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

Abstract

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.

Synergistic Effect of Membrane-Active Peptides Polymyxin B and Gramicidin S on Multidrug-Resistant Strains and Biofilms of Pseudomonas aeruginosa

“…A disk diffusion susceptibility assay on Mueller-Hinton (MH) agar (10) with antibiotic disks from Mast Diagnostica (Reinfeld, Germany) was performed with all 10 isolates and with the DSM 1117 and PAO1 strains using the following antibiotics: 300 U of PMB, 10 g of gentamicin, 5 g of ciprofloxacin, 10 g of imipenem, 30 g of ceftazidime, 30 g of aztreonam, 30 g of amikacin, and 100/10 g of piperacillin-tazobactam. The other P. aeruginosa isolates (49,55,56,59,910,911,912,913,914,965,966,967,968,969, and 987) were enriched from clinical wastewater compartments (33). Their susceptibility to antibiotics was studied using the same disk diffusion assay, with the following antibiotics: 10 g of gentamicin, 5 g of ciprofloxacin, 10 g of imipenem, 10 g of ceftazidime, 20 g of amikacin, 30 g of azlocillin, and 30/10 g of piperacillin-tazobactam.…”

Linear osmoregulated periplasmic glucans are encoded by the opgGH locus of Pseudomonas aeruginosa

“…The opportunistic pathogen Pseudomonas aeruginosa can be found in a variety of moist environments, including natural and man-made environments (Hardalo & Edberg, 1997;Schwartz et al, 2006;Spiers et al, 2000). This bacterium can cause infections in a variety of animals and plants, and can cause acute infections or chronic biofilm infections in the lungs of cystic fibrosis patients (Burns et al, 1993;Costerton, 1995;Costerton et al, 1999;Hoiby, 1993;Singh et al, 2000;Smith & Iglewski, 2003).…”

Real-Time PCR Analysis of the Intestinal Microbiotas in Peritoneal Dialysis Patients

“…The primers and probes for Pseudomonas aeruginosa and Enterococcus species were based on 23S rRNA gene sequences. Species-specific sequences were used to design primers and probes for Bifidobacterium breve, B. bifidum, B. catenulatum, B. longum (11), Lactobacillus plantarum, L. rhamnosus, L. paracasei (10), E. coli (14), K. pneumoniae (28), P. aeruginosa (26), Enterococcus species (6), and B. fragilis (33). All lactobacilli, all bifidobacteria, and total bacteria were also detected using previously designed primers and probes (10,11).…”