Development of a real-time PCR method for the detection of fossil 16S rDNA fragments of phototrophic sulfur bacteria in the sediments of Lake Cadagno.

Lake Cadagno is a crenogenic meromictic lake situated in the southern range of the Swiss Alps characterized by a compact chemocline that has been the object of many ecological studies. The population dynamics of phototrophic sulfur bacteria in the chemocline has been monitored since 1994 with molecular methods such as 16S rRNA gene clone library analysis. To reconstruct paleo-microbial community dynamics, we developed a quantitative real-time PCR methodology for specific detection of 16S rRNA gene sequences of purple and green sulfur bacteria populations from sediment samples. We detected fossil 16S rDNA of nine populations of phototrophic sulfur bacteria down to 9-m sediment depth, corresponding to about 9500 years of the lake's biogeological history. These results provide the first evidence for the presence of 16S rDNA of anoxygenic phototrophic bacteria in Holocene sediments of an alpine meromictic lake and indicate that the water column stratification and the bacterial plume were already present in Lake Cadagno thousands of years ago. The finding of Chlorobium clathratiforme remains in all the samples analyzed shows that this population, identified in the water column only in 2001, was already a part of the lake's biota in the past.

Evaluation of recA sequencing for the classification of Aeromonas strains at the genotype level.

AIMS: To evaluate the usefulness of partial recA sequences for the identification of Aeromonas strains at the genotype level. METHODS AND RESULTS: A partial recA sequence was obtained from 21 type or reference strains and 33 Aeromonas isolates, collected in the South of Switzerland from human, animal and aquatic environments. The 272 bp long recA fragments showed a mean interspecies divergence of 7.8% and allowed the classification of strains at genotype level. However, some discrepancies could be observed with other gene sequence based analyses in the classification of some strains. CONCLUSIONS: The 272 bp long recA fragment is a good molecular marker to infer taxonomy of members of the genus Aeromonas, even if the primers we chose for the amplification did not allow its direct sequencing. SIGNIFICANCE AND IMPACT OF THE STUDY: In the genus Aeromonas, nucleotide sequences of some protein-encoding genes have already been evaluated as molecular markers to be used in taxonomical and epidemiological researches. This study suggests the usefulness of a recA fragment as a further sequence to investigate for these purposes.

Molecular identification of Bacillus thuringiensis var. israelensis to trace its fate after application as a biological insecticide in wetland ecosystems.

AIMS: To determine the fate of viable Bacillus thuringiensis var. israelensis (Bti) spores dispersed in the environment, using a universally applicable molecular detection methodology. METHODS AND RESULTS: Soil samples were spread on growth medium, after a temperature selection of the spores. A PCR amplification of the cry4Aa and cry4Ba insecticidal genes was applied on the colonies. Ribotyping was performed subsequently. This combined molecular method proved to be very specific for Bti, which was easily differentiated from the other B. thuringiensis serovars. A site regularly treated with Vectobac-G was chosen within the 'Bolle di Magadino' natural reserve, and monitored throughout 1 year for the detection of Bti spores. The results showed that the numbers were relatively high after insecticidal applications (1.4 x 10(5) CFU g(-1)), and decreased approx. 10-fold after 220 days. A successive treatment induced a new increase. CONCLUSIONS: The results show that yearly repeated use of Vectobac-G does not seem to have a major ecological impact on the 'Bolle di Magadino' natural reserve. Bti spores followed a trend leading to their eventual disappearance from the ecosystem, despite the seasonal application of this biological insecticide for more than a decade. SIGNIFICANCE AND IMPACT OF THE STUDY: The molecular identification of Bti cells through the PCR analysis of the delta-endotoxins genes coupled to ribotyping, is an innovative method, that has enabled the identification of this organism into wetland environments.

Molecular typing of Yersinia frederiksenii strains by means of 16s rDNA and gyrB genes sequence analyses.

The phenospecies Yersinia frederiksenii is known to comprise three genospecies, indistinguishable on the basis of phenotypic characteristics. In previous works, 13 strains, identified biochemically as Y. frederiksenii, were characterized using Multilocus enzyme electrophoresis and Ribotyping. In order to elucidate the phylogenetic position of these strains we performed their molecular typing by means of 16S rDNA and gyrB sequences analyses. Results demonstrated that gyrB is a better phylogenetic marker than 16S rDNA. The classification achieved by gyrB sequences analyses was in agreement with results obtained with more laborious methods. Moreover, a good phylogenetic identification could be reached also with partial gyrB sequences of only 350 bp.

Current situation of human diphyllobothriasis in Europe.

Diphyllobothriasis, a parasitosis caused by the flatworm Diphyllobothrium latum, is contracted by consuming raw or undercooked freshwater fish. The aim of this study was to evaluate the situation of this parasitosis during the past 20 years in Europe through the analysis of databases and search engines (Medline, Cabi Helminthological abstracts,Yahoo, Google), and through a questionnaire sent to a network of European parasitologists and to microbiological laboratories located on the shores of the large Alpine lakes. This study has shown that several dozen cases have been reported each year in Finland and Sweden, that there have been numerous cases in the French or Italian speaking areas of subalpine lakes, and that sporadic cases only have been observed in Austria, Spain, Greece, Romania, Poland and Norway. Over 30 cases have been identified on the Swiss shores of Lake Maggiore since 1990, and 70 cases on the Swiss and French shores of Lake Leman between 1993 and 2002. Eight to 12% of perch fillets from Lake Leman and 7.8 % of perch from Lake Maggiore were infested with larvae. Contamination sources include marinated fish fillets in northern Europe, 'carpaccio di persico' in northern Italy, and perch and charr consumed raw or undercooked around Lake Leman. Factors allowing the continuation of the parasitic cycle include the continued dumping of wastewater into lakes, yachtsmen who also fish, and a possible animal reservoir.

Current situation of human diphyllobothriasis in Europe.

Diphyllobothriasis, a parasitosis caused by the flatworm Diphyllobothrium latum, is contracted by consuming raw or undercooked freshwater fish. The aim of this study was to evaluate the situation of this parasitosis during the past 20 years in Europe through the analysis of databases and search engines (Medline, Cabi Helminthological abstracts,Yahoo, Google), and through a questionnaire sent to a network of European parasitologists and to microbiological laboratories located on the shores of the large Alpine lakes. This study has shown that several dozen cases have been reported each year in Finland and Sweden, that there have been numerous cases in the French or Italian speaking areas of subalpine lakes, and that sporadic cases only have been observed in Austria, Spain, Greece, Romania, Poland and Norway. Over 30 cases have been identified on the Swiss shores of Lake Maggiore since 1990, and 70 cases on the Swiss and French shores of Lake Léman between 1993 and 2002. Eight to 12% of perch fillets from Lake Leman and 7.8 % of perch from Lake Maggiore were infested with larvae. Contamination sources include marinated fish fillets in northern Europe, 'carpaccio di persico' in northern Italy, and perch and charr consumed raw or undercooked around Lake Léman. Factors allowing the continuation of the parasitic cycle include the continued dumping of wastewater into lakes, yachtsmen who also fish, and a possible animal reservoir.

Persistence of viral pathogens and bacteriophages during sewage treatment: lack of correlation with indicator bacteria.

The effects of different sewage treatments on the viral contamination in rivers which receive water from treatment plants without a final sand filtration step were investigated. They were all heavily contaminated with bacteriophages and human enteric viruses (detected by single step reverse transcription amplification followed by a nested polymerase chain reaction). Bacteriophages, but not faecal indicator organisms, were correlated with viral contamination.

Intestinal secretory immunoglobulin A (sIgA) response to Aeromonas exoproteins in patients with naturally acquired Aeromonas diarrhea.

The secretory immunoglobulin A (sIgA) response at the intestinal mucosa is one of the primary defense mechanisms protecting against enteric infections and may therefore be used as an indicator of bacterial enteropathogenicity. In order to understand the role played by Aeromonas strains as gastrointestinal infectious agents, the sIgA response in fecal specimens obtained from patients with naturally acquired Aeromonas diarrhea was examined. Our results demonstrated a specific sIgA response which was directed against the exoproteins produced by Aeromonas strains. The specific Aeromonas sIgA reacted with extracellular products showing molecular masses similar to those of the Aeromonas hemolytic toxins such as aerolysin and AHH1. Some reactions were directed against other proteins that are known to be important factors in the pathogenicity of Aeromonas. The specific responses highlighted are in support of the view that one should consider at least certain biotypes of Aeromonas enteropathogenic.

Genotyping of rotaviruses in environmental water and stool samples in Southern Switzerland by nucleotide sequence analysis of 189 base pairs at the 5' end of the VP7 gene.

Stool specimens from children (<4 years old) with diarrhea were collected over a 1-year period in Ticino (southern region of Switzerland). During the same period, environmental samples were collected from surface waters in the proximity of major water treatment plants. From treatment plants, samples were collected from the raw sewage and before the release of the treated water. From rivers, samples were collected before and after receiving the treated waters. A single-step reverse transcription (RT)-PCR amplification of the entire VP7 gene from extracted double-stranded RNA was developed. For the water samples, a further nested PCR was necessary to increase sensitivity. All amplified viral products were sequenced, and the sequence profile was compared to that of the VP7 genes of human and animal rotaviruses from GenBank. Rotavirus strains are characterized by outer capsid proteins G (glycoprotein) and P (protease-cleaved protein). Correct G genotyping of viral sequences from stool and water samples was possible by analyzing only 189 bp at the 5' end of the VP7 gene. In the Ticino region, the most predominant G genotype among clinical and water samples was G1. Genotypes G2 and G4 were found only among clinical samples. We also detected rotavirus G1-type sequences in feces from a healthy adult. This finding corroborates the hypothesis that healthy adults act as potential reservoirs for the spread of rotavirus in the environment. In our experiments, this RT-PCR-based method for rotavirus genotyping has proven to be a useful tool for epidemiological investigations.

Epidemiological relationships between Aeromonas strains isolated from symptomatic children and household environments as determined by ribotyping.

Ribotyping was used to study the epidemiology of Aeromonas associated gastro-enteritis in young children. Ribotyping patterns of 29 Aeromonas strains (16 Aeromonas caviae, 8 Aeromonas hydrophila, 3 Aeromonas eucrenophila, 1 Aeromonas veronii, and 1 Aeromonas encheleia) isolated from primary stool cultures of sick children were compared using the GelCompare software with patterns of 104 strains (39 Aeromonas eucrenophila, 29 Aeromonas caviae, 11 Aeromonas encheleia, 10 Aeromonas hydrophila, 6 Aeromonas bestiarum, 3 Aeromonas veronii, 3 Aeromonas popoffii and 3 Aeromonas media) isolated from their household environment in order to investigate the route of transmission of these bacteria. Fifteen strains (approximately 47%) isolated from stool cultures of patients showed the same riboprofile as strains found in contacts or environment. In particular, three strains isolated from patients shared the same riboprofile with strains found in their domestic environment. The wide diffusion of potentially pathogenic Aeromonas strains in our household samples, and the high rate of asymptomatic carriers among family members, suggested that predisposing factors of the host could make children prone to an Aeromonas-related intestinal disease.

In situ analysis of sulfate-reducing bacteria related to Desulfocapsa thiozymogenes in the chemocline of meromictic Lake Cadagno (Switzerland).

Comparative sequence analysis of a 16S rRNA gene clone library from the chemocline of the meromictic Lake Cadagno (Switzerland) retrieved two clusters of sequences resembling sulfate-reducing bacteria within the family Desulfovibrionaceae. In situ hybridization showed that, similar to sulfate-reducing bacteria of the family Desulfobacteriaceae, bacteria of one cluster with similarity values to the closest cultured relatives of between 92.6 and 93.1% resembled free cells or cells loosely attached to other cells or debris. Bacteria of the second cluster closely related to Desulfocapsa thiozymogenes DSM7269 with similarity values between 97. 9 and 98.4% were generally associated with aggregates of different small-celled phototrophic sulfur bacteria, suggesting a potential interaction between the two groups of bacteria.

PCR detection, characterization, and distribution of virulence genes in Aeromonas spp.

We found 73.1 to 96.9% similarity by aligning the cytolytic enterotoxin gene of Aeromonas hydrophila SSU (AHCYTOEN; GenBank accession no. M84709) against aerolysin genes of Aeromonas spp., suggesting the possibility of selecting common primers. Identities of 90 to 100% were found among the eight selected primers from those genes. Amplicons obtained from Aeromonas sp. reference strains by using specific primers for each gene or a cocktail of primers were 232 bp long. Of hybridization group 4/5A/5B (HG4/5A/5B), HG9, and HG12 or non-Aeromonas reference strains, none were positive. PCR-restriction fragment length polymorphism (PCR-RFLP) with HpaII yielded three types of patterns. PCR-RFLP 1 contained two fragments (66 and 166 bp) found in HG6, HG7, HG8, HG10, and HG11. PCR-RFLP 2 contained three fragments (18, 66, and 148 bp) found in HG1, HG2, HG3, and HG11. PCR-RFLP 3, with four fragments (7, 20, 66, and 139 bp), was observed only in HG13. PCR-amplicon sequence analysis (PCR-ASA) revealed three main types. PCR-ASA 1 had 76 to 78% homology with AHCYTOEN and included strains in HG6, HG7, HG8, HG10, and HG11. PCR-ASA 2, with 82% homology, was found only in HG13. PCR-ASA 3, with 91 to 99% homology, contained the strains in HG1, HG2, HG3, and HG11. This method indicated that 37 (61%) of the 61 reference strains were positive with the primer cocktail master mixture, and 34 (58%) of 59 environmental isolates, 93 (66%) of 141 food isolates, and 100 (67%) of 150 clinical isolates from around the world carried a virulence factor when primers AHCF1 and AHCR1 were used. In conclusion, this PCR-based method is rapid, sensitive, and specific for the detection of virulence factors of Aeromonas spp. It overcomes the handicap of time-consuming biochemical and other DNA-based methods.

Signature region within the 16S rDNA sequences of Aeromonas popoffii.

To identify a group of eight Aeromonas strains of our collection showing ribotyping patterns similar to those described for the species Aeromonas popoffii, 16S rRNA gene sequence analysis was performed. Results were in agreement with the DNA binding values, and allowed the identification of a 'signature region' differentiating the A. popoffii strains from all other members of the genus Aeromonas.

In situ analysis of phototrophic sulfur bacteria in the chemocline of meromictic Lake Cadagno (Switzerland).

Comparative sequence analysis of a 16S rRNA gene clone library from the chemocline of the meromictic Lake Cadagno (Switzerland) revealed the presence of a diverse number of phototrophic sulfur bacteria. Sequences resembled those of rRNA of type strains Chromatium okenii DSM169 and Amoebobacter purpureus DSM4197, as well as those of four bacteria forming a tight cluster with A. purpureus DSM4197 and Lamprocystis roseopersicina DSM229. In situ hybridization with fluorescent (Cy3 labeled) oligonucleotide probes indicated that all large-celled phototrophic sulfur bacteria in the chemocline of Lake Cadagno were represented by C. okenii DSM169, while small-celled phototrophic sulfur bacteria consisted of four major populations with different distribution profiles in the chemocline indicating different ecophysiological adaptations.

[Group A beta-hemolytic streptococcal meningitis in a pregnant woman]. / Méningite à streptocoque beta-hémolytique du groupe A chez une femme enceinte.

A case of group A streptococcal meningitis in a normally healthy pregnant woman is described, with a review of the literature. Streptococcus pyogenes is a very rare cause of bacterial meningitis and especially affects immunocompetent subjects. The evolution is often associated with severe neurologic deficits, but the prognosis is favourable under treatment with penicillin. In the light of the recrudescence of invasive infections, this germ could assume a more important role in bacterial meningitis comparable to that in the pre-antibiotic era.

Use of amplified fragment length polymorphism in molecular typing of Legionella pneumophila and application to epidemiological studies.

A novel method for molecular typing of organisms, amplified fragment length polymorphism analysis, was tested for its suitability in epidemiological studies in medical microbiology. Amplified fragment length polymorphism analysis, originally developed for typing crop plants, consists of a simple restriction-ligation reaction and a subsequent PCR amplification. In a single-step reaction, the genomic DNA is digested and the restriction fragments are ligated to specially constructed adapters. PCR amplification of such tagged restriction fragments with primers complementary to the adapters allows the detection of restriction fragment length polymorphisms upon resolution on agarose gels. The method is fast, efficient, and reproducible for typing strains of Legionella pneumophila isolated from both humans and the environment. The accuracy of the method was tested by comparison with standard restriction fragment length polymorphism typing performed with both a ribosomal and a genomic probe.

Epidemiological typing of Legionella pneumophila with ribotyping. Report of two clinical cases.

The ribotyping method, adapted to the strains of Legionella pneumophila in our possession, was tested in two separate cases of legionellosis and in the associated finding of Legionella pneumophila in the water, from different sources, with which these patients had come into contact. Determination of the serogroup enabled us to carry out a preliminary analysis of the strains, which was then confirmed by application of the ribotyping procedure: the ribosomal profile of the strains found in the two patients correspond to that of the strains isolated from the water with which they had come into contact. These results provide important information concerning the probable sources of infection involved in these two cases of Legionnaires' disease. We consider ribotyping to be a very useful tool, which is easy and simple to perform and is applicable to the Legionella genus as the method of choice for epidemiological studies.

Bacteroides ureolyticus in men consulting for infertility.

A screening of 3196 semen analyses performed in our clinic from January 1986 to December 1990 revealed 314 (9.8%) patients whose semen was infected with Bacteroides ureolyticus. Investigating the relationship between the presence of B. ureolyticus, the seminal microflora and the conventional semen parameters, we observed that the presence of this micro-organism in the semen was coupled (1) to an increased presence of Enterococcus species, (2) to an increased number of short-tailed spermatozoa and epithelial cells, and (3) to a decreased total fructose concentration (mg ejaculate-1). These results suggest that B. ureolyticus or its toxins may influence sperm morphology and function by yet unknown mechanisms and may also increase the number of epithelial cells by soft tissue infection in vivo. The decreased fructose levels suggest that this anaerobic micro-organism might specifically colonize the seminal vesicles, while the normal zinc values recorded suggest a normal prostatic function. Overall, our data support the hypothesis that the presence of B. ureolyticus is not associated with nongonococcal urethritis.