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Deciphering the Contribution of Biofilm to the Pathogenesis of Peritoneal Dialysis Infections: Characterization and Microbial Behaviour on Dialysis Fluids.

ABSTRACT: Infections are major complications in peritoneal dialysis (PD) with a multifactorial etiology that comprises patient, microbial and dialytic factors. This study aimed at investigating the contribution of microbial biofilms on PD catheters to recalcitrant infections and their interplay with PD related-factors. A prospective observational study was performed on 47 patients attending Centro Hospitalar of Porto and Vila Nova de Gaia/Espinho to whom the catheter was removed due to infectious (n = 16) and non-infectious causes (n = 31). Microbial density on the catheter was assessed by culture methods and the isolated microorganisms identified by matrix-assisted laser desorption/ionization time-of-flight intact cell mass spectrometry. The effect of conventional and three biocompatible PD solutions on 16 Coagulase Negative Staphylococci (CNS) and 10 Pseudomonas aeruginosa strains planktonic growth and biofilm formation was evaluated. Cultures were positive in 87.5% of the catheters removed due infectious and 90.3% removed due to non-infectious causes. However, microbial yields were higher on the cuffs of catheters removed due to infection vs. non-infection. Staphylococci (CNS and Staphylococcus aureus) and P. aeruginosa were the predominant species: 32% and 20% in the infection and 43.3% and 22.7% in the non-infection group, respectively. In general, PD solutions had a detrimental effect on planktonic CNS and P. aeruginosa strains growth. All strains formed biofilms in the presence of PD solutions. The solutions had a more detrimental effect on P. aeruginosa than CNS strains. No major differences were observed between conventional and biocompatible solutions, although in icodextrin solution biofilm biomass was lower than in bicarbonate/lactate solution. Overall, we show that microbial biofilm is universal in PD catheters with the subclinical menace of Staphylococci and P. aeruginosa. Cuffs colonization may significantly contribute to infection. PD solutions differentially impact microbial species. This knowledge is important for the development of infection diagnosis, treatment and preventive strategies.

SUBMITTER: Sampaio J

PROVIDER: S-EPMC4918928 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:IntroductionPeritoneal dialysis fluids (PDF) differ with respect to osmotic and buffer compound, and pH and glucose degradation products (GDP) content. The impact on peritoneal membrane integrity is still insufficiently described. We assessed global genomic effects of PDF in primary human peritoneal mesothelial cells (PMC) by whole genome analyses, quantitative real-time polymerase chain reaction (RT-PCR) and functional measurements.MethodsPMC isolated from omentum of non-uremic patients were incubated with conventional single chamber PDF (CPDF), lactate- (LPDF), bicarbonate- (BPDF) and bicarbonate/lactate-buffered double-chamber PDF (BLPDF), icodextrin (IPDF) and amino acid PDF (APDF), diluted 1:1 with medium. Affymetrix GeneChip U133Plus2.0 (Affymetrix, CA, USA) and quantitative RT-PCR were applied; cell viability was assessed by proliferation assays.ResultsThe number of differentially expressed genes compared to medium was 464 with APDF, 208 with CPDF, 169 with IPDF, 71 with LPDF, 45 with BPDF and 42 with BLPDF. Out of these genes 74%, 73%, 79%, 72%, 47% and 57% were downregulated. Gene Ontology (GO) term annotations mainly revealed associations with cell cycle (p = 10(-35)), cell division, mitosis, and DNA replication. One hundred and eighteen out of 249 probe sets detecting genes involved in cell cycle/division were suppressed, with APDF-treated PMC being affected the most regarding absolute number and degree, followed by CPDF and IPDF. Bicarbonate-containing PDF and BLPDF-treated PMC were affected the least. Quantitative RT-PCR measurements confirmed microarray findings for key cell cycle genes (CDK1/CCNB1/CCNE2/AURKA/KIF11/KIF14). Suppression was lowest for BPDF and BLPDF, they upregulated CCNE2 and SMC4. All PDF upregulated 3 out of 4 assessed cell cycle repressors (p53/BAX/p21). Cell viability scores confirmed gene expression results, being 79% of medium for LPDF, 101% for BLPDF, 51% for CPDF and 23% for IPDF. Amino acid-containing PDF (84%) incubated cells were as viable as BPDF (86%).ConclusionIn conclusion, PD solutions substantially differ with regard to their gene regulating profile and impact on vital functions of PMC, i.e. on cells known to be essential for peritoneal membrane homeostasis.

Project description:Loss of ultrafiltration capacity in peritoneal dialysis (PD) patients is often associated with peritoneal vascular changes, manifest as diabetes-like vasculopathy and angiogenesis. The endothelial monolayer lining the vessel lumen controls vessel barrier function and thereby influences peritoneal substrate transport and ultrafiltration. In this study, we investigated the influence of exposure of human umbilical vein endothelial cells (HUVEC) to different PD fluids. HUVECs were exposed to experimental solutions for up to 24 h. All test ﬂuids were sterile-ﬁltered before usage. Each experiment consisted of three independent samples in biological replicates on separate culture plates. For PD fluid incubation, cells were first exposed to pure PD fluids (Dianeal PD4 3.86% glucose, Physioneal 3.86% glucose, Extraneal, all Baxter, Castlebar, Ireland), or to a lab-made GDP-free PD fluid or to normal medium without growth factors as control. Cells were subsequently exposed for 24 h to the above solutions diluted 1:1 with culture medium and brought to 2% FCS. Cells were then washed three times (250 mM sucrose, 10 mM Tris/HCl, pH 7) and lysed in 125 µL per well of lysis buffer (30 mM Tris, pH 8.5, 7 M urea, 2 M thiourea, 4% CHAPS, 1 mM EDTA, one tablet of Complete Protease Inhibitor (Roche, Basel; Switzerland) per 100 ml, and one tablet of PhosStop Protease Inhibitor (Roche) per 100 mL). Total protein concentration was determined with the Pierce 660 Kit (Thermo) per manufacturer’s manual. Lysates were stored at -80°C until further processing. We investigated the molecular mechanisms of endothelial cell pathology during in vitro PD fluid exposure by proteomic and bioinformatic analysis of the endothelial cell response to the PD fluid induced stress, integrating current understanding of PD-related cellular injury and stress responses (Bender et al., Nephrol Dial Transplant, 2011, doi:10.1093/ndt/gfq484; Kratochwill et al., BioMed research international, 2015, doi:10.1155/2015/628158; Kratochwill et al., J Proteome Res, 2009, doi:10.1021/pr800916s; Lechner et al., J Proteome Res, 2010, doi:10.1021/pr9011574). We then evaluated the effect of AlaGln addition to conventional PD fluid on these parameters in endothelial cells. This strategy allowed characterization of endothelial cell injury and stress responses during exposure to different PD fluid.

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Deciphering the Contribution of Biofilm to the Pathogenesis of Peritoneal Dialysis Infections: Characterization and Microbial Behaviour on Dialysis Fluids.

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