Porównanie lipopolisacharydów (LPS) z form planktonicznych i tworzących biofilm wybranych szczepów Proteus spp.

Abstract

Rod-shaped, Gram-negative bacteria of the genus Proteus most often cause urinary tract infections, especially among catheterised patients. These infections are associated with formation of the encrustated and usually multispecies biofilm on the catheter. Biofilm is a sessile form of bacterial growth – microorganisms are attached to the surface and surrounded by the extracellular matrix (ECM). Lipopolysaccharide is an integral component of Gram-negative bacterial cells and an important virulence factor of the bacteria. In many microorganisms the LPS structure plays a significant role in the biofilm formation process (e.g. adhesion to the surface). The aim of this study was to compare LPSs of selected clinical Proteus spp. strains derived from planktonic and biofilm cultures. In this study three P. mirabilis strains were selected (among five clinical Proteus spp. isolates), which showed most intensive growth on the polystyrene microtiter plates. These isolates belonged to O78 (P. mirabilis 1 B-m), O11a (P. mirabilis 9 B-m) and O8a,b (P. mirabilis 12 B-r) serogroups. In order to acquire substantial amounts of biofilm biomasses of these three strains, a glass bioreactor was built. This device was equipped with vertically aligned glass plates, which were covered with biofilm. After multiple biofilm cultures of the three selected strains in the bioreactor, lipopolysaccharides were isolated from the bacterial cells using a modified Westphal method. Afterwards, LPSs from biofilm cultures were compared with LPSs of homological isolates from planktonic cultures using SDS-PAGE, ELISA and Western blot techniques. The studies revealed some differences between LPSs samples from both types of the cultures – in case of P. mirabilis 1 B-m and P. mirabilis 9 B-m strains (partial OPS truncation and core structural changes in the samples from biofilm) and none for P. mirabilis 12 B-r strain (therefore excluded from further studies). Purpald assay revealed increased amounts of Kdo in the LPS P. mirabilis 1 B-m and P. mirabilis 9 B-m samples from biofilm (this suggested increase in the amount of low-weight LPS fractions with truncated OPS). Comparative analyses of P. mirabilis 1 B-m and P. mirabilis 9 B-m LPSs (OPS – NMR, lipid A – MALDI-TOF) from planktonic and biofilm cultures did not reveal differences regarding the type of culture. In case of P. mirabilis 1 B-m it was shown that dilution of the microbiological medium influenced the ribitol O-acetylation in the OPS (2,5 x higher O-acetylation was observed in LPS from planktonic culture in 10 x diluted medium in comparison to the LPS from the same type of the culture in non-diluted medium). 107 Chemical analyses of the core region of the LPSs (ESI HR) from two types of cultures showed changes in one P. mirabilis strain (9 B-m) – additional core oligosaccharides (2180 Da, 1978 Da, 1847 Da, 2020 Da) were observed in the LPS sample from biofilm (these oligosaccharides were absent in the LPS sample from planktonic culture). In case of P. mirabilis 1 B-m isolate dilution of the microbiological medium influenced the structure of the LPS core (in LPS from culture in 10 x diluted medium additional core oligosaccharides were detected: 1987 Da, 1725 Da). Lipopolysaccharides of the selected P. mirabilis strains (1 B-m, 9 B–m) from the planktonic and biofilm cultures stimulated the THP-1 cell line (in concentrations 62,50- 1 000 ng/ml) in a similar manner to produce TNF-α (no statistically significant differences were found). Selected P. mirabilis strains were also tested for sensitivity to ten antibiotic / chemotherapeutic agents in planktonic and biofilm cultures using the microdilution method. P. mirabilis microbial resistance in biofilm was 2-64 times higher than in planktonic cultures. Additionally, positive effect of subinhibitory concentrations of some antibiotics / chemioterapeutics on biofilm formation was observed. Performed studies proved that in case of two out of three investigated P. mirabilis strains (1 B-m and 9 B-m), the type of culture (planktonic or biofilm) could affect the LPS synthesis in the bacterial cells.