Virulence factors of Enterococcus strains isolated from patients with inflammatory bowel disease

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

Pseudomonas aeruginosa biofilm is a potent inducer of phagocyte hyperinflammation

Objective Pseudomonas aeruginosa effectively facilitate resistance to phagocyte killing by biofilm formation. However,b the cross talk between biofilm components and phagocytes is still unclear. We hypothesize that a biofilm provides a concentrated extracellular source of LPS, DNA and exopolysaccharides (EPS), which polarize neighbouring phagocytes into an adverse hyperinflammatory state of activation. Methods We measured the release of a panel of mediators produced in vitro by murine neutrophils and macrophages exposed to various biofilm components of P. aeruginosa cultures. Results We found that conditioned media from a high biofilm-producing strain of P. aeruginosa, PAR5, accumulated high concentrations of extracellular bacterial LPS, DNA and EPS by 72 h. These conditioned media induced phagocytes to release a hyperinflammatory pattern of mediators, with enhanced levels of $TNF-\alpha$, IL-6, IL12p40, $PGE_{2}$ and NO. Moreover, the phagocytes also upregulated COX-2 and iNOS with no influence on the expression of arginase-1. Conclusions Phagocytes exposed to biofilm microenvironment, called by us biofilm-associated neutrophils/macrophages (BANs/BAMs), display secretory properties similar to that of N1/M1-type phagocytes. These results suggest that in vivo high concentrations of LPS and DNA, trapped in biofilm by EPS, might convert infiltrating phagocytes into cells responsible for tissue injury without direct contact with bacteria and phagocytosis

Virulence factors of Enterococcus strains isolated from patients with inflammatory bowel disease

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. 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

Analysis of the geological control on the spatial distribution of potentially toxic concentrations of As and F- in groundwater on a Pan-European scale

The distribution of the high concentrations of arsenic (As) and fluoride (F-) in groundwater on a Pan-European scale could be explained by the geological European context (lithology and structural faults). To test this hypothesis, seventeen countries and eighteen geological survey organizations (GSOs) have participated in the dataset. The methodology has used the HydroGeoToxicity (HGT) and the Baseline Concentration (BLC) index. The results prove that most of the waters considered in this study are in good conditions for drinking water consumption, in terms of As and/or F- content. A low proportion of the analysed samples present HGT≥ 1 levels (4% and 7% for As and F-, respectively). The spatial distribution of the highest As and/or F- concentrations (via BLC values) has been analysed using GIS tools. The highest values are identified associated with fissured hard rock outcrops (crystalline rocks) or Cenozoic sedimentary zones, where basement fractures seems to have an obvious control on the distribution of maximum concentrations of these elements in groundwaters.This research was co-funded by the European Union’s Horizon 2020 research and innovation program (GeoERA HOVER project) under grant agreement number 731166. D. Voutchkova, B. Hansen, and J. Schullehner were also supported by Innovation Fund Denmark (funding agreement number 8055- 00073B). N. Rman participation was supported by the Slovenian Research Agency, research program P1-0020 Groundwaters and Geochemistry. A. Felter, J. Cabalska and A. Mikołajczyk participation was supported by the Polish Ministry of Education and Science. E. Giménez-Forcada is grateful for the support received from the CIPROM/2021/032 Project. Valencian Government. University of Valencia (Spain)

In Vitro Studies of Anti-Hemolytic and Cytotoxic Activity of Procyanidin-Rich Extract from the Leaves of Actinidia arguta

The leaves of mini kiwi (Actinidia arguta) are a rich source of phenolic compounds, in particular the B-type procyanidins that exhibit e.g. antioxidant, anticancer, antiviral, and anti-inflammatory activities. The aim of this study was to determine the biological activity of the extract from leaves of kiwi in relation to cells of erythrocytes and lymphoma. This activity was determined by studying kiwi leaves extract anti-hemolytic, cytotoxic and antiproliferative activity, and its ability to change the physical properties of the cell membrane and inhibit multidrug resistance of mouse lymphoma cells. It was shown that the extract ingredients bound to the cells, caused changes in erythrocyte shape and slightly affected the granularity and size of lymphoma cells. They effectively protected the red blood cells from oxidative damage, but were not toxic to lymphoma cells and did not affect their multidrug resistance. The extract of kiwi leaves is an effective antioxidant but it does not exhibit cytotoxic activity. Therefore, it can be used in the prevention of diseases, especially those related to oxidative stress

Aspekty kliniczne i mikrobiologiczne zastosowania szczepów "Lactobacillus rhamnosus PL1" u pacjentów z objawami zapalenia odbytnicy w przebiegu chorób dystalnego odcinka jelita grubego

Nawracające stany zapalne odbytnicy i związane z nimi dolegliwości stanowią problem kliniczny u pacjentów leczonych z powodu chorób proktologicznych. Celem pracy była ocena wpływu probiotycznego szczepu Lactobacillus rhamnosus PL1 na obraz kliniczny i skład mikrobioty jelitowej u pacjentów z objawami zapalenia jelita grubego w przebiegu choroby hemoroidalnej i choroby uchyłkowej. Materiał stanowiła grupa 24 pacjentów, u których nie uzyskano pełnej poprawy klinicznej po leczeniu choroby zasadniczej. Oceniano obecność i nasilenie objawów klinicznych oraz ilościowe i jakościowe zmiany w składzie bakterii obecnych w kale przed 9-tygodniową suplementacją probiotycznym szczepem Lactobacillus rhamnosus PL1, w jej trakcie i po niej. Wyniki: w całej badanej grupie intensywność bólu po 12 tygodniach było znamiennie mniejsza (p=0,011) w porównaniu z wartościami wyjściowymi; intensywność wzdęć i dyskomfort w jamie brzusznej zmniejszał się znamiennie już po 3 tygodniach, a po 12 tygodniach różnica była wysoce znamienna statystycznie (p<0,0001). Nie wykazano znamiennej różnicy w częstości zgłaszanych biegunek, zaparć, a także świądu i pieczenia w okolicy odbytu. Już 3-tygodniowa suplementacja probiotycznym szczepem L. rhamnosus PL1 spowodowała istotne zmiany ilościowe i jakościowe w składzie mikrobioty jelitowej, różne w zależności od choroby zasadniczej. Stwierdzono zwiększenie globalnej liczby bakterii z rodzaju Lactobacillus, ze szczególnym uwzględnieniem szczepu L. rhamnosus PL1, niezależnie od choroby zasadniczej. Wniosek: Probiotyczny szczep Lactobacillus rhamnosus PL1 wydaje się mieć wartość w przywracaniu właściwych proporcji populacji bakteryjnych u pacjentów z objawami zapalenia odbytnicy w przebiegu leczenia niektórych chorób dolnego odcinka przewodu pokarmowego.Recurrent proctitis and the symptoms associated therewith pose significant clinical problem in proctological patients. The objective of this study was to assess the impact of the probiotic Lactobacillus rhamnosus PL1 strain on the clinical presentation and composition of intestinal microbiota in patients with symptoms of proctitis in the course of hemorrhoidal disease and diverticulosis. Material consisted of 24 patients in whom no complete clinical improvement could be obtained after the treatment of the underlying disease. Subject to the assessment was the presence and the intensity of clinical symptoms as well as qualitative and quantitative changes in the composition of bacterial flora detected in the stool before, during and after a 9-week supplementation with the probiotic Lactobacillus rhamnosus PL1 strain. Results: In the entire study group, the intensity of pain after 12 weeks was significantly lower (p=0,.011) compared to baseline; the intensity of flatulence and abdominal discomfort was reduced significantly as early as after 3 weeks, with the difference reaching a highly significant level after 12 weeks (pP<0,.0001). No significant difference was observed in the frequency of the reported episodes of diarrhea, constipation, as well as itching and burning in the anal region. As early as after 3 weeks of supplementation with the probiotic L. rhamnosus PL1 strain, significant qualitative and quantitative changes were observed in the composition of intestinal microbiota; the changes differed depending on the underlying disease. An increase in the total counts of the bacteria of Lactobacillus genus, particularly L. rhamnosus PL1 strain was observed regardless of the underlying disease. Conclusion: Tthe probiotic Lactobacillus rhamnosus PL1 strain appears to be useful in restoring appropriate ratios of bacterial populations in patients presenting with symptoms of proctitis in the course of the treatment of certain diseases of the lower gastrointestinal tract