epidermidis stain harboring PQG56 (spx antisense knock-down plasm

epidermidis stain harboring PQG56 (spx antisense knock-down plasmid) is increased substantially, in accordance with the phenotype in the homologous spx mutant strain of S. aureus (Pamp et al., 2006). This observation further supports that spx is an important regulator mediating the biofilm formation of S. epidermidis. Biofilm formation by S. epidermidis

is generally considered as a two-step process, including primary attachment and biofilm accumulation. To investigate selleck chemical which step is affected by Spx, we first compared the attachment ability of the Spx-overexpressing strain (harboring pQG55) and the vector control strain (harboring pQG53). In primary attachment assays, the Spx-overexpressing strain showed decreased attachment ability (about 34-fold) to polystyrene compared with the WT strain, whereas the strains carrying either pQG53 or pQG54 showed no difference in primary attachment (Fig. 3a and b). To investigate whether the transcription of atlE was affected Belnacasan by Spx, quantitative RT-PCR was performed. The result indicates that the transcriptional level of atlE in the Spx-overexpressing strain carrying pQG55 shows no difference compared with the other three strains (Fig. 3c). This indicates that Spx does not affect the attachment ability by regulating

atlE. We then compared the primary attachment on 96-well polyethylene plates between WT and ica-negative strains isolated from our previous work (Li et al., 2005), and no significant difference was found (data not shown). PIA is a key factor in the biofilm accumulation of S. epidermidis (Rupp et al., 1999). To investigate whether the production of PIA was affected by Spx, immuno-dot blot PDK4 assays were performed. The Spx-overexpressing strain was found to produce significantly less PIA compared with the vector control strain (Fig. 4a). The transcription of the icaADBC operon and its repressor icaR among different strains was further examined by quantitative RT-PCR. Decreased

icaADBC, but comparable icaR transcriptional levels were found in the Spx-overexpressing strain compared with the vector control strain (Fig. 4b and c). This result indicates that Spx affects PIA production by regulating the transcription of icaADBC in an icaR-independent manner. In B. subtilis and S. aureus, Spx plays an important role in the oxidative-stress adaptation. The B. subtilis and S. aureus spx mutant strains were hypersensitive to diamide, a thiol-specific oxidant (Nakano et al., 2003a; Pamp et al., 2006). To study whether the overexpression of Spx affects S. epidermidis in the adaptation to diamide, the diamide sensitivity of the Spx-overexpressing strain (harboring pQG55) and the control strain (harboring pQG53) was compared using disk diffusion tests.


“Although haemolytic factor is known to be a putative viru


“Although haemolytic factor is known to be a putative virulence factor contributing to pathogenicity in Candida species, its production by Candida tropicalis is poorly understood. In this study, we analysed the culture conditions under which C. tropicalis can display haemolytic

factor on plate assay and the secretion of haemolytic factor in liquid medium by clinical isolates obtained from different specimens. All the tested isolates exhibited an internal translucent ring, resembling beta-haemolysis, surrounding by a peripheral greenish-grey halo on sheep blood agar medium. Similar Ivacaftor haemolytic pattern was observed on human blood enriched medium. Furthermore, incubation either under normal atmosphere or under increased CO2 had no effect on haemolysis. Overall, no differences were observed on beta-haemolytic activities (P > 0.05) among tested isolates of C. tropicalis. In glucose-limited medium

(RPMI 1640 with 0.2% glucose), none of the isolates induced haemolysis on red blood cells. Similarly to found on plate assays, there were no significant differences (P > 0.05) in the activity of secreted haemolytic factor in liquid medium among C. tropicalis isolates. However, after growth, the number of yeast Idasanutlin in vivo cells varied among isolates revealing different efficiencies of haemolytic factor production. Haemolytic activity was neither inhibited by heat treatment (100 °C) nor by the addition of pepstatin A. The obtained results extend our knowledge about haemolytic factor production by Candida species. “
“The lungs are common sites for the occurrence of saprophytic or invasive mycosis as well as hydatid cysts. The two diseases seldom coexist, and the manifestation is seen as a fungal ball (usually aspergilloma) formed in the cavity

left behind after hydatid cystectomy. Active invasion and proliferation of the fungi in the laminated ectocyst or sometimes the pericyst of the hydatid is very unusual. We report such a unique coexistence identified in two of the Montelukast Sodium six surgically excised pulmonary hydatid cysts in the past 2 years. Both were immunocompetent males, who had presented with non-specific symptoms of cough, haemoptysis and chest pain. The septate slender hyphae of the invading fungus resembled those of Aspergillus. “
“The purpose of this study was to evaluate a preemptive approach with serum 1,3-beta-d-glucan (BDG) as a marker for treatment stratification of systemic antifungal (AF) therapy in patients with clinical suspected invasive fungal infections (IFI) at intensive care units (ICU), and the impact of surgical procedures. A total of 66 ICU patients with clinical suspected IFI were included in this retrospective analysis. Serum BDG testing was performed prior to initiation of AF treatment and in addition to routine diagnostic measures. Based on the BDG results the initial clinical decision whether or not to start systemic AF therapy was re-evaluated.

Specific

Specific AP24534 order modulatory effects of MSCs from human and experimental animal sources have

been described for the differentiation, activation, proliferation and effector functions of multiple innate and adaptive immune cells 5–11. Among these, MSC-mediated inhibition of primary T-cell activation and proliferation, suppression of DC maturation and promotion of regulatory phenotypes in monocyte/macrophages and T cells have been most extensively characterised 7–9, 11, 12. In keeping with a paracrine or “trophic” model of MSC function in vivo 13, various MSC-produced soluble mediators have been implicated in these immunomodulatory effects including IL-10, IL-6, HGF, TGF-β, chemokine ligand-2 (CCL2), HLA-G, NO, tumor necrosis factor-inducible gene 6 protein (TSG-6), prostaglandin E2 (PGE2) and kyneurenine 1, 2, 7, 9, 12, 14–16. For some such mediators, expression by MSCs may be dependent on pre-exposure to exogenous factors (e.g. IFN-γ, TNF) or on contact-dependent MSC/target cell cross-talk 2, 7, 16–19. The potential for harnessing MSC immunomodulatory

properties has been highlighted by results in pre-clinical models of autoimmunity, allotransplantation, sepsis and acute ischemic injury 1, 4, 7, 14, 15 as well as by outcomes from clinical trials in inflammatory bowel disease, graft-versus-host disease and myocardial infarction 1, 20. T cells represent the primary effector cells for common autoimmune Acetophenone diseases and for rejection of transplanted organs and tissues 21. Furthermore, activated memory T cells have been implicated CDK inhibitor in non-antigen-specific forms of tissue injury such as ischemia-reperfusion 22, 23. In

addition to the investigation of mechanisms underlying MSC inhibition of T-cell activation, attention has also been directed toward their influence on specific T-cell effector phenotypes including CD8+ CTLs and the Th1, Th2 and Treg sub-types of CD4+ T cells which may be more or less prominent in individual immune-mediated diseases 12, 24–26. In vitro and in vivo experimental evidence would suggest that MSCs are consistently suppressive of CTL- and Th1-mediated immune responses while being less inhibitory toward Th2-type responses and actively promoting Treg survival and expansion 9, 12, 27. Less well understood for each of these subsets are the relative effects of MSCs on naïve T cells undergoing primary activation compared with previously activated, or memory-phenotype, T cells. The recent description of an additional CD4+ T-cell subset, termed Th17 cells, has added further complexity to our understanding of cellular adaptive immunity 28. The Th17 effector phenotype is characterised by synthesis of a signature cytokine, IL-17A, in addition to IL-17F, IL-21, IL-22 and CCL20 29.

However, to be sure that isolated B cells do not exhibit a differ

However, to be sure that isolated B cells do not exhibit a different sensitivity to the blocking peptides, we ran the IgA and XTT assays for the optimal conditions only. The results

were not different using PBMC or B cells. Because AID is required for CSR, we examined the impact of either NF-κB p65 or the STAT3 pathways on the transcription of AID. Transcript levels for AID in naive B cells were measured by RT–PCR before or after culturing with sCD40L, IL-10 or sCD40L and IL-10. Messenger RNA encoding for AID was not observed in unstimulated naive B cells JQ1 (Fig. 6a). AID transcript production was induced optimally by addition of sCD40L and IL-10 compared to the other cell culture conditions examined here in terms of signal-enhancing ability. Blocking the NF-κB or STAT3 pathways by incubating the cells for 120 min with blocking peptides (5 µg/ml)

against pNF-κB p65 and/or pSTAT3 suppressed BIBW2992 AID induction. Thus, blocking either the NF-κB p65 or the STAT3 pathway profoundly altered the production of mRNA for AID, an enzyme strictly necessary for CSR [31]. Transcript levels for AID were higher in the presence of sCD40L, IL-10 and sCD40L + IL-10 cell culture conditions (Fig. 6b). Because the blocking peptides against pNF-κB p65 and pSTAT3 blocked AID transcription and IgA production in vitro, we next examined the impact of these peptides on IgG and IgM expression on B cells. First, we examined the B cell switch after 3, 4 and 5 days of incubation in the presence of the blocking peptides against pNF-κB p65 and pSTAT3 and activators (sCD40L + IL-10). The discrete Gefitinib B cell populations (IgD+, IgM+, IgA+, IgG+ or CD27+) were examined by flow cytometry for their individual sensitivity to the blocking peptides (Fig. 7a). Non-viable cells were excluded from the data shown by selective gating on 7-amino-actinomycin D (7AAD)-negative cells. IgM expression on B cells was not affected by the activators (sCD40L + IL-10); in contrast, IgA,

IgG and CD27 expression increased by addition of the activators (Fig. 7b). Although the activators induced CSR towards IgA (and for control – towards IgG in short-term cultures), only the IgA+ population was affected by the blocking peptides against pNF-κB p65 and pSTAT3 (Fig. 7c); this population was decreased significantly in frequency (42·645 ± 0·295 % versus 14·04 ± 0·65 %; P < 0·05) by the inhibitors which caused a return to the baseline level. In addition, we observed that the blocking peptides against pNF-κB p50 decreased IgG expression, while anti-pSTAT3 did not seem to have an effect in this experimental model (Fig. 7d). Incubation of purified blood B cells with blocking peptides against pNF-κB p65 or pSTAT3 (5 µg/ml, 120 min) induced a significant decrease in IgA production compared to the baseline level (Fig. 8a).

HLA-DR3/DR4 alleles were also analysed All T1AD patients satisfi

HLA-DR3/DR4 alleles were also analysed. All T1AD patients satisfied the American Diabetes Association (ADA) classification criteria for type 1A diabetes [37]. This project was approved by the Ethics Committee for Research Project Analysis of Hospital das Clínicas, University of São Paulo School of Medicine. All the buy PLX4032 samples were collected after the patients were provided with guidance and had signed a consent form. Autoantibodies against insulin

(IAA), glutamic acid decarboxylase (GAD65), tyrosine phosphatase (IA2) and 21-hydroxylase (21-OH) were assessed by radioimmunoassay (RSR Limited, Cardiff, UK). Autoantibodies against thyroid peroxidase (TPO) and thyroglobulin (TG) were evaluated by fluorometry (AutoDELPHIA, Turku, Finland). Anti-nuclear antibody (ANA), anti-liver/kidney microsomal

type 1 antibody (LKM1) and anti-smooth muscle (ASM) antibody were quantified using indirect immunofluorescence. Rheumatoid factor (RF) was evaluated using nephelometry, and TSH receptor autoantibody (TRAb) was assessed using iodine radioreceptor assay (RSR Limited). Genomic DNA was extracted by salting-out in blood leucocytes. The region encompassing −448 to +83 base pairs (bp) of the IL-21 gene was amplified and sequenced from samples of 309 Brazilian T1AD patients and 189 control individuals. The following selleck chemicals llc primers were used for the IL-21 gene: (−448) forward: 5′-CCTTATGACTGTCAGAGAGAACA-3′ and (+83) reverse: 5′-CTTGATTTGTGGACCAGTGTC-3′. Direct sequencing of polymerase chain

reaction (PCR)-amplified products was performed using an ABI 3100 capillary sequencer (Applied Biosystems, Tokyo, Pregnenolone Japan) with the ABI PRISM BigDye Terminator version 3·1 cycle sequencing kit (Applied Biosystems) and analysed using an ABI PRISM 3730 genetic analyser (Applied Biosystems). The following PCR amplification reaction primers were used: PTPN22 forward: 5′-TCACCAGCTTCCTCAACCACA-3′ and PTPN22 reverse: 5′-GATAATGTTGCTTCAACGGAATTT-3′. PCR amplification products were digested enzymatically using the Xcml restriction enzyme (Uniscience-New England BioLabs, Inc., Ipswich, MA, USA), which resulted in a 215-bp product for the CC variant (wild-type); 215-bp, 169-bp and 46-bp products for the CT variant; and 169-bp and 46-bp products for the TT variant. PTPN22 genotyping was performed in 689 controls and 434 T1AD patients. All results were confirmed using an RsaI restriction enzyme assay (Uniscience). HLA class II typing for DRB1 was performed using PCR with One Lambda’s SSP™ Generic HLA class II (DRB) DNA typing trays (One Lambda, Canoga Park, CA, USA).

47 Acute dialysis was associated with increased hospitalization (

47 Acute dialysis was associated with increased hospitalization (17.9 vs 9.0 days) and mortality at 90 days (14% vs 6%). In a subsequent prospective study

of 178 patients, use of the algorithm led to increased dialysis access placement and reduction in acute dialysis from 50% to 23%. Holland and Lam studied a retrospective cohort of 201 predialysis patients.48 Independent predictors of in-hospital dialysis initiation were age (OR 1.038, 95% CI: 1.011–1.065), congestive heart failure (OR 2.877, 95% CI: 1.205–6.871) and shorter predialysis follow-up time (OR 0.945, 95% CI: 0.920–0.971). Every month lost due to late referral increased the risk of in-hospital commencement BAY 80-6946 order of dialysis by 5.5%. Jones et al. reviewed the GFR decline of 726 new patients with CKD stages 3–5 referred over a 6-year period.49 The rate of decline slowed from 5.4 mL/min per 1.73 m2 per year to 0.35 mL learn more after nephrological referral. This was associated

with a reduction in blood pressure and improved survival (HR 0.55, 95% CI: 0.40–0.75). Khan et al. analysed a retrospective cohort of 109 321 US Medicaid/Medicare patients who started dialysis between 1995 and 1998.50 Only 50% had received nephrological care in the 24 months preceding dialysis. Higher mortality was associated with age and visits to generalists and non-renal specialists. Compared with patients with three or more ‘months of nephrology care’ in the 6 months preceding commencement of dialysis, mortality was increased in those with no nephrological

care in the 24 months preceding dialysis (HR 1.51), no care in the 6 months preceding dialysis (HR 1.28) and Casein kinase 1 only 1–2 ‘months of nephrology care’ in the 6 months prior to dialysis initiation (HR 1.23). Ledoux et al. defined late referral as presentation to nephrology services less than 3 months prior to starting dialysis.51 In their cohort of 62 patients, biochemical indices were worse and initial duration of hospitalization increased in late referrals, however, 4-year mortality was not increased. Lenz et al., in a retrospective study of 170 patients starting dialysis, found that 92% started with temporary venous access.52 Absence of adequate predialysis care, failure to recover from acute renal failure and non-compliance with scheduled clinic appointments were the main reasons for this. He further suggested that the velocity of eGFR loss rather a given level of renal impairment may be a better trigger for access referral. Lhotta et al. divided a cohort of 75 patients into 33 early referral and 42 late referral (defined as GFR <20 mL/min per 1.73 m2.53 Late referred patients had higher comorbidity. By univariate analysis, comorbidity and age were significantly associated with mortality, whereas in multivariate analysis, only comorbidity was associated with higher 2-year mortality.

Candida non-albicans species predominated (67 7%) The presence o

Candida non-albicans species predominated (67.7%). The presence of acute respiratory distress syndrome (ARDS) was the only independent risk factor for candidaemia development (OR, 2.93; 95% CI 1.09–7.81, P = 0.032). Mortality was 60.6% among patients with candidaemia and 22% among controls (P < 0.001). The presence of candidaemia (OR, 9.37; 95% CI 3.48–25.26, P < 0.001) and the illness severity on admission (acute physiologic and chronic health evaluation II score, OR, 1.17; 95% CI 1.12–1.24, P < 0.001) were independently associated

with mortality. Among candidaemic patients, risk factors for mortality were the severity of organ dysfunction (sequential organ failure assessment score, OR, 1.57; 95% CI 1.00–2.46, P = 0.05) and a low serum albumin level (OR, 0.74; 95% CI 0.59–0.94, P = 0.012) both of them occurred on candidaemia onset. We conclude that in critically ill patients matched for illness Tamoxifen severity

and length of ICU stay, the only independent risk factor for candidaemia was the presence of ARDS. Mortality was independently associated with acquisition of candidaemia and with the illness severity at candidaemia onset. “
“The efficacy of voriconazole (VRC) was evaluated against two strains of each of the two most common species causing sporotrichosis, Sporothrix schenckii sensu stricto and Sporothrix brasiliensis, using a murine model of disseminated infection. Voriconazole was administered at doses of 20 or 40 mg kg−1 per day by gavage. The drug showed some efficacy, especially at 40 mg kg−1 per day, in prolonging the survival and reducing fungal load in spleen and HDAC inhibitor liver in mice infected with S. schenckii, whereas in animals infected with S. brasiliensis the drug did not work. “
“Rapid differentiation of Candida albicans from non-C. albicans species in direct clinical samples is crucial to optimise empirical antifungal therapy at an early stage, which can lead to the reduction in caspofungin usage with an overall cost saving. Traditional phenotypic methods are time-consuming ADP ribosylation factor and difficult to accurately differentiate Candida albicans from non-C. albicans species.

There is an urgent clinical need for a rapid, sensitive and specific method for the differentiation of Candida albicans from non-C. albicans species in clinical specimens. In this study, we established a protocol for the application of a fluorescent in situ hybridisation (FISH) assay on different clinical samples, and analysed the effectiveness of this protocol for discriminating these organisms without prior cultivation. The FISH protocol for differentiating C. albicans from non-C. albicans species showed 95% sensitivity and 100% specificity. The positive predictive value was 100% and the negative predictive value was 94% compared with results obtained using traditional methods. Three clinical samples were FISH negative and culture positive, the percentage of false negatives with FISH was 4.0%.

hADSCs may play a key role in nerve regeneration by acting primar

hADSCs may play a key role in nerve regeneration by acting primarily as support for local neurotrophic mediation and modulation of nerve growth rather than that of a primary neuronal differentiation agent. © 2013 Wiley Periodicals, Inc. Microsurgery 34:324–330, 2014. “
“Microsurgical

revascularized fibula graft is a standard for the reconstruction of mandible or maxilla after major resection. Usually, screwed implants are inserted as a second procedure for dental rehabilitation. A lot has been published about the advantages of vascularized bone grafts, but PF-02341066 mw until now there is only little information about long-term viability of inserted bone grafts. In this study, previously inserted vascularized fibula bone grafts were examined histologically. Bone biopsies were taken during dental implant insertion procedure in average of 19 months after insertion of bone grafts from 10 patients. All bone biopsies showed partially or totally necrotic bone, although clinical examination and postoperative monitoring of the revascularized bone remained

unremarkable. The results of histological examination are surprising, due to the fact of previous insertion of a vascularized bone graft and pretended osseointegration of inserted dental implants with satisfying primary stability. Therefore, one would expect vital bone. For better understanding how much viability is really necessary for sufficient remodeling of HDAC assay inserted bone grafts for adequate functional load, further studies should be performed. © 2011 Wiley-Liss, Inc. Microsurgery, 2011. “
“Background: The Iraq and Afghanistan Wars have presented military reconstructive surgeons with a high volume of challenging extremity injuries. In recent years, a number of upper and

lower extremity injuries requiring multiple tissue transfers for multiple limb salvages in the same casualty have been encountered. Our group will discuss the microsurgical challenges, algorithms, and success and complication rates for this cohort of war injured patients. Methods: during All consecutive limb salvage cases requiring free flaps from 2003 to 2012 were reviewed. Cases involving simultaneous free tissue transfers were identified. Data collected included success rates and complications with comparisons made between the single and multiple free-flap limb salvage cohorts. Results: Seventy-four free flap limb salvage cases were performed over the 10-year period. Of these cases, four patients received two free flaps to separate upper and lower extremity injuries for limb salvage within a single operative setting. The complication rate was 63%, which was significantly higher than those cases in which a single microvascular anastomosis was performed (26%, p = 0.046). However, the higher complication rate did not increase the flap or limb salvage failure rates (p = 0.892 and 0.626).

The distribution of alleles in HIV-1 infected Japanese was simila

The distribution of alleles in HIV-1 infected Japanese was similar to that of the general Japanese population described above (data not shown). We then compared the level of pVL in terms of presence or absence of individual class I alleles (Table 1), and found that five alleles (HLA-A20, B07, B54, Cw01 PD-0332991 supplier and Cw15) were associated with lower or

larger pVL, (P < 0.05 by Fisher's exact probability test). However, after determining q-values (20) none of the associations remained significant, indicating that there are no strongly protective or detrimental alleles in this unique Asian population. Notably, in this cross-sectional analysis, expression of HLA-B51, which is the third most beneficial allele after B57 and B27 in Caucasians (7, 22), proved to be not at all protective in Japan; likewise, HLA-A11, A26 and Cw14, which have also been reported to be protective

in the USA in a study which controlled for ethnicity (7), did not show any protective effects in Japanese, either. Taken together, these results indicate that alleles which have protective effects in a given population do not necessarily behave similarly in other populations. An HLA supertype is defined as a group of class I alleles sharing a similar peptide binding motif, thereby being able to present the same CTL epitopes (23). Some HLA class I supertypes have been reported to be ALK inhibitor clinical trial associated with pVL in the USA: (B7s with larger pVL, and B27s/B58s with lower pVL) (24). We looked for such associations in the Japanese population by classifying alleles observed in our cohort into eight supertypes according to the literature (i.e., A1s, A2s, A3s, A24s, B7s, B27s, B44s, B62s) (23), and found that there were no significant associations between level of pVL and expression of particular class I supertypes in the Japanese population (data not shown). This finding may be due to the Japanese lacking HLA-B27/B57, which are major contributors to the protective supertypes in the USA (24). We further assessed the

impact on pVL of the Bw4/Bw6 motif of HLA class I molecules, which are known to act as ligands of KIR on natural killer cells and to modulate their activity (25, 26). Homozygosity for Bw6 motif has been reported to be associated with rapid disease progression, Amrubicin whereas the subtype of Bw4, which is carried by various alleles including HLA-B27/B57, is associated with slow disease progression (27, 28). However, there was no difference in the level of pVL between Bw4 and Bw6 homozygotes in the Japanese population (median: 26 000 vs. 20 500 RNA copies/ml, P= 0.976, Fig. 2), indicating that the findings reported from the USA cannot reliably be extended to other populations. In the cross-sectional analyses, we did not find any associations between the level of pVL and expression of individual class I alleles, supertypes or Bw motifs in this unique Asian population.

While the aetiology of IBD is not known, it is well established t

While the aetiology of IBD is not known, it is well established that endogenous bacteria, their components and/or antigenic products have a prevailing role in the initiation selleck chemical and perpetuation of the chronic inflammatory response. Indeed, in these genetically susceptible individuals

there is loss of immune tolerance for commensal faecal bacteria and their antigens and a bacteria-specific mucosal and systemic immune response ensues subsequently [4]. In several animal models it has been demonstrated that genetically susceptible animals remain disease-free in a germ-free (axenic) state, but will develop rapid-onset chronic intestinal inflammation when associated with Akt phosphorylation normal endogenous microflora [5–7]. We have demonstrated previously that the acquisition of commensal faecal bacteria in pre-weaned neonatal wild-type mice caused a transient release of cytokines, which was important subsequently for the establishment of tolerance to the individual endogenous microflora later in life [8]. Nevertheless, the intestinal immune and injury response and the systemic response to faecal bacteria and antigen exposure to a sterile intestinal lumen of a healthy post-weaned animal with a mature immune

system are not understood clearly. Understanding the natural immune and injury response in the normal and immune competent animal can

be key to understanding the disease state. We thus examined the effects of normal faecal bacteria Endonuclease and antigen exposure on the intestinal mucosal and systemic immune system in wild-type axenic mice. Experiments were performed in two different mouse strains. Axenic Swiss Webster mice were purchased initially from Taconic Farm (Germantown, NY, USA) and were bred at the University of Alberta in specific sterile isolator bubbles. Axenic 129/SvEv mice were purchased from the Gnotobiotic Core Facility at North Carolina State University. The mice in this experiment were used at approximately 15 weeks of age. Results from analyses performed in both mouse strains had identical outcomes. Faecal material was collected from 129/SvEv mice housed under conventional conditions. For each preparation, 20 fresh faecal pellets were mashed into 3 ml of sterile distilled water. Axenic mice were removed from the sterile environment and 100 µl of this faecal slurry was given orally to the mice with a blue tip (Fisherbrand® General Purpose Redi-Tip™; Fisher Scientific, Ontario, Canada). Mice were forced to swallow by blocking their nasal airways temporarily, forcing the mice to gulp. An additional 100 µl was spread over their abdominal skin. Mice were held subsequently under conventional housing conditions.