A randomized controlled trial assessing infectious disease risks from bathing in fresh recreational waters in relation to the concentration of Escherichia coli, intestinal enterococci, Clostridium perfringens, and somatic coliphages.

We performed epidemiologic studies at public freshwater bathing sites in Germany to provide a better scientific basis for the definition of recreational water quality standards. A total of 2,196 participants were recruited from the local population and randomized into bathers and nonbathers. Bathers were exposed for 10 min and had to immerse their head at least three times. Water samples for microbiological analysis were collected at 20-min intervals. Unbiased concentration-response effects with no-observed-adverse-effect levels (NOAELs) were demonstrated for three different definitions of gastroenteritis and four fecal indicator organisms. Relative risks for bathing in waters with levels above NOAELs compared with nonbathing ranged from 1.8 (95% CI, 1.2-2.6) to 4.6 (95% CI, 2.1-10.1), depending on the definition of gastroenteritis. The effect of swallowing water provided additional evidence for true dose-response relationships. Based on the NOAELs, the following guide values for water quality are suggested: 100 Escherichia coli, 25 intestinal enterococci, 10 somatic coliphages, or 10 Clostridium perfringens per 100 mL. Recreational water quality standards are intended to protect the health of those consumers who are not already immune or resistant to pathogens that may be associated with indicator organisms. In contrast to current World Health Organization recommendations, we concluded that standards should be based on rates of compliance with NOAELs rather than on attributable risks determined above NOAELs, because these risks depend mainly on the unpredictable susceptibility of the cohorts. Although in theory there is no threshold in real concentration-response relationships, we demonstrated that a NOAEL approach would be a more robust and practical solution to the complex problem of setting standards.

Development and test for long-term stability of a synthetic standard for a quantitative Cryptosporidium parvum LightCycler PCR assay.

A recently described quantitative rapid cycle real time PCR (LightCycler) assay detects Cryptosporidium parvum after in vitro excystation, which is a surrogate marker for the viability of the organisms. In the original assay the quantification standard is a dilution series of C. parvum oocysts with a microscopically determined excystation rate. The need to keep suspensions of viable oocysts in stock and to continuously monitor their excystation rate, however, renders the assay impracticable for routine application. A synthetic standard was developed to replace the in vivo standard and was calibrated using oocysts with known excystation rates. The standard consists of a 486 bp DNA segment ranging from 229 bp upstream to 79 bp downstream of the actual PCR target site. Aliquots of the standard were frozen and stored at -20 degrees C and at -70 degrees C or lyophilised and stored at room temperature in the dark. For a period of one year samples preserved with each of the three methods were restored every four or five weeks. They were amplified in the LightCycler and the crossing points (CP) were monitored. No significant trend in the raw CP values could be observed for any of the three storage methods. However, when the methods were compared to each other by calculating the CP ratios (-20 degrees C/-70 degrees C; -20 degrees C/lyophilised; -70 degrees C/lyophilised) at the 10 monitoring dates, the CP ratios -20 degrees C/-70 degrees C and -20 degrees C/lyophilised showed a highly significant positive trend (p < 0.0001) while the CP ratio -70 degrees C/lyophilised did not differ from the null hypothesis (p = 0.53). It can be concluded that the latter two preservation methods are both appropriate, while storage at -20 degrees C is less advisable. Calculations based on the molecular weight of the standard and on the assumption of an average yield of three sporozoites per oocyst led to the conclusion that the target sequence is probably located on a double copy gene.

Improved detection of Salmonella spp. in foods by fluorescent in situ hybridization with 23S rRNA probes: a comparison with conventional culture methods.

This report describes a new technique for the detection and identification of Salmonella species in food with the use of fluorescent in situ hybridization (FISH) with 23S rRNA-targeted oligonucleotide probes. Two species-specific 23S rRNA-targeted oligonucleotide probes (Sal-1 and Sal-3) were selected, and one (Sal-544) was newly designed. The relative specificities of these probes were compared with those of bacterial 23S rRNA sequences from the GenBank database and tested by in situ hybridization with bacterial cell smears of pure cultures. Fifty-one tested reference strains of Salmonella serovars belonging to subspecies I (enterica) hybridized with these probes. No cross-reactions with 46 other strains of the family Enterobacteriaceae or with another 14 bacterial strains from other families were observed. Storage of a Salmonella Panama test strain under various environmental conditions (2, 5, and 15% NaC1; -20 degrees C, 4 degrees C, and room temperature; pHs of 3.3 to 7.4) did not adversely affect the FISH method. No matrix effects were observed with 18 different kinds of foods. FISH was able to detect Salmonella spp. in 52 (probe Sal-1), 56 (probe Sal-3), and 35 (probe Sal-544) of 225 naturally contaminated food samples after 16 h of incubation in a preenrichment broth. When conventional culture and detection methods were used, Salmonella could be isolated from only 30 of these 225 samples. In contrast, FISH failed to identify Salmonella in only two of the culture-positive samples when Sal-1 and Sal-3 were used and in only three of the culture-positive samples when Sal-544 was used.

DNase pretreatment of master mix reagents improves the validity of universal 16S rRNA gene PCR results.

DNase I pretreatment of 16S rRNA gene PCR reagents was tested. The DNase I requirement for the elimination of false-positive results varied between 0.1 and 70 IU per master mix depending on the applied Taq polymerase. PCR sensitivity was mostly maintained when 0.1 IU of DNase I was used.

Effects of reduced mucus oxygen concentration in airway Pseudomonas infections of cystic fibrosis patients.

Current theories of CF pathogenesis predict different predisposing "local environmental" conditions and sites of bacterial infection within CF airways. Here we show that, in CF patients with established lung disease, Pseudomonas aeruginosa was located within hypoxic mucopurulent masses in airway lumens. In vitro studies revealed that CF-specific increases in epithelial O(2) consumption, linked to increased airway surface liquid (ASL) volume absorption and mucus stasis, generated steep hypoxic gradients within thickened mucus on CF epithelial surfaces prior to infection. Motile P. aeruginosa deposited on CF airway surfaces penetrated into hypoxic mucus zones and responded to this environment with increased alginate production. With P. aeruginosa growth in oxygen restricted environments, local hypoxia was exacerbated and frank anaerobiosis, as detected in vivo, resulted. These studies indicate that novel therapies for CF include removal of hypoxic mucus plaques and antibiotics effective against P. aeruginosa adapted to anaerobic environments.

The effect of human serum DNAases on the ability to detect antibiotic-killed Escherichia coli in blood by PCR.

PCR has proved superior to conventional blood culture for diagnosing bacteraemia in the presence of antibiotics. Nevertheless, even PCR might yield false-negative results if the template DNA were to be cleaved by serum DNAases after antibiotics had induced bacterial death. To evaluate the cleavage of bacterial template DNA by human serum DNAase I, serum samples inoculated with purified Escherichia coli DNA were incubated with increasing amounts of recombinant human DNAase (rhDNAase) and then examined by a PCR specific for E. coli. As a prerequisite of potential DNAase attack, the release of E. coli DNA after antibiotic-induced bacterial death was quantified by fluorescence microscopy and flow cytometry. Finally, the influence of rhDNAase on the PCR-based detection of antibiotic-killed E. coli in serum was assessed. The results indicated that purified E. coli DNA is remarkably stable in human serum; positive PCR results did not decrease significantly until the ratio of recombinant human DNAase I:E. coli rose to 106:1. As only 14.8-28.4% of the total E. coli DNA was released after antibiotic killing, the PCR-based detection of E. coli fell by only 10% when cefotaxime-killed E. coli were incubated with rhDNAase. It was concluded that human serum DNAases and antibiotic killing do not compromise the reliability of PCR examinations for bacteraemia.