Marcin T. Schmidt

Intratype HPV16 sequence variation within LCR of isolates from asymptomatic carriers and cervical cancers

BACKGROUND HPV16 is a predominant type of virus identified in genital lesions and strongly associated with the development of genital cancers. Infection with the virus is considered to be the main risk factor in the development of cervical cancer. Based on HPV16 DNA isolated from invasive cancers, a classification of intratype genetic variants was established and the strains were designated according to geographical regions. The HPV16 variants classification was based on isolates derived from cancers. OBJECTIVES Analysis of HPV16 LCR variants isolated from asymptomatic carriers for comparison with cervical cancer isolates to examine whether a correlation can be found between cervical epithelium state and variant of HPV16 it carries. MATERIALS AND METHODS The HPV16 LCR fragments were amplified by PCR using DNA isolated from cervical swabs and tissue sections then screened for nucleotide changes by SSCP. Polymorphic sites were analysed for regulatory protein binding properties by EMSA. RESULTS Comparison of the two groups revealed that isolates from cervical cancers predominantly carry changes in sequences of YY1 binding sites (especially at nucleotide 7519), while variants from asymptomatic carriers contained nucleotide changes within or close to transcription binding sites for AP-1, Oct-1, NF1, Tef-1, Tef-2, Sp1, YY1 and viral E2. EMSA study showed that sequence changes in the segment alter binding and formation of transcriptional complexes in quantitative and/or qualitative manner and so they may inflict viral activity. CONCLUSION The results of our study show that there might be HPV16 variants of decreased oncogenic potential therefore infection with such variants can recede.

Influence of phenolic acids on indole acetic acid production and on the type III secretion system gene transcription in food-associated Pseudomonas fluorescens KM05

The purpose of these investigations was to evaluate the reduction capability of phenolic acids (ferulic, chlorogenic, gallic, and p-coumaric acids) on indole acetic acid synthesis by food-associated Pseudomonas fluorescens KM05. Specific genetic primer for the type III secretion system (TTSS) in P. fluorescens KM05 was designed and the influence of phenolic acids on its expression was investigated. In the work the ferulic and chlorogenic acids at the concentration of 0.02 and 0.04 μg/ml affected on bacterial growth pattern and the signal molecules production. The phenolic acids, that were appreciable effective against P. fluorescens KM05 indole acetic acid production, significantly suppressed TTSS gene.

Role of gallic and p-coumaric acids in the AHL-dependent expression of flgA gene and in the process of biofilm formation in food-associated Pseudomonas fluorescens KM120

BACKGROUND In the process of Pseudomonas fluorescens biofilm formation, N-acyl-l-homoserine lactone (AHL)-mediated flagella synthesis plays a key role. Inhibition of AHL production may attenuate P. fluorescens biofilm on solid surfaces. This work validated the anti-biofilm properties of p-coumaric and gallic acids via the ability of phenolics to suppress AHL synthesis in P. fluorescens KM120. The dependence between synthesis of AHL molecules, expression of flagella gene (flgA) and the ability of biofilm formation by P. fluorescens KM120 on a stainless steel surface (type 304L) was also investigated. RESULTS Research was carried out in a purpose-built flow cell device. Limitations on AHL synthesis in P. fluorescens KM120 were observed at concentrations of 120 and 240 µmol L(-1) of phenolic acids in medium. At such levels of gallic and p-coumaric acids the ability of P. fluorescens KM120 to synthesize 3-oxo-C6-homoserine lactone (HSL) was not observed. These concentrations caused decreased expression of flgA gene in P. fluorescens KM120. The changes in expression of AHL-dependent flgA gene significantly decreased the rate of microorganism colonization on the stainless steel surface. CONCLUSION Phenolic acids are able to inhibit biofilm formation. The results obtained in the work may help to develop alternative techniques for anti-biofilm treatment in the food industry.

Inactivation of Lactobacillus rhamnosus GG by fixation modifies its probiotic properties.

Probiotics are microorganisms that have beneficial effects on the host and are safe for oral intake in a suitable dose. However, there are situations in which the administration of living microorganisms poses a risk for immunocompromised host. The objective of this study was to evaluate the influence of several fixation methods on selected biological properties of Lactobacillus rhamnosus GG that are relevant to its probiotic action. Fixation of the bacterial cells with ethanol, 2-propanol, glutaraldehyde, paraformaldehyde, and heat treatment resulted in a significant decrease of alkaline phosphatase, peroxidase, and β-galactosidase activities. Most of the fixation procedures reduced bacterial cell hydrophobicity and increased adhesion capacity. The fixation procedures resulted in a different perception of the bacterial cells by enterocytes, which was shown as changes in gene expression in enterocytes. The results show that some procedures of inactivation allow a fraction of the enzymatic activity to be maintained. The adhesion properties of the bacterial cells were enhanced, but the response of enterocytes to fixed cells was different than to live bacteria. Inactivation allows maintenance and modification of some of the properties of the bacterial cells.

Exopolysaccharides Produced by Lactobacillus rhamnosus KL 53A and Lactobacillus casei Fyos Affect Their Adhesion to Enterocytes

Abstract Probiotics promote and help to maintain beneficial microbiota composition of the gastrointestinal tract ecosystem and have a positive impact on the host’s health. Production of exopolysaccharides is an important feature of probiotic lactobacilli. It increases the chance of their survival in the gastrointestinal tract and promotes adhesion to the epithelium; therefore, exopolysaccharides are important for the process of colonization. Two lactic acid bacteria strains were used in this study: Lactobacillus rhamnosus KL 53A and Lactobacillus casei Fyos. Exopolysaccharides were isolated from bacterial cells and their monosaccharide composition was examined using liquid chromatography. The influence of exopolysaccharides on lactobacilli adhesion to enterocytes was studied after deglycosylation of the bacterial cells and incubation with the selected intestinal microbiota strains that metabolize polysaccharides – Faecalibacterium prausnitzii DSM 17677 and Blautia luti DSM 14534. Both deglycosylation and incubation with polysaccharide metabolizing strains influenced the ability of probiotic strains to adhere to enterocytes. Enzymatic deglycosylation decreased adhesion efficiency of L. rhamnosus KL 53A; however, co-incubation of both lactobacillus strains with F. prausnitzii DSM 17677 resulted in an increase of their adhesion efficiency. Exopolysaccharides are important adhesins of Lactobacillus spp. that influence their ability to colonize gut epithelium. Other members of gut microbiota can modify the adhesion property in situ; therefore the composition and metabolic state of commensal bacteria may influence their probiotic action.

Lactobacillus strains belonging to Casei group display various adherence to enterocytes and mucus.

BACKGROUND The ability of lactobacilli to adhere to the surface of the intestine is an important functional characteristic which can largely determine the effective colonization of the intestinal tract by probiotics. The following study compares the adhesion efficiency of the twenty strains of Lactobacillus genus belonging to Casei group to the Caco-2 cells and gastrointestinal mucus. METHODS Twenty isolates of lactobacilli belonging to Casei group were tested. The ability of bacterial cells to adhere to mucus was examined using adhesion assay to gastrointestinal mucus. Obtained results were compared with adhesion efficiency to Caco-2 cells. Phylogenetic relationship between isolates was analysed by rep-PCR. RESULTS The results showed large differences in adhesion efficiency between strains, as well as differences in the efficiency of adhesion to the intestinal epithelial cells and mucus. Group similarity highlighted by a rep-PCR technique does not correspond with groups of similarity in terms of the characteristics of the ability to adhere to mucus or the epithelial cells of intestinal tract. CONCLUSIONS Strains having a high adhesion efficiency to enterocytes do not always show a high adhesion efficiency to the mucus. This may indicate the presence of different and multiple factors responsible for adhesion efficiency of Lactobacillus group Casei strains to epithelial cells and mucus.