Agnes E. Wold

Establishment of the gut microbiota in Western infants.

In adult individuals, the intestinal microbiota comprises several hundred, mostly anaerobic, bacterial species. This complex ecosystem is formed through the successive establishment of different bacteria in infancy and early childhood. Facultative and aerotolerant bacteria establish first, followed by more and more strict anaerobes. The bacteria derive from different sources and the colonization pattern is influenced by delivery mode and environmental factors. Commensal microbes provide the major drive for maturation of the immune system. Increased hygiene appears to have changed the gut flora of Western infants, which may affect the risk of developing immune mediated diseases.

Gram-Positive Bacteria Are Potent Inducers of Monocytic Interleukin-12 (IL-12) while Gram-Negative Bacteria Preferentially Stimulate IL-10 Production

ABSTRACT Interleukin-10 (IL-10) and IL-12 are two cytokines secreted by monocytes/macrophages in response to bacterial products which have largely opposite effects on the immune system. IL-12 activates cytotoxicity and gamma interferon (IFN-γ) secretion by T cells and NK cells, whereas IL-10 inhibits these functions. In the present study, the capacities of gram-positive and gram-negative bacteria to induce IL-10 and IL-12 were compared. Monocytes from blood donors were stimulated with UV-killed bacteria from each of seven gram-positive and seven gram-negative bacterial species representing both aerobic and anaerobic commensals and pathogens. Gram-positive bacteria induced much more IL-12 than did gram-negative bacteria (median, 3,500 versus 120 pg/ml at an optimal dose of 25 bacteria/cell; P < 0.001), whereas gram-negative bacteria preferentially stimulated secretion of IL-10 (650 versus 200 pg/ml; P < 0.001). Gram-positive species also induced stronger major histocompatibility complex class II-restricted IFN-γ production in unfractionated blood mononuclear cells than did gram-negative species (12,000 versus 3,600 pg/ml; P < 0.001). The poor IL-12-inducing capacity of gram-negative bacteria was not remediated by addition of blocking anti-IL-10 antibodies to the cultures. No isolated bacterial component could be identified that mimicked the potent induction of IL-12 by whole gram-positive bacteria, whereas purified LPS induced IL-10. The results suggest that gram-positive bacteria induce a cytokine pattern that promotes Th1 effector functions.

Reduced enterobacterial and increased staphylococcal colonization of the infantile bowel: an effect of hygienic lifestyle?

The modern Western lifestyle may have altered the composition of the commensal microflora. Here, we investigated the first years intestinal colonization pattern in 99 vaginally delivered Swedish infants and 17 delivered by cesarean section. Rectal swabs obtained at 3 d of age were cultured for aerobic bacteria and fecal samples obtained at 1, 2, 4, and 8 wk and at 6 and 12 mo of age were cultivated quantitatively for aerobic and anaerobic bacteria. Vaginally delivered infants more often had Escherichia coli compared with cesarean section–delivered infants, whereas the latter more frequently carried other enterobacteria, such as Klebsiella and Enterobacter. Independent of delivery mode, it took 2 mo until most infants were colonized by enterobacteria, traditionally the first colonizers. In contrast, coagulase-negative staphylococci colonized 99% of the infants from d 3 onwards. The poor adaptation of staphylococci to the gut was shown by declining population sizes after some weeks. Dominating anaerobes were initially bifidobacteria and clostridia, whereas Bacteroides initially colonized only 30% of vaginally delivered infants and increased very slowly in prevalence. Bacteroides colonization was delayed up to 1 y in cesarean section–delivered compared with vaginally delivered infants. Our results show that some “traditional” fecal bacteria are acquired late today especially in cesarean section–delivered infants, probably due to limited environmental circulation. In their absence, skin bacteria like staphylococci have become the first gut colonizers.

The hygiene hypotheslis revised: is the rising frequency of allergy due to changes in the intestinal flora?

The prevalence of atopy is steadily rising in modern, highly industrialized countries (1). This is true of hay fever (2-5), asthma (6-4, and eczema (3, 4, 9). The incidence is much higher in Western Europe and Australia than in the former Eastern Europe and the developing countries. For example, the incidence of positive skin prick tests in children 4-14 years of age was 9% in Guinea-Bissau, as compared with 29% in 9-10-year-old children in Australia (10). In the former West Germany, 37% are atopically sensitized, as compared with 18% in the former East Germany (11). A much higher incidence of atopy is also seen in Sweden than in Poland (12). The rising allergy frequency in the West seems to have started with people born in the 1960s (13). In 1983, Gregg concluded that the “Western lifestyle” evolved among well-situated people was responsible for the rise in allergies after the 1960s (14).

Lactobacilli from human gastrointestinal mucosa are strong stimulators of IL‐12 production

Interaction of macrophages with bacteria is a stimulus for production of cytokines such as IL‐10 and IL‐12. IL‐12 stimulates T cell and natural killer (NK) cell cytotoxicity and interferon‐gamma (IFN‐γ) production. IL‐10 opposes the T cell‐stimulating action of IL‐12, decreases the release of proinflammatory cytokines from macrophages, and stimulates B cells. We have studied the capacity of human intestinal isolates from the three Lactobacillus species dominating on the human gastrointestinal mucosa, L. plantarum, L. rhamnosus and L. paracasei ssp. paracasei, to induce production of IL‐10 and IL‐12 from human blood mononuclear cells, or monocytes. Whole killed lactobacilli were potent stimulators of IL‐12 over a wide range of bacterial concentrations. Lactobacillus paracasei gave the highest levels of IL‐12 (1.5 ng/ml in response to 5 × 106 bacteria/ml), roughly 10 times more than obtained by stimulation with L. rhamnosus or L. plantarum. Escherichia coli induced on average < 50 pg/ml of IL‐12 regardless of the bacterial concentration used. The secretion of free p40 subunit IL‐12 followed the same pattern as the secretion of p70 (bioactive IL‐12) with regard to the efficiency of different bacteria as stimulators. Escherichia coli was the most efficient trigger of IL‐10 production, inducing 0.5 ng/ml IL‐10 after stimulation with 5 × 106 bacteria/ml. Lactobacillus rhamnosus induced the highest levels of IL‐10 among the lactobacilli (0.5 ng/ml) compared with 0.1 ng/ml evoked by L. plantarum or L. paracasei, but 10 times more bacteria were required for optimal stimulation than with E. coli. When neutralizing anti‐IL‐10 antibodies were added to the cultures, the IL‐12‐inducing capacity of L. rhamnosus was increased markedly, while that of E. coli remained low. The results show that mucosa‐associated lactobacilli can be potent stimulators of IL‐12, and thus potentially of cell‐mediated immunity, if they pass over the gut epithelial barrier and interact with cells of the gut immune system.

Intestinal colonization with enterobacteriaceae in Pakistani and Swedish hospital-delivered infants.

ABSTRACT. Rectal cultures from Swedish and Pakistani hospital‐delivered newborn infants were analysed regarding the early aquisition of enterobacteria. Swedish infants were delivered vaginally, Pakistani infants were delivered either vaginally or by caesarean section. The Swedish infants were all breast‐fed, whereas breastfeeding was incomplete and often started late among the Pakistani infants. Both groups of Pakistani infants were more rapidly colonized with enterobacteria than were the Swedish infants. Cultures from Swedish infants seldom yielded more than one kind of enterobacteria; E. coli and Klebsiefla were most frequently isolated. E. coli dominated in both Pakistani groups, but especially caesarean section delivered infants were in addition often colonized with Proteus, Klebsiella, Enterobacter or Citrobacterspecies. Breastfeeding from the first day of life reduced colonization with Klebsiella/Enterobacter/Citrobucter.The results suggest that environmental exposure, delivery mode and early feeding habits all influence the early intestinal colonization with enterobacteria.

Commensal Gram-negative bacteria prime human dendritic cells for enhanced IL-23 and IL-27 expression and enhanced Th1 development

Dendritic cells (DC) are the main orchestrators of specific immune responses. Depending on microbial information they encounter in peripheral tissues, they promote the development of Th1, Th2 or unpolarized Th cell responses. In this study we have investigated the immunomodulatory effect of non‐pathogenic intestinal Gram‐negative (Escherichia coli, Bacteroides vulgatus,Veillonella parvula, Pseudomonas aeruginosa) and Gram‐positive (Bifidobacterium adolescentis, Enteroccocus faecalis, Lactobacillus plantarum and Staphylococcus aureus) bacteria on human monocyte‐derived DC (moDC). None of the Gram‐positive bacteria (GpB) primed for Th1 or Th2 development. In contrast, despite the low levels of IL‐12 they induce, all Gram‐negative bacteria (GnB) primed moDC for enhanced Th1 cell development, which was dependent on IL‐12 and an additional unidentified cofactor. Strikingly, GnB‐matured moDC expressed elevated levels of p19 and p28 mRNA, the critical subunits of IL‐23 and IL‐27, respectively, suggesting that the IL‐12 family members may jointly be responsible for their Th1‐driving capacity. Purified majorcell wall components of either GnB or GpB did not yield Th cell profiles identical to those obtained with whole bacteria, and could not explain the induction of the IL‐12 family members nor Th1 priming by GnB. Importantly, this study gives indications that the expression of the different IL‐12 family members is dictated by different priming conditions of immature DC.

Impaired regulatory T cell function in germ-free mice

Regulatory T cells (Treg) are crucial for the maintenance of tolerance to auto‐antigens and harmless exogenous antigens. Here, we studied the role of the commensal microbiota for the development and function of Treg. CD4+CD25+ T cells were obtained from peripheral lymph nodes (PLN) and mesenteric lymph nodes (MLN) of germ‐free (GF) and conventional (conv) NMRI mice and tested for phenotype and functional suppressive capacity. CD4+CD25+ T cells from GF mice showed a lower relative gene expression of fork head box p3 gene (Foxp3) and were not as potent suppressors in vitro as CD4+CD25+ T cells from conv animals. Intracellular staining for Foxp3 and CTLA‐4 revealed proportional and regional differences in putative Treg subsets between conv and GF mice. Fewer of the CD4+CD25+ T cells in GF MLN expressed Foxp3 and CTLA‐4, while the expression of these markers was similar amongst the CD4+CD25+ T cells in PLN of conv and GF mice. The largest difference between conv and GF Treg was observed in the liver draining celiac lymph node, where GF mice had fewer putative Treg as compared to conv mice. We propose that the presence of a microbial flora favors the development of a fully functional Treg population.

Escherichia coli in Infants' Intestinal Microflora: Colonization Rate, Strain Turnover, and Virulence Gene Carriage

Colonization by Escherichia. coli in infants might have decreased in the last decades, owing to changes in hospital routines and family lifestyle. In this study, the E. coli flora was characterized in 70 healthy Swedish infants followed for the first year of life. E. coli was isolated from rectal swabs obtained at 3 d of age and quantified in fecal samples collected at 1, 2, 4, and 8 wk of age and at 6 and 12 mo of age. Strains were typed using random amplified polymorphic DNA, and their virulence factor genes were identified by multiplex PCR. Colonization by E. coli occurred late; only 61% of the infants were positive by 2 mo of age. The turnover of individual strains in the microflora was slow (1.5 strains per infant during 6 mo, 2.1 during 1 y). Environmental factors, such as siblings, pets, or feeding mode, did not influence colonization kinetics or strain turnover rate. Genes encoding type 1 fimbriae, P fimbriae, and hemolysin were significantly more common in E. coli strains persisting for at least 3 wk in the microflora than in transient strains. The P-fimbrial class III adhesin gene was more common in E. coli from children who had a cat in their homes than in E. coli from children without pets (p = 0.01); this adhesin type is common in E. coli from cats. The late colonization and low E. coli strain turnover rate suggest limited exposure of Swedish infants to E. coli. Our results confirm that P fimbriae and other virulence factors facilitate persistence of E. coli in the human colonic microflora.

Molecular Epidemiology of Mycobacterium abscessus, with Focus on Cystic Fibrosis

ABSTRACT Mycobacterium abscessus has been isolated increasingly often from the respiratory tracts of cystic fibrosis (CF) patients. It is not known whether these organisms are transmitted from person to person or acquired from environmental sources. Here, colony morphology and pulsed-field gel electrophoresis (PFGE) pattern were examined for 71 isolates of M. abscessus derived from 14 CF patients, three non-CF patients with chronic respiratory M. abscessus infection or colonization, one patient with mastoiditis, and four patients with infected wounds, as well as for six isolates identified as environmental contaminants in various clinical specimens. Contaminants and wound isolates mainly exhibited smooth colony morphology, while a rough colony phenotype was significantly associated with chronic airway colonization (P = 0.014). Rough strains may exhibit increased airway-colonizing capacity, the cause of which remains to be determined. Examination by PFGE of consecutive isolates from the same patient showed that they all represented a single strain, even in cases where both smooth and rough isolates were present. When PFGE patterns were compared, it was shown that 24 patients had unique strains, while four patients harbored strains indistinguishable by PFGE. Two of these were siblings with CF. The other two patients, one of whom had CF, had not had contact with each other or with the siblings. Our results show that most patients colonized by M. abscessus in the airways have unique strains, indicating that these strains derive from the environment and that patient-to-patient transmission rarely occurs.