Assessment of intraspecific mtDNA variability of European Ixodes ricinus sensu stricto (Acari: Ixodidae).

The Ixodes ricinus complex is composed of 14 species distributed worldwide. Some members of this complex are involved in the transmission of a number of diseases to animals and humans, in particular Lyme borreliosis, tick-borne encephalitis, ehrlichiosis and babesiosis. While the phylogenetic relationships between species of the I. ricinus complex have been investigated in the past, still little is known about the genetic structure within the species I. ricinus sensu stricto. We have investigated the intraspecific variability among 26 I. ricinus s.s. ticks collected in various European countries, including Switzerland, Italy, Austria, Denmark, Sweden, and Finland by using five mitochondrial gene fragments corresponding to the control region, 12S rDNA, cytb, COI, and COII. The five genes considered here showed a low genetic variability (1.6-5%). Our results based on both statistical parsimony (applied to the COI + COII + cytb + 12S + CR data set, for a total of 3423 bp) and maximum parsimony (applied to the COI + COII + cytb + 12S data set, for a total of 2980 bp) did not provide any evidence for a correlation between the identified haplotypes and their geographic origin. Thus, the European I. ricinus s.s. ticks do not seem to show any phylogeography structure.

Gastroretentive dosage forms: overview and special case of Helicobacter pylori.

The challenge to develop efficient gastroretentive dosage forms began about 20 years ago, following the discovery of Helicobacter pylori by Warren and Marshall. In order to understand the real difficulty of increasing the gastric residence time of a dosage form, we have first summarized the important physiologic parameters, which act upon the gastric residence time. Afterwards, we have reviewed the different drug delivery systems designed until now, i.e. high-density, intragastric floating, expandable, superporous hydrogel, mucoadhesive and magnetic systems. Finally, we have focused on gastroretentive dosage forms especially designed against H. pylori, including specific targeting systems against this bacterium.

Comparison of LightCycler PCR and culture for detection of group B streptococci from vaginal swabs.

Group B streptococci (GBS) are an important cause of neonatal sepsis and meningitis. New rapid, sensitive and specific methods for detection of GBS in pregnant women are needed in order to provide timely treatment of neonates. The sensitivity, specificity and cost of a LightCycler PCR method was compared with selective culture for the detection of GBS from 400 vaginal swabs. In addition, two DNA extraction methods (simple boiling and automated DNA extraction by Roche MagNA Pure LC) were compared for a subgroup of 100 clinical samples. The sensitivity of the LightCycler PCR assay for the detection of GBS from vaginal swabs was significantly higher than that of culture. There were no culture-positive, LightCycler PCR-negative cases. The efficiencies of the two DNA extraction procedures were not significantly different. The detection of GBS from vaginal swabs by the molecular method (including simple boiling extraction) required the same hands-on time, but the procedure was completed in 1.5 h, compared with c. 48 h for the culture-based approach. Disadvantages of the molecular method are the increased costs (45%) and the absence of antibiogram data. The LightCycler PCR is a promising tool for sensitive, specific and rapid detection of GBS directly from clinical specimens of pregnant women.

Molecular typing of Shigella strains using pulsed field gel electrophoresis and genome hybridization with insertion sequences.

The genomes of 18 independent Shigella isolates (9 Shigella sonnei, 5 Shigella dysenteriae and 4 Shigella flexneri) as well as of 4 epidemic S. flexneri strains were analysed by pulsed field gel electrophoresis (PFGE) and by the distribution of insertion sequences (IS1, IS2 and IS911). Despite the close relatedness observed among the 9 independent S. sonnei, all of them could be differentiated from each other. The 4 independent S. flexneri isolates showed clearly distinguishable DNA profiles. Nearly complete genetic identity was detected within the 4 epidemic S. flexneri when analysed by PFGE or for IS1 and IS2 patterns. However, IS911 was found to be too mobile in these epidemic S. flexneri to be used as a typing probe. The 5 S. dysenteriae isolates could also be distinguished by the techniques used. The diversity found within this species is striking: of the 5 investigated isolates, 3 completely different DNA profiles were revealed. In conclusion, both PFGE and IS probing demonstrated their potential usefulness in molecular epidemiology and in typing of Shigella strains. The degree of differentiation given by these two methods was generally comparable, although IS probes showed better discrimination of the isolates.

Association between different clinical manifestations of Lyme disease and different species of Borrelia burgdorferi sensu lato.

Borrelia burgdorferi sensu lato, the aetiological agent of Lyme disease, has been subdivided into three species: B. burgdorferi sensu stricto, B. garinii and B. afzelii. We and other authors have hypothesized an association between the three species of B. burgdorferi sensu lato and some of the different clinical manifestations of Lyme disease. In order to demonstrate this hypothesis, we analysed twenty-nine isolates cultured from patients with different symptoms. The method used was multilocus enzyme electrophoresis: twelve genetic loci were characterized on the basis of the electrophoretic mobility of their products, and twenty-eight distinctive allele profiles (electrophoretic types) were distinguished, among which mean genetic diversity per locus was 0.649. Cluster analysis of a matrix of genetic distances between paired electrophoretic types revealed three primary divisions separated at genetic distances greater than 0.7 and corresponding to the three species of B. burgdorferi sensu lato. Ten strains obtained from skin of patients with erythema chronicum migrans (the primary stage of the disease) were assigned to the three different species. All the six strains isolated from patients with acrodermatitis chronica atrophicans were of the species B. afzelii, which was not found to be associated with another chronic manifestation of Lyme disease. Arthritis was caused prevalently by B. burgdorferi sensu stricto, and neuroborreliosis by B. burgdorferi sensu stricto and B. garinii. In conclusion, our results confirm the association between some of the different chronic manifestations of the disease and the species of B. burgdorferi sensu lato.

TnA transposons can be introduced and maintained in Neisseria gonorrhoeae.

In Neisseria gonorrhoeae, all penicillinase-specifying plasmids isolated so far share homology with each other and carry a 60% deleted sequence of TnA. Plasmid pHD131, an element isolated from Haemophilus ducreyi and carrying an intact copy of the ampicillin resistance transposable element, was introduced from Escherichia coli into N. gonorrhoeae by both transformation and conjugative mobilization. Plasmids were recovered with no detectable deletion. After their transfer back into E. coli, transposition assays onto phage-lambda DNA demonstrated that the TnA elements were still functional. Plasmid pHD131 could be stably maintained in N. gonorrhoeae with or without the presence of penicillin. These results support the hypothesis that the absence in N. gonorrhoeae of plasmids carrying entire and functional TnA transposons cannot be ascribed to incompatibility between the genetic element and the host, but rather to a barrier to introduction of foreign DNA into gonococcus.

Arguments against the involvement of Borrelia burgdorferi sensu lato in Alzheimer's disease.

The involvement of spirochaetes, such as the aetiologic agent of Lyme borreliosis, Borrelia burgdorferi sensu lato, in Alzheimer's disease (AD), a common neuropathology, has been proposed by several groups in the past. In our laboratory, brains from 10 AD patients were analysed for the presence of B. burgdorferi sensu lato by both standard and nested PCR techniques based on various target regions, such as the hbb gene (encoding the histone-like protein HBb), the fla gene (flagellin), the rrl-rrf ribosomal intergenic spacer region and the rrs gene (encoding 16S rRNA). In addition, ELISA and Western blot tests for the detection of antibodies against spirochaetal antigens were performed on 27 sera from clinical AD patients. Using these methods, we did not obtain any evidence of the involvement of B. burgdorferi in Alzheimer's disease.

Eradication of Borrelia burgdorferi infection in primary marginal zone B-cell lymphoma of the skin.

Primary cutaneous B-cell lymphomas have been associated with Borrelia burgdorferi, the spirochete responsible for Lyme disease. Recently, cutaneous marginal zone B-cell lymphoma has been proposed as a distinct clinical-pathological entity. We report a case of primary cutaneous marginal zone lymphoma, associated with B burgdorferi infection. Polymerase chain reaction (PCR) amplification of the third complementarity determining region (CDR3) of the immunoglobulin heavy chain gene showed the presence of a monoclonal lymphoproliferation, therefore strengthening the histological diagnosis of a malignant process. B burgdorfer-specific hbb gene sequences were detected by PCR in the lymphoma tissue at diagnosis but not after antibiotic treatment. A nearly complete clinical and histological regression was observed after B burgdorferi eradication, with immunohistochemistry studies showing disappearance of plasma cell differentiation and a marked decline in the number of CD3+ T cells and Ki-67+ cells. Our case confirms the link between B burgdorferi and some cutaneous lymphomas. The disappearance of the microorganism accompanied by the unequivocal decrease of most indicators of active T- and B-cell immune response strongly supported a pathogenetic role for B burgdorferi in sustaining an antigen-driven development and growth of this cutaneous marginal zone lymphoma. Antibiotic therapy (analogous to Helicobacter pylori infection in gastric MALT lymphoma) might be helpful with the aim of averting or at least deferring the indication for more aggressive treatment.

A nested polymerase chain reaction for the detection of Borrelia burgdorferi sensu lato based on a multiple sequence analysis of the hbb gene.

A highly sensitive nested polymerase chain reaction method was designed for the detection of a wide spectrum of strains from Borrelia burgdorferi sensu lato. This technique allows the detection of as little as 3 fg of total genomic DNA extracted and purified from pure cultures of the organism, this amount corresponds to less than 10 organisms. Two sets of primers homologous to conserved spots in the coding region of the hbb gene, encoding a conserved histone-like protein, were constructed. These were based on a multiple sequence alignment of 39 strains representing all the genomic groups described in B. burgdorferi sensu lato.

Interaction of tetanus toxin and toxoid with cultured neuroblastoma cells. Analysis by immunofluorescence.

The primary interaction of tetanus toxin and toxoid with mouse neuroblastoma cells (C 1300, clone NB2A) in tissue culture was studied using direct immunofluorescence. Experiments were done in standard routine cultures and also those influenced by chemical modulators. There is a difference in the characteristic binding response between the growth culture cells (grown in presence of fetal calf serum) and differentiating culture cells (grown in absence of serum). Exposure to the toxin gives no visible effect on the cell division or viability in growth cultures; whereas in differentiating cells the processes are shortened and the adherence to the glass is diminished without involving significant cell death. The toxoid did not bind at all under the same experimental conditions. Since there was no biological effect in growth cultures we have called this binding ineffective, and in the case of the differentiating cells, effective binding. Stimulation of pinocytosis increases the uptake of toxin in both cultures. Presence of some surface bound toxin still remaining on the differentiating cells indicates the possibility of another sort of mechanism for internalization. Pre-treatment of the cells with neuraminidase or beta-galactosidase to alter the membrane gangliosides eliminates binding in growth cultures but not in differentiating cultures. From these results we suggest that even though the toxin may well bind to gangliosides, at least in the differentiating cultures they are not solely responsible for the fixation. The morphologically observed effective binding is probably that not related to gangliosides.

Risk of infection by Brucella melitensis for people living near infected goats.

Taking advantage of a local outbreak of brucellosis in a herd of goats, we have assessed the risk of infection for people living nearby. By means of serological tests we have shown that such a risk is small provided that direct contact with contaminated tissues or secretions of goats is avoided.

[Multicenter evaluation of oral antibiotics: resistance behavior in 5 Swiss centers]. / Multizentrische Evaluation oraler Antibiotika: Resistenzverhalten in fünf schweizerischen Zentren.

The susceptibility of 2196 fresh clinical isolates to twelve different oral compounds was assessed in five Swiss microbiology institutions during summer 1992. A standardized microdilution system including all other material necessary was employed to assess the antibacterial activity of penicillin G, ampicillin, ampicillin + sulbactam, amoxycillin + clavulanic acid, cefadroxil, cephalexin, cefaclor, cefuroxime, cefetamet, doxycycline, erythromycin and clindamycin. The aminopenicillins (including the beta-lactamase inhibitor combinations) were highly active against the streptococci, in combination with a beta-lactamase inhibitor they covered the majority of the bla+ E. coli and Proteus mirabilis and between 60 to 80% of the Klebsiella spp. and Proteus vulgaris isolates. All the cephalosporins exhibited good activity against the streptococci, they were active against Gram-negative fermentative rods to a varying degree. Cefetamet was also active against many cefaclor and cefuroxime-resistant isolates. A considerable part of the species studied exhibited resistance to doxycycline; the observed resistance of S. agalactia, P. mirabilis, and Morganella morganii agreed with previous findings. Most of the Streptococcus spp. were inhibited by erythromycin and clindamycin. There were only single penicillin resistant S. pneumoniae isolates in the five Swiss centers. Taking account of the above particulars the epidemiology of antimicrobial resistance in Switzerland can be considered satisfactory.

Typing of human, animal, food, and environmental isolates of Listeria monocytogenes by multilocus enzyme electrophoresis.

In order to elucidate some aspects of the epidemiology of listeriosis in Switzerland, 181 strains of Listeria monocytogenes isolated from humans, animals, food, and the environment have been analyzed by multilocus enzyme electrophoresis at 21 enzyme loci. The clone responsible for several recent food-borne outbreaks in Switzerland and in North America (marked by electrophoretic type 1 and serovar 4b) has been found frequently among strains isolated from animals. Thus, animals may represent a major source of diffusion of this clone in the environment and in food, in which it has been found only sporadically, however. Two other unrelated clones (including strains belonging to serovars 1/2b and 1/2c) have often been isolated from meat but not from animals. These findings indicate that contamination of meat with L. monocytogenes might originate mainly from the environment in which it is processed rather than from animals themselves. This could explain the differences in the distribution of L. monocytogenes serovars isolated from meat and from animals.

Analysis of the genetic polymorphism of Borrelia burgdorferi sensu lato by multilocus enzyme electrophoresis.

In recent years, Borrelia burgdorferi sensu lato has been subdivided into three species, Borrelia burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii, and a new species restricted to Japan, Borrelia japonica, has been isolated from Ixodes ovatus. In addition, members of several new genomic groups have been found in America and in Europe, suggesting that there are additional genospecies. In order to study the diversity of B. burgdorferi sensu lato, we analyzed 54 isolates, cultured from humans and from different tick species and obtained from diverse geographic areas, including Europe, the United States, Japan, and the People's Republic of China. In order to investigate the genetic relationship between microorganisms that are transmitted by soft ticks and microorganisms that cause Lyme disease, we also included three strains of relapsing fever spirochetes. The method which we used was multilocus enzyme electrophoresis; 12 genetic loci were characterized on the basis of the electrophoretic mobilities of their products, and 50 distinct allele profiles (electrophoretic types) were distinguished. The mean genetic diversity per locus was 0.747. a cluster analysis of a matrix of genetic distances for pairs of electrophoretic types revealed 11 divisions that were separated at genetic distances greater than 0.65. Five of these divisions corresponded to B. burgdorferi sensu stricto, B. garinii, B. afzelii, B. japonica, and the newly proposed species "Borrelia andersonii." Our results also confirmed that there are two additional genomic groups in Europe and at least one additional group in the United States. The relapsing fever spirochetes were were not clearly separated from the spirochetes associated with Lyme disease. In conclusion, we believe that the previously proposed subdivision of B. burgdorferi sensu lato into only four species should be reconsidered.

Binding of chloramphenicol and its acetylated derivatives to Escherichia coli ribosomal subunits.

Chloramphenicol-acetylated derivatives (1-monoacetoxy-; 3-monoacetoxy-; 1,3-diacetoxy-chloramphenicol), produced by a wild type Escherichia coli strain carrying an R factor, have been extracted and purified. None of the acetylated derivatives has been found to bind to E. coli ribosomal subunits.