[Detection of viruses in raw water as a basic tool for risk assessment]. / Nachweis von Viren im Rohwasser als Grundlage einer Risikoabschätzung.

Human pathogenic viruses may end up in surface waters by fecal contamination. However, the German drinking water ordinance requests that pathogens in drinking water should not be present in concentrations constituting a potential danger to human health. Since many viruses do have a very low dose of infection, they have to be sufficiently eliminated in the process of drinking water purification. Waterborne virus outbreaks in Europe, over the last few decades, were mostly linked to noncompliance with the generally accepted codes of practice for drinking water production. The aimed level of protection of drinking water supplies in Germany, however, exceeds prevention of outbreaks by even protecting against sporadic virus infections. Documentation of such a high level of protection is not achieved by end product control alone but requires a process analysis with risk assessment. To do such an analysis, information regarding the presence of viruses in the raw water used for drinking water production, as well as data of virus elimination rates during purification processes, are of major importance. This paper presents suggestions for implementation of such a risk assessment, focusing on the evaluation of raw water quality.

[Viruses in drinking water]. / Viren im Trinkwasser.

Viruses in drinking water can cause infectious diseases. In the past, hepatitis A and E were the most frequently observed drinking- water-borne viral infections, but in recent years several small- and large-scale norovirus epidemics have been described, even in Europe. All virus species spread via drinking water are of fecal origin. They are regularly identified in waste water even after conventional multi-stage water treatment. The approved disinfection methods can cope with these viruses if they are not integrated in larger particles. For this reason particle separation is particularly important in water treatment. Virological tests are not reliable enough to ensure that drinking water is sufficiently virus-free. The examination of 100 mL of water for E. coli and coliform bacteria is not adequate proof either. If potentially contaminated raw water is used, consumer safety must be ensured by calculating the performance of water treatment plants on a case-by-case basis. Such a calculation takes into account the virus load of the raw water, the efficiency of the physical and chemical particle elimination steps and the effect of disinfection. Those factors which determine the effectiveness of disinfection, namely concentration and exposure time or UV radiation strength, must be adjusted according to the risk of viral infection, and calculated settings must be adhered to, even if favorable E. coli levels may make them seem excessive.

Effect of comprehensive validation of the template isolation procedure on the reliability of bacteraemia detection by a 16S rRNA gene PCR.

The influence of the DNA extraction method on the sensitivity and specificity of bacteraemia detection by a 16S rRNA gene PCR assay was investigated. The detection limit of the assay was 5 fg with purified DNA from Escherichia coli or Staphylococcus aureus, corresponding to one bacterial cell. However, with spiked blood samples, the detection limits were 10(4) and 10(6) CFU/mL, respectively. The sensitivity of the S. aureus assay was improved to the level of the E. coli test with the addition of proteinase K to the commercial DNA extraction kit protocol. Ten (16.6%) of 60 amplification reactions were positive with templates isolated from sterile blood, while PCR reagent controls were negative, thereby indicating contamination during the DNA extraction process. Blood samples were spiked with serial dilutions of E. coli and S. aureus cells, and six PCR results were obtained from three extractions for each blood sample. A classification threshold system was devised, based on the number of positive reactions for each sample. Samples were deemed positive if at least four positive reactions were recorded, making it possible to avoid false-positive results caused by contamination. These results indicate that a comprehensive validation procedure covering all aspects of the assay, including DNA extraction, can improve considerably the validity of PCR assays for bacteraemia, and is a prerequisite for the meaningful detection of bacteraemia by PCR in the clinical setting.

Effects of amoxicillin, gentamicin, and moxifloxacin on the hemolytic activity of Staphylococcus aureus in vitro and in vivo.

In Staphylococcus aureus infection hemolysis caused by the extracellular protein alpha-toxin encoded by hla is thought to contribute significantly to its multifactorial virulence. In vitro, subinhibitory concentrations of beta-lactam antibiotics and fluoroquinolones increase the levels of hla and alpha-toxin expression, whereas aminoglycosides decrease the levels of hla and alpha-toxin expression. In the present study we investigated the effects of subinhibitory concentrations of amoxicillin, gentamicin, and moxifloxacin on hla and alpha-toxin expression and total hemolysis of S. aureus strain 8325-4, a high-level alpha-toxin producer, and its alpha-toxin-negative mutant, DU 1090, in vitro and in a rat model of chronic S. aureus infection. The levels of expression of hla and alpha-toxin and total hemolysis did not differ significantly when amoxicillin, gentamicin, or moxifloxacin was added to cultures of S. aureus strain 8325-4. In vivo, strain 8325-4 induced a significantly increased level of hemolysis in infected pouches compared to that in uninfected control pouches, but the hemolysis was reduced to control levels by treatment with doses of amoxicillin, gentamicin, or moxifloxacin that reduced bacterial numbers by 2 orders of magnitude. Additionally, the effects of subinhibitory concentrations of the three antibiotics on total hemolysis of four methicillin-resistant S. aureus and three methicillin-sensitive S. aureus (MSSA) clinical isolates were assessed in vitro. A significant increase in total hemolysis was observed for only one MSSA strain when it was treated with amoxicillin but not when it was treated with moxifloxacin or gentamicin. When purified alpha-toxin was incubated with purified human neutrophil elastase, alpha-toxin was cleaved nearly completely. The results suggest that the penicillin-induced increases in S. aureus alpha-toxin expression are strain dependent, that reduction of bacterial numbers in vivo counteracts this phenomenon effectively, and finally, that in localized S. aureus infections alpha-toxin activity is controlled by neutrophil elastase.

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 building's hot water system was contaminated with Legionella pneumophila. Due to the building's 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.

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.

Adherence of Staphylococcus aureus to endothelial cells: influence of capsular polysaccharide, global regulator agr, and bacterial growth phase.

The adherence of Staphylococcus aureus to human endothelial cells (EC) is probably an important step in the pathogenesis of systemic staphylococcal infections. We examined the influence of type 5 capsular polysaccharide (CP5) production, the global regulator agr, and the bacterial growth phase on S. aureus adherence to EC. Whereas S. aureus Newman showed maximal adherence to EC in the logarithmic phase of growth, an isogenic agr mutant showed maximal adherence in the stationary growth phase. S. aureus adherence to EC and CP5 expression were negatively correlated: a mutation in the agr locus diminished CP5 production and led to increased adherence. Likewise, induction of CP5 expression by addition of NaCl to the growth medium resulted in reduced staphylococcal adherence to EC. S. aureus Newman cells that adhered to EC did not express CP5. A Newman cap5O mutant was acapsular and showed significantly greater adherence to EC than the parental strain did (P<0.005). Complementation of the cap5O mutation in trans restored CP5 expression and reduced EC adherence to a level similar to that of the parental strain. The enhanced adherence shown by the cap5O mutant was similar in magnitude to that of the agr mutant or the cap5O agr double mutant. Cells of the cap5O mutant and cap5O agr double mutant harvested from stationary-phase cultures adhered significantly better than did cells harvested in the exponential growth phase. These data are consistent with the postexponential and agr-independent expression by S. aureus of at least one putative EC adhesin, whose binding domain may be masked by CP5.

Molecular epidemiology of community-acquired Staphylococcus aureus 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.

Direct quantitative transcript analysis of the agr regulon of Staphylococcus aureus during human infection in comparison to the expression profile in vitro.

Bacteria possess a repertoire of distinct regulatory systems promoting survival in disparate environments. Under in vitro conditions it was demonstrated for the human pathogen Staphylococcus aureus that the expression of most virulence factors is coordinated by the global regulator agr. To monitor bacterial gene regulation in the host, we developed a method for direct transcript analysis from clinical specimens. Quantification of specific transcripts was performed by competitive reverse transcription-PCR, and results were normalized against the constitutively expressed gene for gyrase (gyr). Using sputum from cystic fibrosis (CF) patients infected with S. aureus we examined the transcription of the effector molecule RNAIII of agr, of spa (protein A), generally repressed by agr, and of hla (alpha-toxin), generally activated by agr. In the CF lung RNAIII was expressed poorly, indicating an inactive agr in vivo. Despite the low level of RNAIII expression, spa was detectable only in minute amounts and an irregular transcription of hla was observed in all sputum samples. After subculturing of patient strains agr-deficient isolates and isolates with unusual expression profiles, i.e., not consistent with those obtained from prototypic strains, were observed. In conclusion, the agr activity seems to be nonessential in CF, and from the described expression pattern of spa and hla, other regulatory circuits aside from agr are postulated in vivo.

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.

[Comparison of European standards of swimming pool safety]. / Vergleich europäischer Bestimmungen zur Schwimmbeckenwasserhygiene.

European countries follow different procedures to monitor the quality of the water of swimming pools open to the public: some use generally accepted technical standards, some officially recommended guidelines, some rules or regulations by local authorities and some have national laws or regulations. These agree insofar as in every country the water of swimming pools must be disinfected. The microbiological limits are to a certain extent identical with drinking water threshold values, e.g., no faecal coliforms must be present in 100 ml. Other microorganisms as for instance legionellae, staphylococci or Pseudomonas aeruginosa ar not uniformly considered. In all countries the water must be disinfected by chlorination. Big differences exist, however, in respect of the maximum admissible concentrations of free or total chlorine, haloforms and organic carbons. With regard to the common goals, harmonization of the regulations appears possible.

PCR and blood culture for detection of Escherichia coli bacteremia in rats.

Critically ill patients often develop symptoms of sepsis and therefore require microbiological tests for bacteremia that use conventional blood culture (BC) techniques. However, since these patients frequently receive early empirical antibiotic therapy before diagnostic procedures are completed, examination by BC can return false-negative results. We therefore hypothesized that PCR could improve the rate of detection of microbial pathogens over that of BC. To test this hypothesis, male Wistar rats were challenged intravenously with 10(6) CFU of Escherichia coli. Blood was then taken at several time points for detection of E. coli by BC and by PCR with E. coli-specific primers derived from the uidA gene, encoding beta-glucuronidase. In further experiments, cefotaxime (100 or 50 mg/kg of body weight) was administered intravenously to rats 10 min after E. coli challenge. Without this chemotherapy, the E. coli detection rate decreased at 15 min and at 210 min after challenge from 100% to 62% of the animals with PCR and from 100% to 54% of the animals with BC (P, >0.05). Chemotherapy decreased the E. coli detection rate at 25 min and at 55 min after challenge from 100% to 50% with PCR and from 100% to 0% with BC (P, <0.05). Thus, at clinically relevant serum antibiotic levels, PCR affords a significantly higher detection rate than BC in this rat model. The results suggest that PCR could be a useful adjunct tool supplementing conventional BC techniques in diagnosing bacteremia.

[Dust and microorganism count at delivery, sorting and composting of home refuse and home refuse-like industrial waste]. / Staub- und Keimzahlmessungen bei der Anlieferung, Sortierung und Kompostierung von Hausmüll und hausmüllähnlichem Gewerbeabfall.

The exposure of employees to airborne dust and microorganisms was assessed in a waste processing plant established to recover reusable materials from unsorted domestic and industrial waste. Exposure criteria considered relevant were the quantity of the individual size-selected particle fractions, the morphological properties of the particles, their heavy metal content, and the degree of their contamination with various microorganisms and mold. In addition, separate microbiological analyses to determine potential pathogen concentrations in the air were made. The highest concentrations of total and fine dust were measured in the waste delivery area. A close correlation between the frequency of deliveries and the level of dust exposure was observed. In this area, fine dust concentrations exceeded the threshold limit value of 6 mg/m3 repeatedly for shorter periods. The average fine dust concentration during an entire work-shift, however, was considerably lower than this value. Particles with an aerodynamic diameter of 2 to 7 microns predominated both in the waste delivery and the processing areas. Fibrous dust particles were present in smaller numbers than spherical particles and consisted mainly of organic materials. Natural and artificial inorganic fibers were found only occasionally. The concentrations of lead, cadmium, nickel and mercury were considerably lower than the corresponding MAK and TRK values, and were--with the exception of lead--in the range of the respective metal concentrations in the atmosphere of urban areas. Microscopical examinations of used protective masks (protection category P2) revealed that dust particles were deposited even on the inner side of the masks. Most of the particles on this side were very small and carried nickel or titanium. Microorganism concentrations measured in air from the highly dust-exposed areas of the plant showed values up to 6.9 x 10(5) cfu/m3, with a mold content of 6.6 x 10(4) cfu/m3. Approximately 90% of the microorganisms were deposited on particles of the fine dust fraction (particle size < 7 microns), and more than 50% contaminated particles with an aerodynamic diameter of 2 to 4.7 microns. In the compost facility, mold concentrations of up to 8.4 x 10(5) cfu/m3 were measured. In contrast, the level of microbial contamination in the filtered air from the compost facility did not exceed the concentration measured in air outside the plant. The data which were obtained during the winter months are probably at the lower end of the average exposure range over the entire year. In regard to exposure assessment, it should be mentioned that particles with a size of 4 to 7 microns are not really "inert" particles, since they are preferential carriers for heavy metals and microorganisms. More studies in the future should be performed to establish, whether the level of exposure to microorganisms can be estimated indirectly by the determination of dust exposure.

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 [10(2) to 10(5) 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 (> or = 10(3) 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.

Selective detection of viable Cryptosporidium oocysts by PCR.

Although the diagnosis of cryptosporidiosis in diarrheic patients is relatively simple, as large amounts of oocysts are usually shed, environmental samples can contain only few oocysts which have a comparatively high epidemiological relevance. Very sensitive detection methods are therefore requested in environmental hygiene. Additionally these methods should allow a statement about the viability of the detected organisms. The combination of a DNA-digest and a viability assay followed by a PCR might reveal a very promising method. A DNA-digest destroys free DNA in the sample. In the following excystation protocol only viable sporozoites excyst. A PCR carried out now gives definitive proof of viable oocysts. Dead sporozoites within intact oocyst-walls are not detected.

[Occurrence and detection of viruses in drinking water]. / Vorkommen und Nachweis von Viren im Trinkwasser.

Viruses can pass disinfection steps of water treatment plants without being inactivated. Investigations during the last 15 years revealed repeatedly the presence of enteric viruses in finished water meeting standards for coliform bacteria. Methods for the detection of viruses in water which implicate their growth on specific cell cultures are very time consuming and do not cover many viral species. Molecular detection methods including PCR techniques may help in the development of alternate methods for virus detection particularly in drinking water which usually does not contain PCR inhibiting factors.

[Effect of ozone on airborne microorganisms]. / Wirkung von Ozon in der Luft auf Mikroorganismen.

The microbicidal effect of ozone in air was tested at concentrations between 50 and 600 micrograms/m3 against the species: Staphylococcus epidermidis, Micrococcus luteus, Arthrobacter citreus, Bacillus subtilis (veg.), Escherichia coli, Salmonella typhimurium, Serratia marcescens, Pseudomonas fluorescens and Candida albicans. The microorganisms were exposed on membrane filters at 60-75% relative humidity and 21.5-22.5 degrees C. After exposure times between 1 min and 60 min, the filters were incubated on appropriate agar media. The effect of ozone was determined by comparing the number of colonies on exposed filters to the number on nonexposed filters. The die-off curves (colony count against time) proved not to be rectilinear, but to become steeper with increasing time of exposure. Furthermore, the velocity of reduction increased more than proportional with increasing concentration of ozone. Therefore, the bacterial decay seems not to follow first order reaction kinetics. The values presented for k (constant of the velocity of die-off) and D (decimal reduction time) are valid only for narrow ranges of the initial part of the exposure. Concentrations of 50 to 100 micrograms (0)3/m3 for 1 h resulted only in little reduction, whereas 500 to 600 micrograms/m3 for one hour led to 99% reduction in all bacterial species tested. The gram-positive species seemed to be more sensitive than the gram-negative species, C. albicans proved to be more resistant than the bacteria.