Ursula Obst

Detection of antibiotic‐resistant bacteria and their resistance genes in wastewater, surface water, and drinking water biofilms

Abstract In view of the increasing interest in the possible role played by hospital and municipal wastewater systems in the selection of antibiotic-resistant bacteria, biofilms were investigated using enterococci, staphylococci, Enterobacteriaceae, and heterotrophic bacteria as indicator organisms. In addition to wastewater, biofilms were also investigated in drinking water from river bank filtrate to estimate the occurrence of resistant bacteria and their resistance genes, thus indicating possible transfer from wastewater and surface water to the drinking water distribution network. Vancomycin-resistant enterococci were characterized by antibiograms, and the vanA resistance gene was detected by molecular biology methods, including PCR. The vanA gene was found not only in wastewater biofilms but also in drinking water biofilms in the absence of enterococci, indicating possible gene transfer to autochthonous drinking water bacteria. The mecA gene encoding methicillin resistance in staphylococci was detected in hospital wastewater biofilms but not in any other compartment. Enterobacterial ampC resistance genes encoding beta-lactamase activities were amplified by PCR from wastewater, surface water and drinking water biofilms.

A comparison of five rapid direct toxicity assessment methods to determine toxicity of pollutants to activated sludge

Five rapid direct toxicity assessment methods were used in three European partner countries to determine the toxicity of single toxicants, mixed toxicants and real industrial wastes. The final aim was to protect microbial degradation of organic wastes in biological treatment processes and hence enhance the quality of treated effluents to be discharged to the environment. Nitrification inhibition, Respirometry, Adenosine triphosphate luminescence and Enzyme inhibition were tested utilising activated sludge as the testing matrix. The Vibrio fischeri toxicity test was used as a surrogate to compare the various microbial bioassays. The IC50 (toxicant concentration eliciting a 50% inhibitory effect) was determined for a number of pollutants including single toxicants Cd, Cr, Cu, Zn, 3,5-dichlorophenol, toluene and linear alkylbenzenesulphonate (LAS); a standard mixture of metals and LAS; a standard mixture of organics and LAS, and 16 industrial effluents. The V. fischeri bioassay was also chosen in order to assess quality control of toxicant preparation during testing in the different laboratories of the partner countries. Comparisons of sensitivity, cost of implementation, cost per test, relevance, and ease of use were made. The most sensitive bioassays were V. fischeri and Nitrification inhibition, however, this depended in the main on the pollutant and mixtures tested. It is recommended that during assessment of wastewater toxicity a suite of tests be used rather than reliance on one particular test.

Formation and bacterial composition of young, natural biofilms obtained from public bank-filtered drinking water systems

Abstract In Germany, bank-filtered raw water and ground water is mainly used for drinking water conditioning. Microorganisms, which are neither retarded by the subsoil passage of the bank-filtration, nor by the different filtration and disinfection steps at the water works, cause the growth of biofilms on different materials originally used in drinking water distribution systems. The development, the phylogenetical diversity and the bacterial metabolic activities of biofilms on polyethylen (PE-HD), polyvinylchlorid (PVC), steel and copper were analyzed at different sampling points. The incubation experiments were performed under natural conditions using a flow device technique. The devices were installed after the activated carbon filters and disinfection step at the water works and at two different house branch connections within the distribution system of the conditioned drinking water of the water works. The synthetic materials were colonized very rapidly within a few days in significant higher densities than steel and copper. The total bacterial cell counts of the biofilms were measured by DAPI (4′,6-diamidino-2-phenylindole) staining. The metabolic activities of the bacteria were quantified by the use of the redox dye CTC (5-cyano-2,3-ditolyl tetrazolium chloride), which is frequently described as an indicator for respiration. The highest respiratory activities were observed in biofilms from synthetic materials grown after the activated carbon filters at the water works. A significant reduction of the total bacterial cell counts and respiratory activities of about 80% was measured due to the disinfection at the water works. A time dependent growth of bacteria in biofilms was observed at the two sampling points within the distribution system, whereas the percentage of CTC-reducing cells stabilized at 35%. The in situ-hybridizations with fluorescence labelled, group-specific rRNA targeted oligonucleotide probes revealed the following: (i) bacteria of the Beta- and Gamma-subclass of Proteobacteria were found most frequently within the biofilm population, (ii) the percentage of the different subclasses depended on the used material, (iii) there were no significant changes in bacterial subclass composition of the biofilms taken from the water works and house branch connections. In addition, polymerase chain reaction (PCR), southern blot hybridization and in situ hybridization were used to detect facultative pathogenic bacteria in biofilms. Non-pneumophila Legionella were found in a relative high percentage up to about 7% in many biofilms, whereas fecal streptococci were detected only in few biofilms of the drinking water distribution systems

Formation of natural biofilms during chlorine dioxide and u.v. disinfection in a public drinking water distribution system

Aims: The influence of two disinfection techniques on natural biofilm development during drinking water treatment and subsequent distribution is compared with regard to the supply of a high‐quality drinking water.

Induction of Neutrophil Chemotaxis by the Quorum-Sensing Molecule N-(3-Oxododecanoyl)-l-Homoserine Lactone

ABSTRACT Acyl homoserine lactones are synthesized by Pseudomonas aeruginosa as signaling molecules which control production of virulence factors and biofilm formation in a paracrine manner. We found that N-(3-oxododecanoyl)-l-homoserine lactone (3OC12-HSL), but not its 3-deoxo isomer or acyl-homoserine lactones with shorter fatty acids, induced the directed migration (chemotaxis) of human polymorphonuclear neutrophils (PMN) in vitro. By use of selective inhibitors a signaling pathway, comprising phosphotyrosine kinases, phospholipase C, protein kinase C, and mitogen-activated protein kinase C, could be delineated. In contrast to the well-studied chemokines complement C5a and interleukin 8, the chemotaxis did not depend on pertussis toxin-sensitive G proteins, indicating that 3OC12-HSL uses another signaling pathway. Strong evidence for the presence of a receptor for 3OC12-HSL on PMN was derived from uptake studies; by use of radiolabeled 3OC12-HSL, specific and saturable binding to PMN was seen. Taken together, our data provide evidence that PMN recognize and migrate toward a source of 3OC12-HSL (that is, to the site of a developing biofilm). We propose that this early attraction of PMN could contribute to prevention of biofilm formation.

Hydrophobic Liquid-Infused Porous Polymer Surfaces for Antibacterial Applications

Biofilms represent a fundamental problem in environmental biology, water technology, food hygiene as well as in medical and technical systems. Recently introduced slippery liquid-infused porous surface (SLIPS) showed great promise for preventing biofilm formation owing to the low surface energy of such surface in combination with its self-cleaning properties. In this study we demonstrated a novel hydrophobic liquid-infused porous poly(butyl methacrylate-co-ethylene dimethacrylate) surface (slippery BMA-EDMA) with bacteria-resistance in BM2 mineral medium and long-term stability in aqueous environments. We showed that the slippery BMA-EDMA surface prevents biofilm formation of different strains of opportunistic pathogen Pseudomonas aeruginosa for at least up to 7 days in low nutrient medium. Only ∼1.8% of the slippery surface was covered by the environmental P. aeruginosa PA49 strain under investigation. In uncoated glass controls the coverage of surfaces reached ∼55% under the same conditions. However, in high nutrient medium, more relevant to physiological conditions, the biofilm formation on the slippery surface turned out to be highly dependent on the bacterial strain. Although the slippery surface could prevent biofilm formation of most of the P. aeruginosa strains tested (∼1% surface coverage), the multiresistant P. aeruginosa strain isolated from wastewater was able to cover up to 12% of the surface during 7 days of incubation. RAPD-PCR analysis of the used P. aeruginosa strains demonstrated their high genome variability, which might be responsible for their difference in biofilm formation on the slippery BMA-EDMA surface. The results show that although the slippery BMA-EDMA surface has a great potential against biofilm formation, the generality of its bacteria resistant properties is still to be improved.

A Nested Array of rRNA Targeted Probes for the Detection and Identification of Enterococci by Reverse Hybridization

Complete 23S and almost complete 16S rRNA gene sequences were determined for the type strains of the validly described Enterococcus species, Melissococcus pluton and Tetragenococcus halophilus. A comprehensive set of rRNA targeted specific oligonucleotide hybridization probes was designed according to the multiple probe concept. In silico probe design and evaluation was performed using the respective tools of the ARB program package in combination with the ARB databases comprising the currently available 16S as well as 23S rRNA primary structures. The probes were optimized with respect to their application for reverse hybridization in microplate format. The target comprising 16S and 23S rDNA was amplified and labeled by PCR (polymerase chain reaction) using general primers targeting a wide spectrum of bacteria. Alternatively, amplification of two adjacent rDNA fragments of enterococci was performed by using specific primers. In vitro evaluation of the probe set was done including all Enterococcus type strains, and a selection of other representatives of the gram-positive bacteria with a low genomic DNA G+C content. The optimized probe set was used to analyze enriched drinking water samples as well as original samples from waste water treatment plants.

Application of a molecular biology concept for the detection of DNA damage and repair during UV disinfection

As nucleic acids are major targets in bacteria during standardised UV disinfection (254 nm), inactivation rates also depend on bacterial DNA repair. Due to UV-related DNA modifications, PCR-based approaches allow for a direct detection of DNA damage and repair during UV disinfection. By applying different primer sets, the correlation between amplicon length and PCR amplification became obvious. The longer the targeted DNA fragment was, the more UV-induced DNA lesions inhibited the PCR. Regeneration of Pseudomonas aeruginosa, Enterococcus faecium, and complex wastewater communities was recorded over a time period of 66 h. While phases of intensive repair and proliferation were found for P. aeruginosa, no DNA repair was detected by qPCR in E. faecium. Cultivation experiments verified these results. Despite high UV mediated inactivation rates original wastewater bacteria seem to express an enhanced robustness against irradiation. Regeneration of dominant and proliferation of low-abundant, probably UV-resistant species contributed to a strong post-irradiation recovery accompanied by a selection for beta-Proteobacteria.

Application of 23S rDNA-targeted oligonucleotide probes specific for enterococci to water hygiene control.

Identification of enterococci species by hybridization with recently designed species-specific and group-specific 23S rDNA-targeted oligonucleotide probes was superior to results obtained with a common biochemical test panel. Considering these findings, a molecular biological procedure for the detection of enterococci in water samples was developed. A short enrichment is followed by an amplification step and a hybridization reaction in microtiter plate format. The detection limit is about 1 CFU/ml, and results are available within 26 h.

Molecular monitoring of inactivation efficiencies of bacteria during pulsed electric field treatment of clinical wastewater

Aims:  The applicability of an alternative wastewater disinfection concept based on the pulsed electric field (PEF) treatment is tested with molecular biology techniques using clinical wastewaters.