Maria Hedberg

Proximal small intestinal microbiota and identification of rod-shaped bacteria associated with childhood celiac disease

OBJECTIVES:Alterations in the composition of the microbiota in the intestine may promote development of celiac disease (CD). Using scanning electron microscopy (SEM) we previously demonstrated that rod-shaped bacteria were present on the epithelium of proximal small intestine in children with CD but not in controls. In this study we characterize the microbiota of proximal small intestine in children with CD and controls and identify CD-associated rod-shaped bacteria.METHODS:Proximal small intestine biopsies from 45 children with CD and 18 clinical controls were studied. Bacteria were identified by 16S rDNA sequencing in DNA extracted from biopsies washed with buffer containing dithiothreitol to enrich bacteria adhering to the epithelial lining, by culture-based methods and by SEM and transmission electron microscopy.RESULTS:The normal, mucosa-associated microbiota of proximal small intestine was limited. It was dominated by the genera Streptococcus and Neisseria, and also contained Veillonella, Gemella, Actinomyces, Rothia, and Haemophilus. The proximal small intestine microbiota in biopsies from CD patients collected during 2004–2007 differed only marginally from that of controls, and only one biopsy (4%) had rod-shaped bacteria by SEM (SEM+). In nine frozen SEM+ CD biopsies from the previous study, microbiotas were significantly enriched in Clostridium, Prevotella, and Actinomyces compared with SEM− biopsies. Bacteria of all three genera were isolated from children born during the Swedish CD epidemic. New Clostridium and Prevotella species and Actinomyces graevenitzii were tentatively identified.CONCLUSIONS:Rod-shaped bacteria, probably of the indicated species, constituted a significant fraction of the proximal small intestine microbiota in children born during the Swedish CD epidemic and may have been an important risk factor for CD contributing to the fourfold increase in disease incidence in children below 2 years of age during that time.Am J Gastroenterol advance online publication, 15 September 2009; doi:10.1038/ajg.2009.524

Growth inhibition of oral mutans streptococci and candida by commercial probiotic lactobacilli - an in vitro study

BackgroundProbiotic bacteria are suggested to play a role in the maintenance of oral health. Such health promoting bacteria are added to different commercial probiotic products. The aim of the study was to investigate the ability of a selection of lactobacilli strains, used in commercially available probiotic products, to inhibit growth of oral mutans streptococci and C. albicans in vitro.MethodsEight probiotic lactobacilli strains were tested for growth inhibition on three reference strains and two clinical isolates of mutans streptococci as well as two reference strains and three clinical isolates of Candida albicans with an agar overlay method.ResultsAt concentrations ranging from 109 to 105 CFU/ml, all lactobacilli strains inhibited the growth of the mutans streptococci completely with the exception of L. acidophilus La5 that executed only a slight inhibition of some strains at concentrations corresponding to 107 and 105 CFU/ml. At the lowest cell concentration (103 CFU/ml), only L. plantarum 299v and L. plantarum 931 displayed a total growth inhibition while a slight inhibition was seen for all five mutans streptococci strains by L. rhamnosus LB21, L. paracasei F19, L. reuteri PTA 5289 and L. reuteri ATCC 55730. All the tested lactobacilli strains reduced candida growth but the effect was generally weaker than for mutans streptococci. The two L. plantarum strains and L. reuteri ATCC 55730 displayed the strongest inhibition on Candida albicans. No significant differences were observed between the reference strains and the clinical isolates.ConclusionThe selected probiotic strains showed a significant but somewhat varying ability to inhibit growth of oral mutans streptococci and Candida albicans in vitro.

Sugar fermentation in probiotic bacteria – an in vitro study

INTRODUCTION Food supplemented with probiotic bacteria is a rapidly growing sector of the market. The aim of the present study was to evaluate and compare the acid production of selected probiotic strains available in commercial products. METHODS Six Lactobacillus strains (Lactobacillus plantarum 299v and 931; Lactobacillus rhamnosus GG and LB21; Lactobacillus paracasei subsp. paracasei F19, and Lactobacillus reuteri PTA 5289) were cultivated at 37 degrees C in an anaerobic atmosphere on Man, Rogosa, Shape (MRS) agar for 48 h or MRS broth for 16 h. After centrifugation, the cells were washed and resuspended in sterile phosphate-buffered saline and immediately subjected to a fermentation assay with 12 different carbohydrates (nine sugars and three sugar alcohols) in microtiter plates with a pH indicator. The plates were examined for color changes after 24, 48, and 72 h of incubation under aerobic and anaerobic conditions. Three scores were used: negative (pH > 6.8); weak (pH 5.2-6.8), and positive (pH < 5.2). The strains were characterized with the API 50 CH system to confirm their identity. RESULTS L. plantarum fermented all the sugars except for melibiose, raffinose, and xylitol. Both L. rhamnosus strains were generally less active although L. rhamnosus GG was slightly more active than strain LB21 in the 5% CO(2) setting. The latter strain exhibited negative reactions for sucrose, maltose, arabinose, and sorbitol under anaerobic conditions. The assays with L. paracasei and L. reuteri had negative or weak reactions for all tested sugars under both aerobic and anaerobic conditions. CONCLUSION The metabolic capacity to form acid from dietary sugars differed significantly between the various probiotic strains.

Intestinal T-cell responses in celiac disease : impact of celiac disease associated bacteria

A hallmark of active celiac disease (CD), an inflammatory small-bowel enteropathy caused by permanent intolerance to gluten, is cytokine production by intestinal T lymphocytes. Prerequisites for contracting CD are that the individual carries the MHC class II alleles HLA-DQ2 and/or HLA-DQ8 and is exposed to gluten in the diet. Dysbiosis in the resident microbiota has been suggested to be another risk factor for CD. In fact, rod shaped bacteria adhering to the small intestinal mucosa were frequently seen in patients with CD during the “Swedish CD epidemic” and bacterial candidates could later be isolated from patients born during the epidemic suggesting long-lasting changes in the gut microbiota. Interleukin-17A (IL-17A) plays a role in both inflammation and anti-bacterial responses. In active CD IL-17A was produced by both CD8+ T cells (Tc17) and CD4+ T cells (Th17), with intraepithelial Tc17 cells being the dominant producers. Gluten peptides as well as CD associated bacteria induced IL-17A responses in ex vivo challenged biopsies from patients with inactive CD. The IL-17A response was suppressed in patients born during the epidemic when a mixture of CD associated bacteria was added to gluten, while the reverse was the case in patients born after the epidemic. Under these conditions Th17 cells were the dominant producers. Thus Tc17 and Th17 responses to gluten and bacteria seem to pave the way for the chronic disease with interferon-γ-production by intraepithelial Tc1 cells and lamina propria Th1 cells. The CD associated bacteria and the dysbiosis they might cause in the resident microbiota may be a risk factor for CD either by directly influencing the immune responses in the mucosa or by enhancing inflammatory responses to gluten.

European surveillance study on antimicrobial susceptibility of Gram-positive anaerobic cocci

Gram-positive anaerobic cocci (GPAC) are a heterogeneous group of microorganisms frequently isolated from local and systemic infections. In this study, the antimicrobial susceptibilities of clinical strains isolated in 10 European countries were investigated. After identification of 299 GPAC to species level, the minimum inhibitory concentrations of penicillin, imipenem, clindamycin, metronidazole, vancomycin and linezolid were determined by the agar dilution method according to the Clinical and Laboratory Standards Institute. The majority of isolates were identified as Finegoldia magna and Parvimonas micra (formerly Peptostreptococcus micros), isolated from skin and soft tissue infections. All isolates were susceptible to imipenem, metronidazole, vancomycin and linezolid. Twenty-one isolates (7%) were resistant to penicillin (n=13) and/or to clindamycin (n=12). Four isolates were resistant to both agents. The majority of resistant isolates were identified as F. magna and originated from blood, abscesses and soft tissue infections.

Bacteraemia in Patients with Hidradenitis Suppurativa Undergoing Carbon Dioxide Laser Surgery: Detection and Quantification of Bacteria by Lysis-Filtration

Background: Hidradenitis suppurativa (HS) is a cicatrising and persistent disease of apocrine gland-bearing areas in adults. The severity of this condition varies from a few suppurating lesions to widespread, disabling disease. The aetiology is obscure, but suggested contributory factors include a genetic predisposition, comedones occluding the pilosebaceous apparatus, bacterial infections, and hormonal factors. Treatment consists mainly of surgery, while medical therapies serve principally as adjunct therapy. Objectives: The aim of the study was to determine the number and type of bacteria circulating in the bloodstream in patients with HS undergoing surgical treatment with a carbon dioxide laser stripping-secondary intention technique. Methods: Twenty-one patients (20 females and 1 male, mean age 36, range 20–55 years) were included in the study. One blood sample (8.3 ml) was taken before surgery, one during the operation and the last one 10 min after surgery. Five healthy persons (all females, mean age 36, range 23–48 years) not undergoing any operation were used as the controls. The blood was cultured by a lysis-filtration technique which had been shown to be very sensitive. Since the filter catches the microorganisms and colonies are formed during culturing, the number of bacteria in the samples is easily determined. Results: In 6 patients, all samples were negative, which indicates that the method of surgery itself caused no spread of bacteria from the lesions. Bacterial growth in the first blood sample was found in 9 patients, from the second sample in 10 and from the third one in 6. In 1 patient, bacteria were detected in three samples. At least 12 bacterial species were identified. The dominating bacteria were coagulase-negative staphylococci of which most were subtyped as Staphylococcus warneri. Among the anaerobic microorganisms, Propionibacterium acnes and P.granulosum were the most frequently isolated bacteria. The bacterial findings in the blood samples accord well with the results from a previous study in which cultures were taken from the deep parts of the HS lesions. In the 5 controls, no microbial growth was detected. Conclusion: The carbon dioxide laser stripping technique caused no additional spread of bacteria into the bloodstream. The evaluation of cultures containing microorganisms from normal skin flora is controversial. Since the bacteria encountered in this study are in close agreement with the findings in cultures from the deeper parts of HS lesions they seem to be relevant. The growth of bacteria in the first blood sample taken before surgery may indicate that some of these patients have bacteria continuously circulating in their blood.

Prevotella jejuni sp. nov., isolated from the small intestine of a child with coeliac disease.

Five obligately anaerobic, Gram-stain-negative, saccharolytic and proteolytic, non-spore-forming bacilli (strains CD3 : 27, CD3 : 28T, CD3 : 33, CD3 : 32 and CD3 : 34) are described. All five strains were isolated from the small intestine of a female child with coeliac disease. Cells of the five strains were short rods or coccoid cells with longer filamentous forms seen sporadically. The organisms produced acetic acid and succinic acid as major metabolic end products. Phylogenetic analysis based on comparative 16S rRNA gene sequence analysis revealed close relationships between CD3 : 27, CD3 : 28T and CD3 : 33, between CD3 : 32 and Prevotella histicola CCUG 55407T, and between CD3 : 34 and Prevotella melaninogenica CCUG 4944BT. Strains CD3 : 27, CD3 : 28T and CD3 : 33 were clearly different from all recognized species within the genus Prevotella and related most closely to but distinct from P. melaninogenica. Based on 16S rRNA, RNA polymerase β-subunit (rpoB) and 60 kDa chaperonin protein subunit (cpn60) gene sequencing, and phenotypic, chemical and biochemical properties, strains CD3 : 27, CD3 : 28T and CD3 : 33 are considered to represent a novel species within the genus Prevotella, for which the name Prevotella jejuni sp. nov. is proposed. Strain CD3 : 28T ( = CCUG 60371T = DSM 26989T) is the type strain of the proposed novel species. All five strains were able to form homologous aggregates, in which tube-like structures were connecting individual bacteria cells. The five strains were able to bind to human intestinal carcinoma cell lines at 37 °C.

Immunopathology of childhood celiac disease—Key role of intestinal epithelial cells

Background & Aims Celiac disease is a chronic inflammatory disease of the small intestine mucosa due to permanent intolerance to dietary gluten. The aim was to elucidate the role of small intestinal epithelial cells in the immunopathology of celiac disease in particular the influence of celiac disease-associated bacteria. Methods Duodenal biopsies were collected from children with active celiac disease, treated celiac disease, and clinical controls. Intestinal epithelial cells were purified and analyzed for gene expression changes at the mRNA and protein levels. Two in vitro models for human intestinal epithelium, small intestinal enteroids and polarized tight monolayers, were utilized to assess how interferon-γ, interleukin-17A, celiac disease-associated bacteria and gluten influence intestinal epithelial cells. Results More than 25 defense-related genes, including IRF1, SPINK4, ITLN1, OAS2, CIITA, HLA-DMB, HLA-DOB, PSMB9, TAP1, BTN3A1, and CX3CL1, were significantly upregulated in intestinal epithelial cells at active celiac disease. Of these genes, 70% were upregulated by interferon-γ via the IRF1 pathway. Most interestingly, IRF1 was also upregulated by celiac disease-associated bacteria. The NLRP6/8 inflammasome yielding CASP1 and biologically active interleukin-18, which induces interferon-γ in intraepithelial lymphocytes, was expressed in intestinal epithelial cells. Conclusion A key factor in the epithelial reaction in celiac disease appears to be over-expression of IRF1 that could be inherent and/or due to presence of undesirable microbes that act directly on IRF1. Dual activation of IRF1 and IRF1-regulated genes, both directly and via the interleukin-18 dependent inflammasome would drastically enhance the inflammatory response and lead to the pathological situation seen in active celiac disease.