-specific IgM was compared with established reference methods. The MEIA had an acceptable level of sensitivity and reproducibility and was easier to perform than conventional tests, but it required expensive, dedicated equipment and, in our study, false positive results were recorded with 7% of samples. MEIA could be used to investigate immunocompetent patients with suspected toxoplasmosis but positive findings should be confirmed by an alternative form of assay. The technique is not suitable for the investigation of neonates, for which a more sensitive method is required.

A two-stage polymerase chain reaction (PCR) assay employing oligonucleotide primers from the B1 gene of Toxoplasma gondii was developed and assessed for sensitivity and specificity. It was able to detect T. gondii DNA from as little as one parasite/sample in mock-infected rat or mouse leucocyte preparations. Parasitaemia was also identified in animals at five stages between 16 and 66 h after infection with the virulent RH strain, and at 12 stages between 2 and 38 days after infection with the cyst-forming Beverley strain. In the latter case, PCR was more sensitive than animal culture. No cross-reactions were observed in samples containing various opportunist pathogens which may also be found in the blood of immunocompromised patients.

Strains of Aspergillus flavus, Fusarium sp., Rhizopus sp. and Candida albicans all produced inhibitors of β-adrenergic receptor binding; strains of Saccharomyces sp. and Schizosaccharomyces sp. did not. In tests with glutamic acid as the sole nutrient source, a Fusarium sp. produced four-fold larger amounts of inhibitor than the other fungi. The inhibitor from the Fusarium sp. was further purified by lyophilisation and sequential solvent extractinon in chloroform, ethyl acetate and butanol; 60% of the original activity was recovered. The inhibitor had an estimated molecular size of 650 Da, and did not absorb light in the visible or ultraviolet range. When compared with a similar inhibitor from Escherichia coli, the Fusarium sp. inhibitor appeared to be a more potent inhibitor of β-adrenergic and dopaminergic binding to mammalian cells.

Of 97 isolates of Aeromonas spp. that were examined for haemagglutination (HA) and enterotoxigenicity, 35 were from clinical and 62 from environmental sources; 66 of them were also screened for sensitivity to normal human serum (NHS). HA was caused by 44 isolates (45%); it was unrelated to the source of the strain, but it was caused by a higher proportion of the isolates of A. hydrophila than of A. sobria or A. caviae. Of the haemagglutinating strains, 82% were enterotoxigenic, whereas most of the non-haemagglutinating strains were non-toxigenic when tested initially. All the latter became enterotoxin producers after serial passage through rabbit ileal loops, but without change in HA. Most (64%) of the isolates, including 68% of A. caviae (72% of clinical and 65% of environmental), were resistant to the bactericidal action of NHS. Most (92%) of the serum-sensitive strains were killed by activation of both the classical and alternate pathways of complement, the others only by the alternate pathway. Most (74%) of the serum-resistant strains caused fluid accumulation in the initial tests in ileal loops, regardless of species or source. Haemagglutinating and serum-resistant strains caused significantly more accumulation of fluid (p.0.05) than non-haemagglutinating and serum-sensitive strains. This study shows partial correlation between HA or serum sensitivity and enterotoxigenicity, but the properties are probably not genetically linked.

The inhibitory effect of non-ionic, anionic, cationic and ampholytic surfactants on cellular growth of Streptococcus mutans MT8148 and S. sobrinus6715, on glucan synthesis by the purified glucosyltransferase (GTase) from these organisms, and on bacterial adherence to glass surfaces was examined in vitro. Cationic surfactants exhibited marked bactericidal activities. Anionic and ampholytic compounds were less strongly bactericidal and non-ionic surfactants produced only slight inhibition of cell growth under the conditions tested. Some non-ionic compounds had no effect on this. Glucan synthesis by GTase from mutans streptococci was inhibited by anionic and cationic surfactants. Among various GTase proteins, insoluble glucan synthesising GTases, i.e., S. mutans CA-GTase and S. sobrinus GTase-I were those most effectively inhibited by these agents. However, it was noted that whereas lower concentrations of cationic surfactants enhanced these GTase activities, higher concentrations of the surfactants were inhibitory. Non-ionic detergents stimulated soluble glucan synthesis from S. mutans CF-GTase and cationic and ampholytic surfactants enhanced or inhibited glucan synthesis depending on the concentrations of the surfactants. Sucrose-dependent cellular adherence of resting cells of mutans streptococci to glass surfaces was inhibited by the addition of surfactants that annulled the GTase activities.

The resurgence of streptococcal infections in the USA and Europe and their high incidence in other parts of the world prompted an examination of the survival and maintenance of virulence of group A streptococci. Human blood containing group A streptococci was placed on small pieces of sterile paper towelling and allowed to dry at room temperature. At periods of 2, 8, 15 and 20 weeks later, the paper with the dried blood was placed in Todd-Hewitt broth and incubated at 37°C overnight. All the samples tested at 2 weeks grew in broth, and with only one exception, grew in fresh human blood provided by five donors. At 8 weeks only two of the 10 strains failed to grow in broth; seven of the eight viable cultures also grew in blood. At 15 and 20 weeks after drying the eight cultures were still viable. Since seven were able to grow in fresh blood as well as in broth it is assumed that their virulence factor(s) had been retained.

Electronmicroscopy of thin sections of log phase cells of Enterobacter cloacae NCTC 10005 grown for 4 h in the presence of sulphadiazine 250 μg/ml, trimethoprim 12.5μl/ml or the combination of sulphadiazine 250 μg/ml plus trimethoprim 12.5μg/ml indicated that both agents caused marked morphological damage even though the MIC of sulphadiazine for the E. cloacae strian was > 3000μg/ml. The damage took the form of electron-transparent areas devoid of ribosomes in the cytoplasm and detachment of the outer membrane. The latter was most marked with trimethoprim, which also caused damage to the cytoplasmic membrane. It is postulated that the synthesis of the peptidoglycan layer was affected by the antimetabolites since the morphological effects were strikingly similar to those caused by treatment of E. cloacae with disodium edetate plus lysozyme. Viable counts of cultures undergoing the same treatments as those prepared for electronmicroscopy indicated that although sulphadiazine merely partially inhibited growth it nevertheless enhanced the bactericidal action of trimethoprim over a 5-h period.

After sonic disintegration of Clostridium difficile cells, intracellular toxin A was purified to homogeneity by thyroglobulin affinity chromatography (TGAC) followed by anion-exchange (Mono Q) by fast protein liquid chromatography (FPLC). High haemagglutinating (HA) activity was detected in TGAC-unbound fractions (29/50 μl), but not in TGAC thermal eluates (20/50 μl). The low HA titre of the thermal eluates was markedly increased to 25/50 μl) after dialysis against 0.02 M Tris-HCI (pH 7.5). A disparity in the position of the peaks containing cytotoxic and HA activity was observed in the first Mono Q-FPLC step. Intracellular toxin A without HA activity was obtained by a second Mono Q-FPLC step. The Mr of the intracellular toxin A was estimated by polyacrylamide gel electrophoresis (PAGE) to be 580 kDa under non-denaturing conditions. The minimum doses of the toxin causing cytotoxicity, mouse lethality and enterotoxicity were 0.83 ng, 8.7 ng and 5 μg, respectively.

Edited by Y.-M. WEN. 2.-Y. Xu and J. L. MELNICK. 1992. ISBN 3-8055-5364-1. S Karger AG, Basel. Pp. 159. £77.90.

Edited by C. P. HOWSON, C. J. HOW and H. V. FINEBERG. 1991. ISBN 0-309-04499-5. National Academy Press. Washington. Pp. 367. $48.

W. H. NELSON. 1991. ISBN 3-527-28022-7 VCH Publishers, Weinheim. Pp. 263. £45.