Multidrug-resistant Pseudomonas aeruginosa is a major cause of severe hospital-acquired infections. Currently, polymyxin B (PMB) is a last-resort antibiotic for the treatment of infections caused by Gram-negative bacteria, despite its undesirable side effects. The delivery of drug combinations has been shown to reduce the required therapeutic doses of antibacterial agents and thereby their toxicity if a synergistic effect is present. In this study, we investigated the synergy between two cyclic antimicrobial peptides, PMB and gramicidin S (GS), against different P. aeruginosa isolates, using a quantitative checkerboard assay with resazurin as a growth indicator. Among the 28 strains that we studied, 20 strains showed a distinct synergistic effect, represented by a fractional inhibitory concentration index (FICI) of <0.5. Remarkably, several clinical P. aeruginosa isolates that grew as smallcolony variants revealed a nonsynergistic effect, as indicated by FICIs between >0.5 and <0.70. In addition to inhibiting the growth of planktonic bacteria, the peptide combinations significantly decreased static biofilm growth compared with treatment with the individual peptides. There was also a faster and more prolonged effect when the combination of PMB and GS was used compared with single-peptide treatments on the metabolic activity of pregrown biofilms. The results of the present study define a synergistic interaction between two cyclic membrane-active peptides toward 17 multidrug-resistant P. aeruginosa and biofilms of P. aeruginosa strain PAO1. Thus, the application of PMB and GS in combination is a promising option for a topical medication and in the prevention of acute and chronic infections caused by multidrug-resistant or biofilm-forming P. aeruginosa. Pathogenic Gram-negative Pseudomonas aeruginosa possesses both intrinsic and adaptive resistance toward many currently available antibiotics and causes infections that are effectively untreatable (1-3). P. aeruginosa "superbugs" are resistant to fluoroquinolones, expanded-spectrum cephalosporins, carbapenems, aminoglycosides, and in a few cases, even polymyxins, a last-resort class of antibiotics used to treat P. aeruginosa infections (4-7). One important mechanism for the intrinsic antibiotic resistance of Gram-negative pathogens, especially multidrug-resistant (MDR) P. aeruginosa clinical isolates, is their ability to efflux antibacterial agents via tripartite efflux pumps located in the inner and outer membranes, a mechanism that limits the access of the drug to intracellular targets (8). However, the overexpression of the P. aeruginosa efflux pump protein MexAB was unable to confer resistance to the host defense peptides (HDPs) cathelicidin LL-37 and defensins (9). One way in which pathogens acquire adaptive resistance to positively charged antimicrobial peptides is to modify lipid A by substitution with aminoarabinose (6). The emergence of drug-resistant P. aeruginosa strains, which exhibit increased MICs even for polymyxin B (PMB) (10), requires the development...

The occurrence of new chemical and microbiological contaminants in the aquatic environment has become an issue of increasing environmental concern. Thus, wastewater treatment plants (WWTPs) play an important part in the distribution of so-called new emerging pathogens and antibiotic resistances. Therefore, the daily loads released by the WWTP were calculated including a model system for the distribution of these loads within the receiving water body. UV-, as well as ozone-treatment in separate or in combination for wastewater treatment were under investigation aiming at the reduction of these loads. Here, the impact of these treatments on the DNA integrity via antibody staining and PCR efficiencies experiments were included. All three facultative pathogenic bacteria [enterococci (23S rRNA), Pseudomonas aeruginosa (ecfX), and Escherichia coli (yccT)] and seven clinically relevant antibiotic resistance genes (ARGs) (mecA (methicillin resistance gene), ctx-M32 (β- lactame resistance gene), ermB (erythromycine resistance gene), blaTEM (β- lactame resistance gene), sul1 (sulfonamide resistance gene), vanA (vancomycin resistance gene), and intI1 (Integrase1 gene) associated with mobile genetic elements were detected in wastewaters. Different reduction efficiencies were analyzed during advanced wastewater treatments. ARGs were still found to be present in the effluents under the parameters of 1.0 g ozone per g dissolved organic carbon (DOC) and 400 J/m2, like ctx-M32, ermB, blaTEM, sul1, and intI1. Especially UV radiation induced thymidine dimerization which was analyzed via antibody mediated detection in the metagenome of the natural wastewater population. These specific DNA alterations were not observed during ozone treatment and combinations of UV/ozone treatment. The dimerization or potential other DNA alterations during UV treatment might be responsible for a decreased PCR efficiency of the 16S rRNA amplicons (176, 490, and 880 bp fragments) from natural metagenomes compared to the untreated sample. This impact on PCR efficiencies was also observed for the combination of ozone and UV treatment.

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Antibiotic resistant bacteria and antibiotic resistance genes (ARGs) are major human-health threats, widely distributed in the environment. Quantitative PCR (qPCR) is a standard approach to detect and quantify ARGs in environmental compartments. However, the comparison of gene quantification reported by different laboratories is challenging since data are predominantly obtained under nonharmonized conditions, using different qPCR protocols. Objectives The aim of this study was to develop and calibrate standardized qPCR procedures for quantification of key ARGs, analyzing the same samples with common protocols and distinct equipment, reagents batches and operators. Methods Treated wastewater from three European countries were processed immediately after collection and transported to the laboratory for total DNA extraction. DNA extracts from each sample were pooled and aliquots were distributed by five partners involved in the calibration procedure. The genes 16S rRNA, vanA, blaTEM, qnrS, sul1, blaCTXM-32 and intI1 were analyzed using harmonized qPCR protocols and the constructed pNORM1 plasmid, which contains fragments of the seven targeted genes, was used for generating standard curves. Conclusions The 16S rRNA gene was the most abundant, followed by sul1, intI1, qnrS and blaTEM. Quantifications made by different partners were reproducible and inter-laboratory variation was < 20%. The notorious exception was for the qnrS gene, and therefore protocol improvement is recommended. The genes blaCTXM-32 and vanA were below the limit of quantification in most or all of the samples analyzed. The inter-laboratory calibration is an adequate approach to reliably assess ARG abundance and environmental contamination in different environments and geographic locations.