Resting energy expenditure and gut microbiota in obese and normal weight subjects.

OBJECTIVES: It is suggested that gut microbiota play a role in the pathogenesis of obesity enhancing energy utilization from digested food. The influence of gut microbiota on resting energy expenditure (REE) has not been evaluated yet. AIM: The aim of the study is to assess the composition on gut microbiota and its association with REE in obese and normal weight subjects. SUBJECTS AND METHODS: REE measurement and semi-quantitative analysis of gut microbiota composition in aerobic and anaerobic conditions were performed in 50 obese and 30 normal weight subjects without concomitant diseases. RESULTS: A count of bacterial colony was greater in obese than in normal weight subjects. However, the proportion of Bacteroides spp. and Firmicutes was similar in both study groups. A positive correlation between REE (kcal/d) and total bacterial count (r = 0.26, p < 0.05), as well as between REE and the percentage of Firmicutes (r = -0.24, p < 0.05) was found. The multiple regression analysis did not prove an independent impact of total bacterial as well as Bacteroides spp. and Firmicutes counts on REE. CONCLUSIONS: The composition of gut microbiota is not associated with the level of resting energy expenditure. The proportion of Bacteroides and Firmicutes in gut microbiota is not related to body mass.

Characterization of vaginal lactobacilli in women after kidney transplantation.

Limited number of publications described vaginal microflora after kidney transplantation. Our PubMed search revealed only 18 publications including words "vaginal bacteria & kidney transplant" in the period of 1978-2011. The aim of this study was to characterize lactobacilli isolated from vaginal swabs of women after kidney transplantation, compared with healthy women. Eighteen renal transplant recipients (mean age 36.1) and 20 healthy women (mean age 36.0) were evaluated. Lactobacilli were cultured on MRS and Columbia blood agars. Biochemical identification with API 50 CHL (bioMerieux, Marcy L'Etoile, France) and multiplex PCR according to Song et al. was performed. Lactobacilli were tested for production of H(2)O(2). Minimal inhibitory concentrations (MICs) of selected antimicrobial agents were determined with E-tests (bioMerieux, Marcy L'Etoile, France) and interpreted with CLSI and EUCAST criteria. No bacterial vaginosis was found among studied women. Two strains of group I were identified as Lactobacillus delbrueckii; 18 strains as Lactobacillus gasseri and 15 strains as Lactobacillus crispatus. Only 3 strains from group II were not identified by species-specific mPCR. Group IV was represented with 2 unidentified strains. Vaginal lactobacilli isolated from healthy women represented more homogenous group compared with heterogenous renal transplant recipients. Biochemical identification of lactobacilli by API 50 CHL kits was concordant with mPCR results only in 7 cases (17.5%), all 7 strains were identified as L. crispatus. Majority (93%) of lactobacilli were H(2)O(2) producers. All isolated lactobacilli (100%) demonstrated high resistance to metronidazole (MIC > 256 µg/ml). Only 2 strains resistant to vancomycin (MICs: 32 and 256 µg/ml respectively), in the study and control group, and one to moxifloxacin (MIC = 32 µg/ml), were found. Resistance to metronidazole and vancomycin was concordant in CLSI and EUCAST (2010) criteria. Although significant differences between lactobacilli isolated from vaginas of kidney transplant and healthy women were not demonstrated, we demonstrated strains resistant to metronidazole, vancomycin and moxifloxacin in groups of examined women. Our study was performed on a small group of kidney transplant recipients and further more detailed molecular studies on a larger group of patients are required to confirm our results.

Pathogenicity of Helicobacter pylori infection.

Numerous Helicobacter pylori virulence factors, including various enzymes (urease, catalase, lipase, phospholipase and proteases), vacuolating cytotoxin (a product of expression of the vacA gene), and the immunogenic protein CagA, encoded by the cagA gene localised in the H. pylori pathogenicity island, are involved in the pathomechanism of infection caused by these organisms. This review presents the current state of knowledge concerning the molecular mechanisms and epidemiology of H. pylori infection, based on the published literature and recent unpublished observations.

Clonal dissemination of a toxin-A-negative/toxin-B-positive Clostridium difficile strain from patients with antibiotic-associated diarrhea in Poland.

OBJECTIVE: To determine the incidence of toxin-A-negative/toxin-B-positive Clostridium difficile strains and their genetic relatedness in the feces of patients suffering from antibiotic-associated diarrhea (AAD) in Polish hospitals. METHODS: C. difficile strains were cultured from patients' stool samples. The present study characterises these strains with respect to their cytopathogenicity on McCoy cells and the absence of toxin A despite a functional toxin B as determined with commercial test kits (Culturette Brand Toxin CD-TCD toxin A test and C. difficile Tox A/B test). In addition, PCR using different primer pairs aiming at non-repeating or repeating regions of the toxin A and B genes were used to confirm the findings. All toxin A(-)B(+) strains were genetically characterised by random amplification of polymorphic DNA (RAPD) analysis, PCR ribotyping and, in part, pulsed-field gel electrophoresis (PFGE) of DNA macrorestriction fragments. RESULTS: We here present the presence of 17 toxin A(-)B(+) strains among 159 C. difficile strains (11%) isolated from fecal samples from 413 patients with antibiotic-associated diarrhea. All 17 strains possessed the toxin B gene, demonstrated a cytopathogenic effect on the McCoy cells, and were positive in the Tox A/B test. Molecular typing of these 17 C. difficile strains revealed that 7 of 17 (41%) toxin A(-)/B(+) C. difficile strains could not be discriminated. It appeared that these strains had a genotype that could not be distinguished from that of a Japanese control strain. CONCLUSION: Our observations imply that a particular genotype of toxin A(-)B(+) C. difficile has spread extensively, not only in Poland but possibly even worldwide.

Acute appendicitis: the role of enterotoxigenic strains of Bacteroides fragilis and Clostridium difficile.

BACKGROUND: The aim of this study was to investigate whether there is a relationship between enterotoxin-producing B. fragilis strains and toxigenic C. difficile strains and the pathogenesis of acute appendicitis. MATERIAL AND METHODS: Post-appendectomy tissues from 34 patients with histopathologically confirmed phlegmonous or gangrenous appendicitis were studied. RESULTS: Among 86 anaerobes isolated, the B. fragilis group was most frequently isolated: 34 B. fragilis strains were cultured from 21 post-appendectomy tissues. Two enterotoxin-producing B. fragilis strains were found. Enterotoxin titers (1:10 and 1:160, respectively) were measured on HT29/C cells. The presence of the enterotoxin gene was confirmed by PCR in DNA extracted from both strains. Among 21 DNA samples isolated from those post-appendectomy tissues from which B. fragilis strains were cultured, the presence of the enterotoxin gene was confirmed in only one case (the corresponding B. fragilis strain enterotoxin titer was 1:160). A unique toxigenic C. difficile strain was also cultured from the tissue of an adult patient with gangrenous non-perforated appendicitis. The presence of toxin A and toxin B genes was confirmed by PCR in DNA extracted from the C. difficile strain, but these genes were not found in the DNA extracted from the corresponding tissue. CONCLUSION: The presence of enterotoxigenic B. fragilis and toxigenic C. difficile strains was shown in post-appendectomy tissue from patients with phlegmonous and gangrenous appendicitis, and the B. fragilis enterotoxin gene was detected directly in the corresponding tissue. Further investigations (including immunologic aspects) require to confirm the role of these toxins in pathogenesis of acute appendicitis.

Search for enterotoxin gene in Bacteroides fragilis strains isolated from clinical specimens in Poland, Great Britain, The Netherlands and France.

BACKGROUND: Bacteroides fragilis is a member of normal human flora and well known pathogenic agent. This bacterium produces many virulence factors. In 1984 new virulence factor--enterotoxin was described. The aim of the study was to search for enterotoxin gene in B. fragilis strains isolated from clinical specimens. MATERIAL AND METHODS: Strains isolated in Poland, Great Britain, France and the Netherlands were cultured on BBE medium. For DNA isolation Genomic DNA PREP PLUS isolation kit manufactured by A&A Biotechnology (Poland) was used. In order to detect enterotoxin (fragilysin) gene, polymerase chain reaction (PCR) was applied utilizing the following primers: 404 (GAG CGG AAG ACG GTG TAT GTG ATT TGT) and 407 (TGC TCA GCG CCC AGT ATA TGA CCT AGT). DNA obtained from bacterial cells was amplified in thermocycler Techne. The amplification products were detected by the electrophoresis in 1% agarose gel. RESULTS: Among 65 investigated B. fragilis strains, the enterotoxin gene was detected in DNA isolated from 12 strains. CONCLUSION: The enterotoxin producing B. fragilis strains were detected among strains isolated from different clinical specimens in Poland, Great Britain, the Netherlands and France.

[Enterotoxin-producing Bacteroides fragilis strains isolated from horses]. / Enterotoksynotwórcze szczepy Bacteroides fragilis izolowane od koni.

Seven Bacteroides fragilis strains were cultured from samples collected from horses. From all the tested strains, as well as from the reference B. fragilis strains: enterotoxigenic NCTC 11925 and nonenterotoxigenic IPL 323 strain, DNA was isolated using Genomic DNA PREP PLUS isolation kit manufactured by A&A Biotechnology (Poland). To detect the enterotoxin (fragilysin) gene, polymerase chain reaction (PCR) was applied, using the following starters: 404 (GAG CCG AAG ACG GTG TAT GTG ATT TGT) and 407 (TGC TCA GCG CCC AGT ATA TGA CCT AGT). DNA obtained from bacterial cells was amplified in a thermocycler (Techne). The temperature profile was as follows: 1 cycle (4 min. 94 degrees C), 40 cycles (1 min. 94 degrees C, 1 min. 52 degrees C, 1 min. 74 degrees C). Amplification products were detected by electrophoresis in agarose gel (1%) with ethidium bromide added. The presence of the fragilysin gene was detected in two strains. Among the strains isolated from horses enterotoxin gene-possessing Bacteroides fragilis strains (ETBF) can be detected.

In vitro antibiotic susceptibility of Bacteroides fragilis strains isolated from excised appendix of patients with phlegmonous or gangrenous appendicitis.

Out of 34 studied after-appendectomy tissues of adult and child patients 86 different strains of anaerobes were isolated. The antibiotic susceptibility of 30 isolated B. fragilis strains was tested using E tests. All studied strains were sensitive to imipenem, clindamycin and penicillin/tazobactam. Sensitivity to penicillin and cefoxitin was variable among these strains. One strain resistant to metronidazole (MIC--256 mg/L) and 3 strains with increased MIC to metronidazole were detected. Most of isolated strains were beta-lactamase producers.

[Intestinal flora of patients with suspected antibiotic associated diarrhea (AAD). I. Clostridium perfringens]. / Flora przewodu pokarmowego chorych z podejrzeniem biegunki poantybiotykowej (AAD). I. Clostridium perfringens.

Stool samples of 158 patients suspected of antibiotic-associated diarrhoea (AAD) were studied. Toxin A of C. difficile and enterotoxin of C. perfringens were detected in stool samples by immunoenzymatic assays and PCR. In 35 stool samples toxin A of C. difficile was detected and in 48 cases (30%) C. difficile strains were cultured from 21 stool samples (13%). The presence of the cpe gene of C. perfringens, enabling the production of enterotoxin, could not be detected by PCR, both in stool samples and in isolated strains, using ent 1 and ent 2 primer pairs. C. difficile and C. perfringens were isolated from the same stool samples in 4 cases. From stool samples of two patients with AAD C. perfringens strains, thermoresistant spores were cultured.

Structure of an antigenic teichoic acid shared by clinical isolates of Enterococcus faecalis and vancomycin-resistant Enterococcus faecium.

A shared antigenic teichoic acid, previously found to be a surface capsule-like polysaccharide, was isolated from clinical isolates of Enterococcus faecalis and vancomycin-resistant E. faecium. It was composed of glucose, glycerol, and phosphate as determined by chemical and GC-MS analysis. The repeating-unit structure was elucidated by a series of 1H, 13C, and 31P NMR spectroscopy to be the following: [formula: see text]

Isolation and chemical characterization of a capsular polysaccharide antigen shared by clinical isolates of Enterococcus faecalis and vancomycin-resistant Enterococcus faecium.

Enterococci are a common cause of serious infections, especially in newborns, severely immunocompromised patients, and patients requiring intensive care. To characterize enterococcal surface antigens that are targets of opsonic antibodies, rabbits were immunized with various gentamicin-killed Enterococcus faecalis strains, and immune sera were tested in an opsonophagocytic assay against a selection of clinical isolates. Serum raised against one strain killed the homologous strain (12030) at a dilution of 1:5,120 and mediated opsonic killing of 33% of all strains tested. In addition, this serum killed two (28%) of seven vancomycin-resistant Enterococcus faecium strains. Adsorption of sera with the homologous strain eliminated killing activity. The adsorbing antigens were resistant to treatment with proteinase K and to boiling for 1 h, but were susceptible to treatment with sodium periodate, indicating that the antigen inducing opsonic activity is a polysaccharide. Antibodies in immune rabbit sera reacted with a capsule-like structure visualized by electron microscopy both on the homologous E. faecalis strain and on a vancomycin-resistant E. faecium strain. The capsular polysaccharides from E. faecalis 12030 and E. faecium 838970 were purified, and chemical and structural analyses indicated they were identical glycerol teichoic acid-like molecules with a carbohydrate backbone structure of 6-alpha-D-glucose-1-2 glycerol-3-PO4 with substitution on carbon 2 of the glucose with an alpha-2-1-D-glucose residue. The purified antigen adsorbed opsonic killing activity from immune rabbit sera and elicited high titers of antibodies (when used to immunize rabbits) that both mediated opsonic killing of bacteria and bound to a capsule-like structure visualized by electron microscopy. These results indicate that approximately one-third of a sample of 15 E. faecalis strains and 7 vancomycin-resistant E. faecium strains possess shared capsular polysaccharides that are targets of opsonophagocytic antibodies and therefore are potential vaccine candidates.

Evaluation of different methods for detection of Clostridium difficile toxins in Poland.

The aim of this study was to compare different methods for C. difficile toxins detection. Fifty three stool samples taken from patients with antibiotic-associated diarrhoea were studied. TCD toxin A EIA (Becton Dickinson, USA), Tox A/B ELISA test (TechLab, USA), cytotoxicity and neutralization assay on McCoy cells and PCR for detection of both toxin A and B genes were performed in vivo (in stool samples) and in vitro (in isolated strains). Reference toxigenic and nontoxigenic and two Japanese toxin A-negative and toxin B-positive C. difficile strains were used as a controls. TCD toxin A EIA detected in vivo only 19 positive samples. Tox A/B test detected 52 positive samples out of 53 studied. All 53 stool samples were C. difficile culture positive (53 strains were cultured). Toxin B was detected in 52 strain-supernatants and in all controls (except the nontoxigenic one). Both toxin A and B genes were detected by PCR in all 53 isolated strains, Japanese and reference strain (except the nontoxigenic one). In vitro toxin A was detected by TCD toxin A EIA in 42 strains. These results were compared with those obtained in Tox A/B ELISA test. We observed 52 positive strains. Toxigenic reference strain and two Japanese toxA(-)/toxB(+) strains were also positive. Only 2 negative results were obtained with the nontoxigenic reference strain and unique nontoxigenic isolated strain. Tox A/B ELISA test seems to be the best for detection of C. difficile toxins in vivo and in vitro. Test avoids the false-negative results in the case of presence of toxin A-negative and toxin B-positive strain.

[Detection of toxin producing strains of Clostridium difficile using rapid diagnostic methods]. / Wykrywanie toksynotwórczosci szczepów Clostridium difficile przy pomocy szybkich metod diagnostycznych.

Feces of 53 patients from different hospital wards suffering from long term post-antibiotic therapy diarrhea were tested. For direct detection of C. difficile toxin A, in samples TCD (Becton-Dickinson), and C. difficile Toxin A Test (Oxoid) tests were used. Toxin A was detected in 16 samples (29.6% tested). C. difficile strains were isolated from 40% of the fecal samples. Toxin A was detected in 25 Clostridium difficile strains with commercial tests and toxin B was detected using McCoy cell line. Toxin A was not produced by 3 C. difficile strains in vitro, but toxin B was produced by all strains. The polymerase chain reaction (PCR) test showed that all isolated strains possess genes of toxins A and B.

Dioctahedral smectite neutralization activity of Clostridium difficile and Bacteroides fragilis toxins in vitro.

The neutralization activity of dioctahedral smectite for ten toxigenic Clostridium difficile and eight enterotoxigenic Bacteroides fragilis strains was studied using McCoy and HT 29/C1 cell lines, respectively. Minimalization of the cytopathic effect of C. difficile toxin B on McCoy cell lines by dioctahedral smectite dissolved in PBS was observed. After incubation with dioctahedral smectite the toxic effects of B. fragilis enterotoxins on HT/29C1 (human colon adenocarcinoma cell line) were eliminated. Best neutralization of B. fragilis toxin was achieved using dioctahedral smectite dissolved in BHI.

PCR ribotyping and arbitrarily primed PCR for typing strains of Clostridium difficile from a Polish maternity hospital.

Detection of the source of Clostridium difficile strains is of importance for the control of the nosocomial spread of this microorganism. For this purpose, vaginal and rectal swabs from 183 mothers, duplicate fecal samples (taken on days 1 and 4 after birth) from 183 neonates, and 94 environmental samples were cultured for C. difficile. The microorganism was never detected in the meconium obtained on day 1 after birth. On the other hand, an incidence of 17% C. difficile positivity was noted in the fecal samples obtained on day 4 after birth. Forty-two percent of the 31 colonized neonates had been delivered with complications. The bacteria were never encountered in the rectal swabs of the mothers, and C. difficile was identified in only one vaginal swab. In contrast, 13% of the environmental samples were positive for C. difficile. No major difference was encountered between patient and environmental isolates with respect to toxigenicity (58 to 65% toxigenic isolates). All strains were subsequently typed by PCR amplification of the 16S-23S ribosomal intergenic spacer regions and by arbitrarily primed PCR (AP-PCR) with different primers and combinations thereof. All environmental isolates and 11 of 31 neonatal strains were of a single type. The vaginal strain was unique, and among the maternity ward- and neonate-related isolates, only two additional AP-PCR types were identified. When a collection of C. difficile strains from patients hospitalized in other institutions and suffering from antibiotic-associated diarrhea or pseudomembranous colitis was analyzed in a similar manner, it appeared that the strain from the maternity ward was unique. The other strain commonly encountered among the neonates was also identified frequently among the isolates from patients with antibiotic-associated diarrhea or pseudomembranous colitis, indicating its general occurrence. On the basis of both epidemiological studies and PCR-mediated genotyping, it was shown that the environment and not the birth canal is the major source of C. difficile acquisition by neonates in this maternity hospital setting. Furthermore, AP-PCR appears to be a fast and useful method for epidemiologically relevant typing of C. difficile isolates.