Original Article
Copyright ©2013 Baishideng Publishing Group Co., Limited. All rights reserved.
World J Gastroenterol. Jun 21, 2013; 19(23): 3562-3572
Published online Jun 21, 2013. doi: 10.3748/wjg.v19.i23.3562
Virulence factors of Enterococcus strains isolated from patients with inflammatory bowel disease
Edyta Golińska, Anna Tomusiak, Tomasz Gosiewski, Grażyna Więcek, Agnieszka Machul, Diana Mikołajczyk, Małgorzata Bulanda, Piotr B Heczko, Magdalena Strus
Edyta Golińska, Anna Tomusiak, Tomasz Gosiewski, Grażyna Więcek, Agnieszka Machul, Diana Mikołajczyk, Małgorzata Bulanda, Piotr B Heczko, Magdalena Strus, Department of Microbiology, Jagiellonian University Medical College, 31-121 Cracow, Poland
Author contributions: Golińska E performed the majority of the experiments, including the detection of gelatinase activity, measurement of hydrogen peroxide production and the determination of hydrogen peroxide decomposition, and wrote the manuscript; Tomusiak A collected and analysed the data; Gosiewski T performed PCR and multiplex PCR; Więcek G evaluated the adherence to human gut epithelium cells; Machul A and Mikołajczyk D evaluated the biofilm production; Heczko PB and Bulanda M supervised the experiments; and Strus M designed the experiments and supervised the project.
Supported by The Polish Ministry of Science and Higher Education Grants No. 2 PO5A 094 29, 3 P05E 091 25, N N402 0861 and N N401 144638
Correspondence to: Magdalena Strus, PhD, Chair of Microbiology, Department of Microbiology, Jagiellonian University Medical College, 18 Czysta Str., 31-121 Cracow, Poland. mbstrus@cyf-kr.edu.pl
Telephone: +48-12-6332567 Fax: +48-12-4233924
Received: December 18, 2012
Revised: March 5, 2013
Accepted: March 22, 2013
Published online: June 21, 2013
Processing time: 184 Days and 11.5 Hours
Abstract

AIM: To determine the features of Enterococcus that contribute to the development and maintenance of the inflammatory process in patients with inflammatory bowel disease (IBD).

METHODS: Multiplex polymerase chain reaction (PCR) was applied to assess the presence of genes that encode virulence factors [surface aggregating protein (asa1), gelatinase (gelE), cytolysin (cylA), extracellular surface protein (esp) and hyaluronidase (hyl)] in the genomic DNA of 28 strains of Enterococcus isolated from the intestinal tissues of children with IBD (n = 16) and of children without IBD (controls; n = 12). Additionally, strains with confirmed presence of the gelE gene were tested by PCR for the presence of quorum sensing genes (fsrA, fsrB, fsrC) that control the gelatinase production. Gelatinase activity was tested on agar plates containing 1.6% gelatin. We also analysed the ability of Enterococcus strains to release and decompose hydrogen peroxide (using Analytical Merckoquant peroxide test strips) and tested their ability to adhere to Caco-2 human gut epithelium cells and form biofilms in vitro.

RESULTS: A comparison of the genomes of Enterococcus strains isolated from the inflamed mucosa of patients with IBD with those of the control group showed statistically significant differences in the frequency of the asa1 gene and the gelE gene. Furthermore, the cumulative occurrence of different virulence genes in the genome of a single strain of Enterococcus isolated from the IBD patient group is greater than in a strain from the control group, although no significant difference was found. Statistically significant differences in the decomposition of hydrogen peroxide and adherence to the Caco-2 epithelial cell line between the strains from the patient group and control group were demonstrated. The results also showed that profuse biofilm production was more frequent among Enterococcus strains isolated from children with IBD than in control strains.

CONCLUSION: Enterococcus strains that adhere strongly to the intestinal epithelium, form biofilms and possess antioxidant defence mechanisms seem to have the greatest influence on the inflammatory process.

Keywords: Enterococcus; Virulence factors; Inflammatory bowel disease; Hydrogen peroxide; Biofilm

Core tip: In this research we have attempted to show which features make Enterococcus strains contributing to the development and maintenance of the inflammatory process in patients with inflammatory bowel disease. The outcome of this research may have an impact on better understanding of the pathomechanisms of this disease, as its etiology is not fully known. The study results suggest that Enterococcus strains which adhere strongly to the intestinal epithelium, form biofilm as well as possess the enzymatic mechanisms protecting them against the effects of reactive oxygen species, seem to have the highest chances to survive and influence the inflammatory process.