Water quality assessment of a Central European River - does the Directive 2000/60/EC cover all the needs for a comprehensive classification?

Over the last decades, European rivers have mostly shared the same fate. Until the mid 1980s many of them were in rather bad shape. Nobody would have thought about using the waters for recreation. However, continuous efforts over the last three decades have achieved success and today's water quality has changed to the better. This obvious change in water quality, which was proved by investigations done on the basis of the European Directive 2000/60/EC framework for Community action in the field of water policy, made people consider using the rivers for recreation or irrigation. In the present study a Central European River was investigated from hygienic perspectives (faecal indicators), and its toxicological status was examined - both of which are parameters which are not included in the Directive 2000/60/EC. The pivotal question was whether these parameters provide essential and additional information about the river's status and its quality. In general, the monthly values for all sampling points from the toxicological tests correlated with the determined physical and chemical statuses according to the national water management plan. Once, however, the toxicological values did increase significantly. This detection of a single toxicological burst within a year of observation underlines the applicability of the tests as screening methods for the detection of toxic substances. Moreover, our microbiological investigations show high numbers of faecal indicators and Salmonellae. These results are not consistent with the apparently good quality of the river. Therefore faecal indicators, which are directly related to the discharge of waste water, should be an integral part of the procedure of water classification.

Comparison of extended-spectrum-ß-lactamase (ESBL) carrying Escherichia coli from sewage sludge and human urinary tract infection.

For many years, extended-spectrum-beta-lactamase (ESBL) producing bacteria were a problem mainly located in medical facilities. Within the last decade however, ESBL-producing bacteria have started spreading into the community and the environment. In this study, ESBL-producing Escherichia coli from sewage sludge were collected, analysed and compared to ESBL-E. coli from human urinary tract infections (UTIs). The dominant ESBL-gene-family in both sample groups was bla(CTX-M), which is the most prevalent ESBL-gene-family in human infection. Still, the distribution of ESBL genes and the frequency of additional antibiotic resistances differed in the two sample sets. Nevertheless, phenotyping did not divide isolates of the two sources into separate groups, suggesting similar strains in both sample sets. We speculate that an exchange is taking place between the ESBL E. coli populations in infected humans and sewage sludge, most likely by the entry of ESBL E. coli from UTIs into the sewage system.

Concentrations of viable airborne fungal spores and trichloroanisole in wine cellars.

In wineries, unwanted microorganisms present not only hygienic problems but also have a negative influence on wine quality. An evaluation of Austrian/Styrian wine cellars with regard to the volume and the composition of the mycoflora is very important both for the process of wine production and for occupational safety. Thirty-six wine cellars of 20 vintners were investigated with regard to microorganisms in the air and on material surfaces. Moreover, the presence of trichloroanisole in the air was determined by means of solid-phase micro-extraction. Microorganisms were sampled using the six-stage Andersen-Cascade impactor. The results showed that the concentrations of xerophilic fungi in the air of cellars with large visible mold areas (> 80%) reached values up to 1.4 × 104 colony forming units per m³. In the wine cellars fourteen predominant fungal genera were found in the indoor air, the most frequent was Penicillium. Trichloroanisole was detected in the air of wine cellars with large visible moldy patches. The spore concentrations in the cellar air were two times higher in cellars with Zasmidium cellare growth than in cellars without Z. cellare. These results will serve as a database for further studies.

Antimicrobial resistance of Streptococcus pneumoniae in Southeast Austria, 1997-2008.

Antibiotic resistance in Streptococcus pneumoniae has increased worldwide but varies within geographical regions. We conducted a retrospective analysis of resistance in S. pneumoniae over a 12-year period to assess local and temporal trends in antibacterial resistance. From 1997 to 2008, a total of 1814 non-duplicate S. pneumoniae isolates were identified at the Institute of Hygiene, Microbiology and Environmental Medicine, Medical University of Graz, Austria. Antibiotic resistance was determined by the Clinical and Laboratory Standards Institute (CLSI) disk diffusion test. For penicillin, the minimum inhibitory concentration was determined by Etest. Susceptibility was defined according to CLSI interpretive criteria. For penicillin, resistance rates were consistently low at 0.2% over the 12-year study period. An increase in resistance was remarkable for erythromycin (3.5% in 1997; 14.7% in 2008), clindamycin (1.8% in 1997; 10.6% in 2008) and tetracycline (1.8% in 2000; 11.0% in 2008). For trimethoprim/sulfamethoxazole, resistance increased slightly to 9.2% in 2008. Quinolones showed a low resistance rate of 0.2% that persisted over the whole study period. In contrast to previously published national data, resistance to penicillin was observed to remain at a remarkably low and constant level. Although international surveillance programmes have set up sustainable and interlinked data networks, our results suggest that regional surveillance may still be needed as decision support for appropriate empirical antibiotic therapy in the local health setting.

Use of automated repetitive-sequence-based PCR for rapid laboratory confirmation of nosocomial outbreaks.

OBJECTIVE: Rapid and reliable diagnosis of genetic relatedness of clinical isolates in microbiologic laboratory is essential in case of nosocomial outbreak investigation. Most molecular techniques used to type microorganisms are technically demanding and time consuming. Currently repetitive-sequence-based PCR (rep-PCR) technique has been adapted to an automated format on the DiversiLab system (bioMérieux, Marcy l'Etoile, France). Aim of this study was to compare the performance of the DiversiLab system to that of pulsed-field gel electrophoresis (PFGE) in nosocomial outbreaks. METHODS: 122 clinical isolates (28 Methicillin-resistant Staphylococcus aureus (MRSA), 26 Acinetobacter baumannii, 45 extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae and 13 ESBL-producing Klebsiella oxytoca) were investigated. 70 isolates originated from six well-documented outbreaks, 52 were non-outbreak isolates. RESULTS: Concordant results for identification of outbreak and non-outbreak MRSA, A. baumannii and ESBL-producing K. pneumoniae strains were achieved with both methods. In the outbreak of ESBL-producing K. oxytoca automated rep-PCR was slightly more discriminatory than PFGE. Rep-PCR identified investigated ESBL-producing K. oxytoca outbreak-strains as indistinguishable or closely related, showing similarity of >90%, while PFGE identified these strains as indistinguishable. CONCLUSION: Automated rep-PCR assays on the DiversiLab system were used for MRSA, A. baumannii and for the first time ESBL-producing Klebsiella spp. and proved as a rapid and reliable method for molecular analysis of nosocomial outbreaks.

Occurrence and hygienic relevance of fungi in drinking water.

Fungi, above all filamentous fungi, can occur almost everywhere, even in water. They can grow in such a quantity in water that they can affect the health of the population or have negative effects on food production. There are several reports of fungal growth in water from different countries, but to our knowledge none from Austria so far. The aim of this study was to gain an overview of the spectrum of filamentous fungi and yeasts in drinking water systems. Thirty-eight water samples from drinking water and groundwater were analysed. Fungi were isolated by using membrane filtration and plating method with subsequent cultivation on agar plates. The different taxa of fungi were identified using routine techniques as well as molecular methods. Fungi were isolated in all water samples examined. The mean value for drinking water was 9.1 CFU per 100 ml and for groundwater 5400 CFU per 100 ml. Altogether 32 different taxa of fungi were found. The taxa which occurred most frequently were Cladosporium spp., Basidiomycetes and Penicillium spp. (74.6%, 56.4% and 48.7%, respectively). This study shows that drinking water can be a reservoir for fungi, among them opportunists, which can cause infections in immunosuppressed patients.

Evaluation of pelvic floor muscle function in a random group of adult women in Austria.

Despite an increasing clinical interest in female pelvic floor function, there is a lack of data with respect to the knowledge of average adult women about the physiological role of the pelvic floor and their ability to contract pelvic floor muscles (PFM) voluntarily. It was the aim of our study to evaluate the percentage of PFM dysfunction in adult women and the impact of risk factors, such as age, body mass index (BMI), number of children delivered, and the influence of previous PFM training. A total of 343 Austrian adult women (mean age, 41.2 +/- 14.6 years; range, 18-79 years), selected at random, were examined to test their ability to contract the PFM. The examination was carried out by three independent gynecologists during the course of a routine gynecological visit. The ability to contract the PFM voluntarily or involuntarily was assessed by digital intravaginal palpation with the patients in a supine position. The muscle strength was graded according to the Modified Oxford Grading Scale by Laycock. A high percentage (44.9%) of the women was not able to voluntarily perform a normal PFM contraction. In only 26.5%, an involuntary contraction of the pelvic floor was present before an increase in intra-abdominal pressure. The inability to contract the PFM did not correlate with women's age but revealed a weak relationship with the number of childbirths and the patient's BMI. A significant correlation was found between the Oxford Grading Scale rating and the patient's report about previous PFM training.

Isolation of Gordonia terrae from a patient with catheter-related bacteraemia.

A cornyeform bacterium was isolated from a blood culture from a 24-year-old man with familial hypertrophic non-obstructive cardiomyopathy, chronic abuse of anabolic steroids and prior admission to hospital because of clinical signs of sepsis. 16S rRNA gene analysis unambiguously identified Gordonia terrae.

A 5-year (2000-2004) epidemiological survey of Candida and non-Candida yeast species causing vulvovaginal candidiasis in Graz, Austria.

Vulvovaginal candidasis (VVC) is a common disease. The majority of cases is caused by Candida albicans, but in recent years an increase has been observed in the frequency of non-albicans Candida infections, especially due to C. glabrata and C. tropicalis. The aim of the study was to assess the prevalence of non-albicans Candida infections in patients suffering from VVC. Therefore, the statistical data of culture-confirmed VVC ascertained at the Institute of Hygiene (Medical University Graz) have been studied. Altogether, 10,463 samples from patients with vulvovaginal complaints were analysed in the years 2000-2004, a number of 3184 proved to be culture-positive for yeast. Candida albicans was the most prevalent cause in 87.9% of all cases. Non-albicans Candida yeast were detected in 12.1%, mainly C. glabrata and Saccharomyces cerevisiae. During a 1-year period 185 patients showed more than one episode of VVC. Patients aged 21-40 years were significantly more prone to suffer from VVC compared with other age-related groups.

Bioavailability and pharmacokinetics of Echinacea purpurea preparations and their interaction with the immune system.

Echinacea is a widely used herbal remedy for the prevention and treatment of the common cold. Recently, many new insights concerning the molecular mode of action of the main lipophilic constituents, the alkamides, have renewed interest in this plant. In order to compare the bioavailability of alkamides from liquid and tablet preparations of E. purpurea (Echinaforce) in humans and to study the effects on ex vivo stimulated blood cells, a randomized, single-dose, crossover study with 10 (8 test, 2 placebo) volunteers has been performed. They received either 4 ml of the standardized E. purpurea (Echinaforce) tincture or 12 E. purpurea (Echinaforce) tablets or placebo. Both doses contained the same amount (0.07 mg) of the major alkamides, dodeca-2E,4E,8Z, 10E/Z-tetraenoic acid isobutylamides. Liquid chromatography electrospray ionization ion-trap mass spectrometry was used to determine the content of alkamides in serum. It was found that the arithmetic mean C(max) of dodeca-2E,4E, 8Z,10E/Z-tetraenoic acid isobutylamides absorbed after oral application of the Echinaforce tincture appeared after 30 min (0.40 ng/ml serum). In comparison, the t(max) of tablets was 45 min with a C(max) of 0.12 ng/ml. An ex vivo stimulation of blood by LPS was carried out to measure the influence of E. purpurea on the innate and adaptive immune system. Both E. purpurea preparations led to the same effects on the immune system according to the concentration of pro-inflammatory cytokines TNF-alpha and IL-8. 23 hours after oral application a significant down-regulation of TNF-alpha and IL-8 in LPS pre-stimulated whole blood was found. However, no significant changes in the concentration of IL-6 were observed. Although a quarter of the dodeca-2E,4E,8Z, 10E/Z-tetraenoic acid isobutylamides was absorbed from the tablets, the study shows that the formulations trigger the same effects on the measured immune parameters.

Lethal brain abscess due to the fungus Scedosporium apiospermum (teleomorph Pseudallescheria boydii) after a near-drowning incident: case report and review of the literature.

A 39-year-old healthy man developed a brain abscess weeks after a near-drowning incident. Scedosporium apiospermum, the anamorph of Pseudallescheria boydii, was isolated from the abscess. The patient died 153 days after the accident despite antifungal therapy. We discuss the role of antifungals and review the literature for comparable cases.

Transmission of Campylobacter spp. in a poultry slaughterhouse and genetic characterisation of the isolates by pulsed-field gel electrophoresis.

1. Contamination of retail products with Campylobacter spp. during the slaughter of poultry is a well-known problem of product hygiene. Mechanical evisceration often leads to intestinal rupture and discharge of gut contents, which can contain zoonotic and human pathogens. Processes along the slaughter line cause aerosols and airborne droplets, containing bacterial loads. 2. To estimate the possible transmission routes of intestinal Campylobacter, 36 measurements of the bioaerosol (Andersen sampler and SKC BioSampler), 30 cloacal (of three flocks), 10 equipment and 4 sedimentation samples were tested for the presence of Campylobacter species. 3. The results imply that, in addition to contaminated equipment, which was Campylobacter-positive in 80% of cases, aerosols with peak values of 4.0 x 10(4) (test series 1) and 1.4 x 10(4) (test series 2) CFU/m3 also provide a potential vector for horizontal transmission. 4. To explore the genetic similarities of isolates from different origins, 18 isolates recovered from air, 26 cloacal, 8 equipment and 4 sedimentation isolates were analysed by pulsed-field gel electrophoresis (PFGE), using the restriction enzymes Sma I and Sal I. The similarity of cloacal isolates with isolates from equipment, air and sediment, suggest that the contamination is of intestinal origin. 5. There were direct links between Campylobacter-positive flocks and the presence of the same strains in the aerosol of the slaughter hall. Air as a potential source for microbial transmission must be taken into account.

Antibiotic resistance of E. coli in sewage and sludge.

The aim of the study is the evaluation of resistance patterns of E. coli in wastewater treatment plants without an evaluation of basic antibiotic resistance mechanisms. Investigations have been done in sewage, sludge and receiving waters from three different sewage treatment plants in southern Austria. A total of 767 E. coli isolates were tested regarding their resistance to 24 different antibiotics. The highest resistance rates were found in E. coli strains of a sewage treatment plant which treats not only municipal sewage but also sewage from a hospital. Among the antimicrobial agents tested, the highest resistance rates in the penicillin group were found for Ampicillin (AM) (up to 18%) and Piperacillin (PIP) (up to 12%); in the cephalosporin group for Cefalothin (CF) (up to 35%) and Cefuroxime-Axetil (CXMAX) (up to 11%); in the group of quinolones for Nalidixic acid (NA) (up to 15%); and for Trimethoprime/Sulfamethoxazole (SXT) (up to 13%) and for Tetracycline (TE) (57%). Median values for E. coli in the inflow (crude sewage) of the plants were between 2.0 x 10(4) and 6.1 x 10(4)CFU/ml (Coli ID-agar, BioMerieux 42017) but showed a 200-fold reduction in all three plants in the effluent. Nevertheless, more than 10(2)CFU E. coli/ml reached the receiving water and thus sewage treatment processes contribute to the dissemination of resistant bacteria in the environment.

The significance of sunlight for the elimination of indicator bacteria in small-scale bathing ponds in central Europe.

In order to determine the impact of sunlight on the reduction rate of indicator bacteria, bathing waters of different turbidity were artificially contaminated with indicator bacteria and exposed to sunlight. The reduction rates determined for the indicator bacteria showed a significant correlation with radiation intensity and, thus, also the time of day as well as the turbidity of the water. Under conditions of strong radiation intensity and low water turbidity (< 5 FTU), indicator bacteria were eliminated within 1-2 h of contamination with an initial concentration of 10(5) CFU/mL. Moreover, differences between different indicator bacteria were found. Enterococcus faecalis proved to be significantly more resistant to sunlight than the other indicator bacteria tested (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus). The results of the present study, namely the complete elimination of indicator bacteria within a few hours, showed that sunlight, given an appropriate intensity and good water transparency, was the most important factor in the reduction of hygienically-relevant microorganisms in surface waters and, thus, also in small-scale bathing ponds.

Qualitative detection of Legionella species in bronchoalveolar lavages and induced sputa by automated DNA extraction and real-time polymerase chain reaction.

Molecular assays for qualitative detection of Legionella spp. in clinical specimens were evaluated. DNA extraction was done either with a fully automated DNA extraction protocol on the MagNA Pure LC System or with manual DNA extraction. Amplification and detection were done by real-time polymerase chain reaction (PCR) on the LightCycler (LC) instrument. Oligonucleotides were derived from the 16S rRNA gene of Legionella spp. The assays included a specially designed DNA fragment as Legionella-specific internal control. For both molecular assays, the detection limit was determined to be 5 CFU per LC PCR run. Sixty-one clinical specimens were tested with the molecular assays. Results were compared to culture. Five samples were found to be positive with the molecular assays. Three of them were positive in culture. No inhibition was found throughout the whole study. In conclusion, the molecular assays described may lead to safe and early diagnosis of Legionnaires' disease. They proved to be suitable for the routine molecular diagnostics laboratory.

Comparative investigation of airborne culturable microorganisms in sewage treatment plants.

The present study investigated emissions and emmissions of airborne microorganisms (mesophilic bacteria, Escherichia coli, molds, Aspergillus fumigatus, thermophilic actinomycetes/bacilli) in sewage treatment plants. For the aerobiological investigations three sewage treatment facilities with an activated-sludge process, capacities between 2000 and 28,000 PE and different cleaning steps were selected. The measurements of microorganism emission were conducted in the area of the intake (screen), in the area of biological treatment (activated sludge tank) and at a distance of 10 m from the activated sludge tanks. In order to determine the emmission, additional measurements were conducted leeward of the plant at a distance of 200 m. Samples were taken using four parallel six-stage Andersen 1 AFCM volumetric samplers. In the area of the intake counts for bacteria were 7.4 x 10(2) CFU/m3 (median), for thermophilic actinomycetes 1.8 x 10(1) CFU/m3, for thermophilic bacilli 7.1 x 10(1) CFU/m3, for molds 2.4 x 10(3) CFU/m3 and for Aspergillus fumigatus 1.8 x 10(1) CFU/m3. Only isolated airborne coliform recoveries, i.e. E. coli, were detected. In the area of the activated sludge tank, in the adjoining area (10 m) and in the vicinity of the plants (200 m), the counts for all microorganism groups investigated corresponded to natural conditions. The results show that the counts of culturable aerogenic microorganisms in and in the immediate surrounding of the sewage plants investigated are low. Although the possibility of an infection through inhalation cannot be ruled out, the direct contact with sewage is much more critical.

Albumin is a necessary stabilizer of TBE-vaccine to avoid fever in children after vaccination.

A thiomersal-free and also an albumin-free tick-borne encephalitis-vaccine (TBE-vaccine) was developed. This vaccine was approved by the Austrian health authorities in the year 2000. Contrary to previous experience, 779 cases of fever attacks occurred following the first vaccination of children under 15 years of age. The induction of the immune system by different TBE virus (TBEV) vaccines (FSME-Immun [1999], Ticovac [2000] and FSME-Immun [2001] all from Baxter Hyland Immuno, Vienna) was compared in an in vitro immune stimulation test in order to find an explanation for the unexpected fever attacks. It was shown that only Ticovac, which contains no albumin as a stabilizer, can induce relative high amounts of TNF-alpha (P < or = 0.0001) and lower amounts of IL-1 beta (P < or = 0.05). Increase of both cytokines is first observed following an incubation of 4 h. The maximum is reached after 15 h. After 26 h, it has reverted to the original value. The course of concentration of both cytokines corresponds to the time of observed febrile phases. Albumin or immunoglobulin prevents a rise of cytokines so that it is recommended to add the albumin again to the vaccine.

Determination of human immunodeficiency virus type 1 subtypes by a rapid method useful for the routine diagnostic laboratory.

The existence of human immunodeficiency virus type 1 (HIV-1) subtypes has many important implications for the global evolution of HIV and for the evaluation of pathogenicity, transmissibility, and candidate HIV vaccines. The aim of this study was to establish a rapid method for determination of HIV-1 subtypes useful for a routine diagnostic laboratory and to investigate the distribution of HIV-1 subtypes in Austrian patients. Samples were tested by a subtyping method based on a 1.3-kb sequence of the polymerase gene generated by a commercially available drug resistance assay. The generated sequence was subtyped by means of an HIV sequence database. Results of 74 routine samples revealed subtype B (71.6%) as the predominant subtype, followed by subtype A (13.5%) and subtype C (6.8%). Subtypes E, F, G, and AE (CM240) were also detected. This subtyping method was found to be very easy to handle, rapid, and inexpensive and has proved suitable for high-throughput routine diagnostic laboratories. The specific polymerase gene sequence, however, must be existent.