Tomasz Gosiewski

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.

Genetic characterization and diversity of Streptococcus agalactiae isolates with macrolide resistance.

Macrolide resistance in 169 Streptococcus agalactiae [group B streptococcus (GBS)] isolates originating from pregnant carriers was investigated. Using multiplex PCR the presence of genes encoding erythromycin resistance and capsular polysaccharides, as well as surface proteins, was determined. Random amplification of polymorphic DNA (RAPD) and PFGE were used to characterize specific clones among the isolates. In the examined population of women, erythromycin-resistant strains were found in 4.5 % of patients, whereas clindamycin-resistant strains were found in 3 % of patients, which was 16 % of strains resistant to erythromycin and 10 % of strains resistant to clindamycin among GBS isolates, respectively. Among the isolates, the largest percentage was represented by the constitutive macrolide-lincosamide-streptogramin B (cMLS(B)) phenotype (63 %), then the inductive macrolide-lincosamide-streptogramin B (iMLS(B)) phenotype (26 %) and the macrolide resistance (M) phenotype (11 %). The ermB gene was indicated in all isolates with the cMLS(B) phenotype and V serotype, whereas mefA/mefE genes were found in isolates with the M phenotype and Ia serotype. Among resistance isolates, serotype V was predominant (67 %), followed by serotypes II (15 %), Ia (11 %) and III (7 %). The most common surface protein encoding genes were alp3 (70 %), then rib (11 %), epsilon (7.5 %), bca (7.5 %) and alp2 (4 %). A statistically significant relationship between macrolide resistance, serotype V and the alp3 gene was demonstrated. PFGE, in comparison to the RAPD method, gave better genetic discrimination of GBS isolates. A relatively high genetic diversity among investigated strains was shown. In addition, the largest genetic homogeneity was found in serotype V.

Group B streptococcus colonization of pregnant women and their children observed on obstetric and neonatal wards of the University Hospital in Krakow, Poland.

The study was arranged to assess the actual rates of colonization of pregnant women and their children with group B streptococcus (GBS) in a Polish university hospital. Resistance of these cocci to macrolides and clindamycin was also tested and routes of transmission of GBS were followed in some cases using molecular typing. Colonization with GBS was checked in 340 pregnant women living in the south-eastern region of Poland (Małopolska) in the years 2004-2006. Women with a complicated pregnancy were more often colonized than those with a normal pregnancy (20.0 % versus 17.2 %). Moreover, women with a complicated pregnancy were twice as often colonized with GBS strains with the MLS(B) phenotype indicating resistance to macrolides and clindamycin. Regarding neonatal colonization by GBS, we found that neonates born from the colonized mothers with a complicated pregnancy were more often colonized with GBS than those from the mothers with a normal pregnancy (35 % versus 26.7 %). By molecular typing of the GBS strains isolated from mothers and their newborns we have been able to suggest the possibility of horizontal transmission of the strains from the hospital environment to newborns. Our results clearly indicate that rates of GBS colonization among pregnant women and neonates in a Polish university hospital have reached levels comparable to those reported in other European clinical centres.

Possible role of Escherichia coli in propagation and perpetuation of chronic inflammation in ulcerative colitis

BackgroundThis study investigated a possible role of Escherichia coli in propagation and perpetuation of the chronic inflammation in ulcerative colitis (UC). The lesions of UC are located superficially on the rectal and/or colonic mucosa. It is suggested that the commensal bacteria of the digestive tract may play a role in the pathogenesis of UC. Several studies have demonstrated proliferation of E. coli in the gut of UC patients. An increase in the number of E. coli in the inflamed tissue is most probably related to the abundance of iron ions produced by the bacteria.MethodsColon mucosal biopsies were collected from 30 patients with acute-phase UC, both from tissues with inflammatory changes (n = 30) and unchanged tissue with no inflammatory changes (n = 30) from the same patient. Biopsies were also taken from 16 patients with irritable bowel syndrome diarrhea who comprised the control group. Quantitative and qualitative analysis of the biopsy specimens was performed using culture methods and real-time polymerase chain reaction (PCR). Genotyping of the E. coli isolates was done using pulsed-field gel electrophoresis. Multiplex PCR was used to compare the E. coli strains for the presence of genes responsible for synthesis of iron acquisition proteins: iroN, iutA, iha, ireA, chuA, and hlyA.ResultsWe demonstrated that there was a significant increase in the number of E. coli at the sites of inflammation in patients with UC compared to the control group (P = 0.031). Comparative analysis of the restriction patterns of E. coli isolated from inflammatory and unchanged tissues showed that the local inflammatory changes did not promote specific E. coli strains. There was a significant difference in the frequency of the iroN gene in E. coli isolated from patients with UC as compared to the control group.ConclusionsThe increase in the numbers of E. coli in the inflammatory tissues is related to the presence of chuA and iutA genes, which facilitate iron acquisition during chronic intestinal inflammatory processes.

Quantitative evaluation of fungi of the genus Candida in the feces of adult patients with type 1 and 2 diabetes - a pilot study

BackgroundGastrointestinal tract microbiota, particularly bacterial microflora, seem to have a different qualitative and quantitative composition in both type 1 (T1DM) and type 2 diabetes (T2DM) mellitus cases as compared to non-diabetic individuals. So far, there are no data from diabetes research concerning the prevalence of fungi, particularly the most common genus, i.e. Candida, which are important components of human colon microflora.We aimed to examine whether there are quantitative changes of Candida fungi in the feces of patients with T1DM and T2DM as compared to healthy controls.FindingsOverall, we included 44 diabetic patients (27 patients with T1DM and 17 with T2DM) as well as 17 healthy, non-diabetic controls. Feces and blood samples were collected from all study individuals. DNA was isolated from fecal samples and quantitative real time PCR (qPCR) was applied in order to determine the number of fungal cells. Statistical association with selected clinical and biochemical features was examined.There was a difference in the amount of Candida in the feces among the three examined groups (p = 0.007). Candida spp. populations in T1DM and T2DM subjects were larger as compared to controls (p = 0.017 and p = 0.037, respectively). However, no difference was found between T1DM and T2DM. No association was identified between the quantity of fungi and examined patients’ characteristics, except for negative correlation with blood lipid parameters in T2DM group.ConclusionsCandida fungi appear to be more prevalent in the feces of patients with T1DM and T2DM. Their amount seems to be associated with serum lipids in T2DM patients. This initial finding requires further confirmation.

Molecular characterization of capsular polysaccharides and surface protein genes in relation to genetic similarity of group B streptococci isolated from Polish pregnant women.

Serotyping, subtyping and genotyping are important tools for epidemiological studies of group B streptococci (GBS). We investigated the genotype distribution of 353 GBS isolates originating from vaginal or rectal carriage to identify capsular serotypes and subtypes based on the surface protein genes of the alpha-like protein (Alp) family. GBS were recovered from 30% of 1176 pregnant women during the period 2007-2009, with a predominance of capsular genotypes III (35%), Ia (20%), V (17%), II (15%), Ib (8%) and IV (5%). The most common Alp gene was epsilon (26%), followed by rib (22%), alp2 (21%), bca (17%) and alp3 (14%). Several protein genes were significantly associated (G(2)=249·635, P<0·0001) with particular serotypes: epsilon with Ia, Ib, IV; bca with Ib, II; rib with II, III; alp3 with V; alp2 with III. High genetic diversity within GBS strains was observed using DNA macrorestriction. Serotypes Ib, II and III demonstrated the greatest genetic heterogeneity and serotype V the lowest heterogeneity (relative frequency coefficient ≥0·03 vs. -0·46, respectively). Macrolide-resistant strains with serotype V and alp3 gene, showed higher uniformity in genetic profile. The distribution of serotypes and surface proteins of GBS strains are necessary data to inform the design and formulation of new GBS vaccines for use in Poland and other countries.

Antibacterial activity of selected standard strains of lactic acid bacteria producing bacteriocins--pilot study.

INTRODUCTION In this paper, an attempt was made to evaluate the antibacterial potential of standard strains of lactic acid bacteria (LAB) producing bacteriocins of various classes, thus demonstrating various mechanisms of cell membrane damages against the Streptococcus agalactiae strains (Group B Streptococcus, GBS), depending on surface polysaccharides and surface alpha-like protein genes. MATERIALS/METHODS Antimicrobial property of the strains of L. plantarum C 11, L. sakei DSMZ 6333, and L. lactis ATCC 11454 producing bacteriocins: JK and EF plantaricins, sakacin and nisin, respectively, against the GBS strains was evaluated. The chosen to the study GBS strains were represented by serotypes Ia, Ib, II, III, V and they had bca, epsilon, rib, alp2 or alp3 alpha-like protein genes. The experiment was conducted by means of suspension culture and the bacteria count was determined using the serial dilution method. RESULTS A great ability of L. plantarum C 11 strain was proven to inhibit the GBS growth. The strain of L. sakei DSMZ 6333 did not demonstrate any ability to inhibit the growth of GBS, whereas L. lactis ATCC 11454 inhibited the growth of S. agalactiae indicator strains to a minor extent. Statistically significant differences were demonstrated between the GBS strains representing various serotypes against the antimicrobial activity of model LAB strains. The least sensitive to the activity of bacteriocins were the strains representing serotypes Ib and III, whereas the strains representing serotype II were the most sensitive. The sensitivity of the GBS strains to the antimicrobial activity of LAB was not dependent on alpha-like protein genes. DISCUSSION Among the LAB standard strains producing bacteriocins, the strongest antimicrobial property was observed in the strain of L. plantarum C 11. Because of the generally known and verified strong antagonistic property of the strains of L. plantarum species against indicator bacteria, it is necessary to further pursue the research presented in this paper.

Horizontal distribution of the fecal microbiota in adolescents with inflammatory bowel disease.

Background and Aims: The commensal microbiota of the gastrointestinal tract plays an important role in the pathogenesis of inflammatory bowel disease. We examined the horizontal structure of the fecal microbiota in the colon in adolescents with Crohn disease or ulcerative colitis and a control group. Patients and Methods: Fecal samples were collected in 3 fractions from patients with Crohn disease (n = 22), ulcerative colitis (n = 12), and controls (n = 24) during preparation for colonoscopy. Additionally, biopsies from colon tissue were taken. Samples were examined using a culture technique and a fluorescent in situ hybridization method. The mucin degradation assay was carried out. Results: Quantitative composition of the microbiota was different in the consecutive 3 fecal fractions and in the colon tissue of the study groups, but in patients from the control group, the composition of microbiota in the consecutive fractions was similar. Statistical analyses showed that the total distribution of the studied bacterial taxons in the contents in all 3 fecal fractions and in the colon tissue in the given disease group, and in the control group was characteristic for the studied patient group. Differences in species distribution among the cohorts studied were highly significant (P < 0.0001). Moreover, it was shown that in the fecal fraction I and in the colon tissue samples, there is no significant difference for any of the analyzed bacterial groups, using the culture methods or fluorescent in situ hybridization, but significant results were demonstrated in the II and III fractions for specific bacterial groups. The bacterial flora attached to the mucus layer in the UC group had significantly more degraded mucus in comparison with the control group (P = 0.045). Conclusions: Distribution of the microbiota in the colon is layered, which can be called horizontal distribution of the fecal flora. Only in the ulcerative colitis group, the bacterial flora attached to the mucous layer exerts action on the mucin.

The application of genetics methods to differentiation of three Lactobacillus species of human origin

In recent decades, the interest in probiotics as diet supplements or drugs has increased. In order to determine a specific bacterial isolate to be probiotic, it is necessary to describe precisely its probiotic characteristics and taxonomic properties, including the strain level. Most of the well-known genotyping methods were designed for the commonly-found pathogenic bacteria. The objective of this study is to undertake an attempt at standardization of FISH, RAPD and PFGE methods to genotype and identify the bacteria belonging to Lactobacillus fermentum, L. gasseri and L. plantarum species. The FISH probes have been designed and tested for Lactobacillus fermentum, L. gasseri and L. plantarum species and an endeavor has been made at standardization of RAPD and PFGE methods for these bacterial species. Moreover, the MLST method was applied to differentiate Lactobacillus plantarum strains. L. plantarum isolated from humans could not be genetically diversified with the use of RAPD, PFGE or MLST methods; only the strains originating from plants have displayed diversification among themselves and have been different from the strains of human origin.

Comparison of Methods for Isolation of Bacterial and Fungal DNA from Human Blood

AbstractThe study aimed at optimization of DNA isolation from blood of representatives of four microbial groups causing sepsis, i.e., Gram negative: Escherichia coli, Gram positive: Staphylococcus aureus, yeast: Candida albicans, and filamentous fungus: Aspergillus fumigatus. Additionally, the five commercial kits for microbial DNA isolation from the blood were tested. The developed procedure of DNA isolation consisted of three consecutive steps, i.e., mechanical disruption, chemical lysis, and thermal lysis. Afterward, DNA was isolated from the previously prepared samples (erythrocyte lysis) with the use of five commercial kits for DNA isolation. They were compared paying heed to detection limit, concentration, DNA purity, and heme concentration in samples. The isolation of DNA without preliminary erythrocyte lysis resulted in far higher heme concentration than when lysis was applied. In the variant with erythrocyte lysis, two of the commercial kits were most effective in purifying the DNA extract from heme. Designed procedure allowed obtaining microbial DNA from all four groups of pathogens under study in the amount sufficient to conduct the rtPCR reaction, which aimed at detecting them in the blood.