Pulsed electric field treatment for bacteria reduction and its impact on hospital wastewater.

During the last years the pulsed electric field (PEF) method entered several fields of application. A promising application is the decontamination of hospital wastewater effluents, which are loaded with pathogenic and increasingly with antibiotic-resistant bacteria. For this study, Pseudomonas putida suspended in buffer solution or wastewater from university hospital was used as reference strain. To prove whether the descendent of the survival bacteria develop an adaptation to electric field, surviving PEF treated bacteria were recultivated and pulsed in serial experiments with 10 pulses (100kVcm(-1) and 600ns pulse duration). This procedure was repeated for 30 generations. The inactivation rate was calculated with 3.5+/-0.8 log of colony forming units and remained constant over 30 cycles. Investigations of the variable intergenic spacer region of the ribosomal operon demonstrated no visible changes in this highly variable part of the genome structure during the serial PEF treatment experiments. The mutagenicity of PEF treated hospital wastewater, buffer solutions and tap water was analyzed by the umu-test. Most hospital wastewater samples exhibit a considerable genotoxicity already before PEF treatment, but this was not increased by the PEF treatment, not even for higher treatments energies over 250JmL(-1). No genotoxicity was induced in buffer solutions and tap water by PEF treatment. This study supports, that PEF treatment is a sustainable non-chemical method for bacterial decontamination without any adverse effects.

Evaluation of inhibition and cross-reaction effects on real-time PCR applied to the total DNA of wastewater samples for the quantification of bacterial antibiotic resistance genes and taxon-specific targets.

In order to evaluate the applicability of six primer and probe sets for TaqMan real-time RCR on DNA of wastewater samples, effects of the sample matrix and DNA background on target quantification were studied with respect to differences between functional genes and taxonomically used rDNA targets. Primer/probe assays for real-time PCR (TaqMan) designed to quantitatively detect the antibiotic resistance genes bla(VIM), vanA, ampC, mecA, and taxon-specific 23S rDNA sequences for Pseudomonas aeruginosa and Enterococcus faecium/faecalis were tested for their sensitivity and amplification robustness. The amplification of their gene targets in DNA extracts of wastewater ("wastewater DNA") and reference strains ("reference DNA") was compared with their amplification in "model DNA" which was composed of wastewater DNA and reference DNA. Target detection was quantifiable along up to seven decimal orders of magnitude. For the detection of the resistance genes bla(VIM), vanA, ampC, and mecA as well as the enterococci directed PCR only weak or no inhibition due to the impurities or wastewater DNA matrix were demonstrated for the applied target concentrations. The taxonomically applied detection system for the quantification of P. aeruginosa showed a limited performance. For the analysis of the amplification dynamics of possibly similar nucleotide sequences of organisms related to P. aeruginosa a SYBR Green assay was employed. Competitive amplification of similar sequences was identified to be a major mechanism of reduced sensitivity. Hence, the primers were modified for an optimised detection. With the resulting reduction of cross reactions an increased sensitivity was achieved for the detection and quantification of P. aeruginosa in wastewater DNA.

Real-time PCR detection of Pseudomonas aeruginosa in clinical and municipal wastewater and genotyping of the ciprofloxacin-resistant isolates.

Real-time quantification of Pseudomonas aeruginosa was performed in various wastewater systems including clinical, municipal wastewaters and inflow from a wastewater treatment plant. The highest concentrations of P. aeruginosa-specific targets were detected in clinical wastewaters. Limitations of the detection system resulting from inhibition or cross-reaction were identified. Ciprofloxacin-resistant P. aeruginosa strains were isolated after specific enrichment from clinical and municipal wastewaters. In some cases they were also cultivated from effluent of a wastewater treatment plant, and from its downstream river water. A total of 119 isolates were phenotypically characterized as ciprofloxacin-resistant via antibiogram testing. Subsequently, the fluoroquinolone-resistance-mediating mutations in the genes gyrA codon positions 83 and 87, gyrB codon position 466 and parC codon positions 87 and 91 were determined by mini-sequencing. Ciprofloxacin resistance was mainly associated with mutations in gyrA codon position 83 and parC mutation in codon positions 87 or 91 of the bacterial gyrase and topoisomerase II genes. All ciprofloxacin-resistant P. aeruginosa strains were compared with genotypes from clinical data of fluoroquinolone-resistant P. aeruginosa infections. The results were in agreement with data from clinical analyses, with the exception that no gyrA 87 and no gyrB mutations were found in ciprofloxacin-resistant P. aeruginosa wastewater isolates.

Detection of clinically relevant antibiotic-resistance genes in municipal wastewater using real-time PCR (TaqMan).

Real-time PCR assays were developed for the quantifiable detection of the antibiotic-resistance genes vanA of enterococci, ampC of Enterobacteriaceae, and mecA of staphylococci in different municipal wastewater samples. Primer and probe designs for these resistance genes were constructed and optimised for application in standardised TaqMan PCR assays. Using reference strains, the linear measurement ranges of the assays were defined and covered concentration ranges of five to seven exponential values. Wastewater isolates of vancomycin-resistant enterococci (VRE) and beta-lactam-resistant Enterobacteriaceae were cultivated from municipal wastewaters in order to verify the specificity and sensitivity of the primer-probe systems. Additionally, clinical strains of staphylococci resistant to methicillin (MRSA) confirmed the applicability of the mecA-specific detection system. Total DNAs were extracted from five different wastewater treatment plants and used for direct TaqMan PCR detection of the resistance genes without prior cultivation. In municipal wastewater, the resistance gene vanA was detected in 21% of the samples, and ampC in 78%. The gene mecA was not found in municipal wastewater, but in two clinical wastewater samples.