Konrad Botzenhart

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 non-bathers. 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.

Distribution and transmission of Pseudomonas aeruginosa and Burkholderia cepacia in a hospital ward

Genotyping and antibiotic susceptibility testing were used to analyze Pseudomonas aeruginosa and Burkholderia cepacia strains from sink drain from 14 pediatric patients with cystic fibrosis (CF) and from hospital personnel as part of a 4 week prospective study of strain transmission in a pediatric ward. A total of 87.5% of all washbasin drains were contaminated with P. aeruginosa [102 to 105 colony forming units (CFU)/ml sink fluid], whereas B. cepacia was found only once in a sink drain. From the eight CF patients already infected with P. aeruginosa upon entering the ward, we isolated six genotypes that were identical with strains found in sink drains of the ward. Four of the 16 members of the personnel had one positive P. aeruginosa hand culture. B. cepacia was never found in patients or on personnel hands. Hand washing in contaminated sinks (≥ 103 CFU/ml) led to positive P. aeruginosa or B. cepacia hand cultures. P. aeruginosa or B. cepacia embedded in sputum were transmissable by hand shaking for up to 180 min, whereas both pathogens suspended in physiological saline were transmissable to other hands only up to 30 min. Genotyping of P. aeruginosa revealed strain transmission from CF patients or the environment to other patients or the personnel, as well as one transmission from the environment to a CF patient. The ability of CF sputum to prolong survival of P. aeruginosa and B. cepacia may be important for strain transmission. The results suggest that improved hygienic measures are required to prevent routes of bacterial transmission via the hands and sink drains. Pediatr Pulmonol. 1996; 21:90–100.

Pseudomonas aeruginosa cross-colonization and persistence in patients with cystic fibrosis. Use of a DNA probe

To investigate cross-colonization with and persistence of Pseudomonas aeruginosa in cystic fibrosis (CF), 181 isolates from 76 CF patients were typed using a P. aeruginosa-specific DNA probe. Whereas sibling pairs predominantly harboured genotypically identical P. aeruginosa strains, all of the other patients harboured different strains. Seventy-nine per cent (22/31) of the infected CF patients harboured the same strains at the beginning and the end of a summer camp. A change of strains was seen in 10% (3/31) of the patients at the end of the camp. Forty-six per cent (6/13) of the patients who were apparently initially uninfected, acquired P. aeruginosa by the end of the period. Genotyping proved that strain change or acquisition was due to cross-colonization in four of nine cases. Very little P. aeruginosa was isolated from the inanimate environment. Persistence of P. aeruginosa after a temporary loss due to antibiotic therapy was seen in 12/16 paired patient strains before and after antibiotic therapy. Thus, suppression followed a flare-up seemed to occur in these patients rather than eradication and a new infection. When 35 patients were followed over a period of 6 months, 7 (20%) changed the strain in their sputum. Only one of 43 patients harboured two different P. aeruginosa strains simultaneously over a long period.

Trefoil factor family domain peptides in the human respiratory tract

Trefoil factor family domain peptides (TFF) are thought to be involved in mucosal epithelial restitution and wound healing of the gastrointestinal tract and are up‐regulated in ulceration and in a variety of solid tumours. It was hypothesized that TFFs are also expressed on mucosal surfaces of the human respiratory tract. Lung tissue, nasal polyps, and sputum samples from seven patients with cystic fibrosis (CF), two with chronic and acute bronchitis, and non‐dysplastic material from two cases of bronchial adenocarcinoma were analysed for TFF expression by immunohistochemistry, immunofluorescence, western blot and RT‐PCR. Expression of TFF1 and TFF3 was observed in material from all patients. TFFs were localized in goblet and ciliated cells, as well as in some submucosal cells of tracheobronchial tissues and nasal polyps from normal and CF individuals. In sputa of patients with CF and with chronic or acute bronchitis, TFF1 and TFF3 were detected by western blotting. Freshly cultivated nasal epithelial cells transcribed and secreted TFFs and mucins, whereas nasal cells cultivated for 6 weeks still expressed mucins, but not TFFs. Secreted TFFs and mucins also bound to the surface of Staphylococcus aureus in infected CF airways. In conclusion, TFF1 and TFF3 are expressed and secreted in normal and inflamed airways. The association of TFFs with bacteria may contribute to the anti‐microbial mucociliary defence system. Copyright

Ecology and Epidemiologyof Pseudomonas aeruginosa

The first description of Pseudomonas aeruginosa as a distinct bacterial species was made at the end of the nineteenth century, after Pasteur’s development of sterile culture media. Screening for dyes provided the stimulus for the first scientific study on P. aeruginosa published by pharmacist Carle Gessard in 1882 and entitled “On the blue and green coloration of bandages.”1 This characteristic pigmentation, later attributed to a phenazine derivative, pyocyanine, is reflected in the old names Bacillus pyocyaneus, Pseudomonas polycolor, Bakterium aeruginosa and Pseudomonas pyocyaneus. Although the ability of P. aeruginosa to produce infections was noticed by 1889,2 its pathogenicity was doubted,3 and P. aeruginosa was regarded mainly as a source of potent antimicrobial substances.4 Before 1947 only 91 cases of septicemia attributable to P. aeruginosa were reported in the literature.5 Its importance as a human pathogen, especially in hospitalized patients, did not emerge until the second half of the twentieth century,6 although the organism was certainly present in the inanimate and human environment before then. Because P. aeruginosa is easy to culture and identify it is unlikely that it was missed by clinical microbiologists. Thus, the considerable change in the significance of P. aeruginosa as a nosocomial pathogen probably reflects advances made in the life sciences as well as changes in the susceptibility of patients.

Molecular Epidemiology of Community-Acquired Staphylococcus auveus in Families with and without Cystic Fibrosis Patients

The molecular epidemiology of Staphylococcus aureus nasal commensal strains and community-acquired infecting strains was assessed by comparison of prevalence, persistence, transmission rate, and clonal distribution of S. aureus in families with and without cystic fibrosis (CF) patients. Isolates were typed by pulsed-field gel electrophoresis. CF patients without antibiotic treatment had a significantly higher nasal prevalence (66%) of S. aureus than did treated patients (29%; P<.001) or healthy controls (32%; P<.001), suggesting that persons with CF have a higher susceptibility to this organism. Strain transmission was frequent within both CF (55%) and non-CF (62%) families. After 3 and 19 months, 57% and 21%, respectively, of all persons still harbored the same S. aureus strain. Most of the isolates (78%) belonged to 8 of 38 genome types common in CF patients and in healthy persons. The predominant occurrence of a limited number of S. aureus clones within the community suggests evolutionary mechanisms for the selection of certain strains without an obvious association with disease.

PCR detection of Cryptosporidium parvum in environmental samples—a review of published protocols and current developments

Since 1991 more than 30 PCR protocols have been published, which show a potential to replace the current microscopic detection method for Cryptosporidium parvum in environmental samples and food. This review provides a synoptic comparison of these protocols with respect to the following features: isolation and purification of oocysts from tested matrices, elimination of free DNA, viability and infectivity assessment, release of nucleic acids, nucleic acid extraction, type of PCR (PCR, RT-PCR, internal-standard-PCR, in situ PCR, TaqMan-PCR), primary product detection, additional specificity control, secondary product detection, reported sensitivity, cross-reaction with other Cryptosporidium species, and target and sequence information such as amplicon length, primer sequences, multiple copy target, presence of strain-specific differences in the amplicon, GenBank accession numbers and gene function. The results demonstrate that problems like PCR inhibition, viability assessment, and the requirement of an extreme sensitivity have been solved. PCR assays would be most valuable to control presence-absence standards in defined matrix volumes, and the setup of such standards would very much contribute to a rapid introduction of this awaited technology into routine monitoring of environmental, water and food samples, and to a further standardization of the various protocols. It can be expected that satisfactory solutions for quantification will be found for a growing number of PCR-based assays. Systematic field evaluation and interlaboratory studies will complement our present knowledge of these methods in the near future.

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.

Rapid identification of Enterobacteriaceae using a novel 23S rRNA-targeted oligonucleotide probe.

The aim of this study was to rapidly identify bacteria of the family of Enterobacteriaceae using fluorescent in situ hybridization (FISH). A comparative sequence analysis was carried out and a 23S rRNA signature sequence for Enterobacteriaceae was identified. A 23S rRNA-targeted oligonucleotide probe (EBAC1790) was constructed and subsequently tested against 40 reference strains. Nearly all of the Enterobacteriaceae used in this study yielded positive results with EBAC1790, except for Edwardsiella tarda (ATCC 15947). None of the non-Enterobacteriaceae reference strains gave positive signals with the probe. The possibility of a rapid detection of Enterobacteriaceae in groundwater was demonstrated using colony hybridization.

A case report of false negative Legionella test results in a chlorinated public hot water distribution system due to the lack of sodium thiosulfate in sampling bottles

We examined samples from the showers and the central water distribution system of a public building with an indoor swimming pool. The pool was used for school and recreational activities and as a sports therapy facility for patients with coronary heart disease. The buildings hot water system was contaminated with Legionella pneumophila. Due to the buildings intricate piping system, several attempts to completely eliminate legionellae by thermal and chemical disinfection had failed, so an external sanitation company was charged with the installation of a continuous chlorination device in order to keep Legionella concentrations low. The laboratory which was contracted by the sanitation company to monitor bacteria levels after installation of the chlorination device used sampling bottles without sodium thiosulfate and repeatedly reported an absence of Legionella. However, up to 69,000 colony forming particles (CFP) of Legionella pneumophila (Lp) per litre and up to 171 CFP/ml of heterotrophic bacteria could be detected when parallel samples were collected in bottles containing sodium thiosulfate at standard concentrations. Laboratories, epidemiologists, public health officials and technical staff who may be in charge of delivering, preparing or using sterile sampling devices for the collection of environmental samples to be tested for legionellae should be aware that cultures can return false negative results if the sampling containers used to collect chlorinated drinking water or chlorinated pool water samples do not contain a neutralizing agent to instantly inactivate residual halogen biocides. False negative results may lead to a false sense of security regarding the safety of water systems or the success of disinfection measures, and may thus endanger public health or even hinder the epidemiological clarification of outbreaks.