Live-dead discrimination analysis, qPCR assessment for opportunistic pathogens, and population analysis at ozone wastewater treatment plants.

In respect to direct and indirect water reuse, the microbiological quality of treated wastewater is highly important. Conventional wastewater treatment plants are normally not equipped with advanced technologies for the elimination of bacteria. Molecular biology analyses were combined with live-dead discrimination analysis of wastewater population using Propidium monoazide (PMA) to study population shifts during ozonation (1 g ozone/g DOC) at a municipal wastewater treatment plant. Escherichia coli, enterococci, and Pseudomonas aeruginosa were quantified by polymerase chain reaction (qPCR) and the whole wastewater population was analyzed by metagenomic sequencing. The PMA-qPCR experiments showed that the abundances of P. aeruginosa didn't change by ozone treatment, whereas a reduction was observed for E. coli and enterococci. Results comparing conventional cultivation experiments with PMA-qPCR underlined the presence of viable but not culturable cells (VBNC) and their regrowth potential after ozone treatment. Illumina HiSeq sequencing results with and without PMA treatment demonstrated high population similarities in water samples originating from ozone inflow sampling sides. Upon using PMA treatment after ozonation, population shifts became visible and also underlined the importance of PMA treatment for the evaluation of elimination and selection processes during ozonation at WWTPs. Amongst a number of 14 most abundant genera identified in the inflow samples, 9 genera were found to be reduced, whereas 4 genera increased in relative abundance and 1 genus almost remained constant. The strongest increase in relative abundance after ozonation was detected for Oscillatoria spp., Microcoleus spp. and Nitrospira spp. Beside this, a continuous release of Pseudomonas spp. (including P. aeruginosa) to the downstream receiving body was confirmed. Regrowth experiments demonstrated a high prevalence of P. aeruginosa as part of the surviving bacterial population. Summing up, molecular biology analyses in combination with live-dead discrimination are comprehensive methods to evaluate the elimination processes targeting specific species and/or whole microbial populations.

Whole genome and transcriptome analyses of environmental antibiotic sensitive and multi-resistant Pseudomonas aeruginosa isolates exposed to waste water and tap water.

The fitness of sensitive and resistant Pseudomonas aeruginosa in different aquatic environments depends on genetic capacities and transcriptional regulation. Therefore, an antibiotic-sensitive isolate PA30 and a multi-resistant isolate PA49 originating from waste waters were compared via whole genome and transcriptome Illumina sequencing after exposure to municipal waste water and tap water. A number of different genomic islands (e.g. PAGIs, PAPIs) were identified in the two environmental isolates beside the highly conserved core genome. Exposure to tap water and waste water exhibited similar transcriptional impacts on several gene clusters (antibiotic and metal resistance, genetic mobile elements, efflux pumps) in both environmental P. aeruginosa isolates. The MexCD-OprJ efflux pump was overexpressed in PA49 in response to waste water. The expression of resistance genes, genetic mobile elements in PA49 was independent from the water matrix. Consistently, the antibiotic sensitive strain PA30 did not show any difference in expression of the intrinsic resistance determinants and genetic mobile elements. Thus, the exposure of both isolates to polluted waste water and oligotrophic tap water resulted in similar expression profiles of mentioned genes. However, changes in environmental milieus resulted in rather unspecific transcriptional responses than selected and stimuli-specific gene regulation.

Sub-inhibitory concentrations of antibiotics and wastewater influencing biofilm formation and gene expression of multi-resistant Pseudomonas aeruginosa wastewater isolates.

Sub-inhibitory concentrations of antibiotics, which are found in environmental water systems and sewage plants due to an increased use in therapeutical and preventive fields, influence bacterial behavior in biofilms. The application of sulfamethoxazole, erythromycin, and roxithromycin induced changes in biofilm dynamics regarding biomass formation, spatial structure and specific gene expression in different Pseudomonas aeruginosa isolates. Exposing multi-resistant environmental isolated strains for 17 h to environmental concentrations of antibiotics or wastewater, directly, an increase in biofilm biomass and thickness could be observed for each strain. Additionally, multi-resistant strains responded to the applied growth conditions with changes in transcriptional activity. Here, sub-inhibitory concentrations of macrolides specifically upregulated expression of quorum sensing genes (rhlR, lasI), whereas sulfonamides and municipal wastewater, instead upregulated expression of specific resistant genes (sul1) and efflux pumps (mexD). Antibiotic sensitive isolates demonstrated an overall higher transcriptionally activity, but did not show a specific gene response to the applied exogenous stimuli. Furthermore, the presence of low concentrated antibiotics induced also phenotypical change in the biofilm architecture observed by 3D-imaging.

Intracellular localization of membrane-bound ATPases in the compartmentalized anammox bacterium 'Candidatus Kuenenia stuttgartiensis'.

Anaerobic ammonium-oxidizing (anammox) bacteria are divided into three compartments by bilayer membranes (from out- to inside): paryphoplasm, riboplasm and anammoxosome. It is proposed that the anammox reaction is performed by proteins located in the anammoxosome and on its membrane giving rise to a proton-motive-force and subsequent ATP synthesis by membrane-bound ATPases. To test this hypothesis, we investigated the location of membrane-bound ATPases in the anammox bacterium 'Candidatus Kuenenia stuttgartiensis'. Four ATPase gene clusters were identified in the K. stuttgartiensis genome: one typical F-ATPase, two atypical F-ATPases and a prokaryotic V-ATPase. K. stuttgartiensis transcriptomic and proteomic analysis and immunoblotting using antisera directed at catalytic subunits of the ATPase gene clusters indicated that only the typical F-ATPase gene cluster most likely encoded a functional ATPase under these cultivation conditions. Immunogold localization showed that the typical F-ATPase was predominantly located on both the outermost and anammoxosome membrane and to a lesser extent on the middle membrane. This is consistent with the anammox physiology model, and confirms the status of the outermost cell membrane as cytoplasmic membrane. The occurrence of ATPase in the anammoxosome membrane suggests that anammox bacteria have evolved a prokaryotic organelle; a membrane-bounded compartment with a specific cellular function: energy metabolism.

Morphological, bacterial, and secondary metabolite changes of Aplysina aerophoba upon long-term maintenance under artificial conditions.

The aim of this study was to analyze successional changes in the bacterial community over a period of 6 months of cultivation of Aplysina aerophoba sponges under different artificial cultivation conditions by use of denaturing gradient gel electrophoresis (DGGE). The cultivation conditions varied concerning the water temperature (20 +/- 2 degrees C and 25 +/- 2 degrees C) of the aquaria, additional illumination of one aquarium, and feeding of the sponges. Amplicons from DGGE separation of dominant colonizing or variably appearing bacteria were sequenced and aligned for taxonomical identification. In addition, secondary metabolites typically found in A. aerophoba were analyzed to investigate changes in the natural product profile during cultivation. The cultivation of sponges under any given condition did not lead to a depletion of their bacterial community in the course of the experiment. On the contrary, the distinctive set of associated bacteria was maintained in spite of a dramatic loss of biomass and morphological degradation during the cultivation period. Generally, all sequences obtained from the DGGE gels were related to bacteria of five phyla: Actinobacteria, Cyanobacteria, alpha-Proteobacteria, gamma-Proteobacteria, and Chloroflexi. Despite the overall stability of the bacterial community in A. aerophoba, an unambiguous variability was detected for the Cyanobacteria "A. aerophoba clone TK09". This variability was ascribed to the predominant light conditions. The analysis of the metabolic pattern revealed that the concentration of a class of characteristic-brominated compounds typically found in A. aerophoba, like aeroplysinin-1, aerophobin-1, aerophobin-2, and isofistularin-3, increased over the 6 months of cultivation.

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.