The commensal microbiome is associated with anti-PD-1 efficacy in metastatic melanoma patients.

Anti-PD-1-based immunotherapy has had a major impact on cancer treatment but has only benefited a subset of patients. Among the variables that could contribute to interpatient heterogeneity is differential composition of the patients' microbiome, which has been shown to affect antitumor immunity and immunotherapy efficacy in preclinical mouse models. We analyzed baseline stool samples from metastatic melanoma patients before immunotherapy treatment, through an integration of 16S ribosomal RNA gene sequencing, metagenomic shotgun sequencing, and quantitative polymerase chain reaction for selected bacteria. A significant association was observed between commensal microbial composition and clinical response. Bacterial species more abundant in responders included Bifidobacterium longum, Collinsella aerofaciens, and Enterococcus faecium. Reconstitution of germ-free mice with fecal material from responding patients could lead to improved tumor control, augmented T cell responses, and greater efficacy of anti-PD-L1 therapy. Our results suggest that the commensal microbiome may have a mechanistic impact on antitumor immunity in human cancer patients.

Enterococcus faecium FC-K Derived from Kimchi Is a Probiotic Strain That Shows Anti-Allergic Activity.

A rise in the occurrence of allergic diseases is attributed to the dysregulated balance of type 1/type 2 immunity, where type 2 T-helper (Th2) cells predominate over type 1 T-helper (Th1) cells, leading to an abnormally increased production of IgE in response to unharmful antigens. Kimchi, a traditional Korean fermented food, is a rich source of beneficial lactic acid bacteria. In this study, we investigated the ability of Enterococcus faecium FC-K derived from kimchi to induce type I immunity in the presence of Th2 polarizing conditions in vitro and in vivo. Stimulation of mouse peritoneal macrophages with E. faecium FC-K induced the production of tumor necrosis factor alpha, interleukin (IL)-6, and IL-12. Under the in vitro Th2 conditions in which splenic T cells were activated in the presence of IL-4, E. faecium FC-K enhanced the ability of T cells to produce interferon (IFN)-γ. Using the ovalbumin (OVA)-induced allergy model, male BALB/c mice receiving E. faecium FC-K reduced the serum level of total IgE, but not that of OVA-specific IgE. Furthermore, the population of activated splenic B cells during OVA immunization was decreased in E. faecium FC-K-treated mice, accounting for a reduction of total IgE in the serum. Restimulating splenocytes from OVA-immunized mice with OVA ex vivo resulted in an increased production of IFN-γ, with no effect on IL-4, in E. faecium FC-Ktreated mice. These observations provide the evidence that E. faecium FC-K can be a beneficial probiotic strain that can modulate the Th2-mediated pathologic response.

Using In Vitro Immunomodulatory Properties of Lactic Acid Bacteria for Selection of Probiotics against Salmonella Infection in Broiler Chicks.

Poultry is known to be a major reservoir of Salmonella. The use of lactic acid bacteria has become one of successful strategies to control Salmonella in poultry. The purpose of this study was to select lactic acid bacteria strains by their in vitro immunomodulatory properties for potential use as probiotics against Salmonella infection in broiler chicks. Among 101 isolated lactic acid bacteria strains, 13 strains effectively survived under acidic (pH 2.5) and bile salt (ranging from 0.1% to 1.0%) conditions, effectively inhibited growth of 6 pathogens, and adhered to Caco-2 cells. However, their in vitro immunomodulatory activities differed significantly. Finally, three strains with higher in vitro immunomodulatory properties (Lactobacillus plantarum PZ01, Lactobacillus salivarius JM32 and Pediococcus acidilactici JH231) and three strains with lower in vitro immunomodulatory activities (Enterococcus faecium JS11, Lactobacillus salivarius JK22 and Lactobacillus salivarius JM2A1) were compared for their inhibitory effects on Salmonella adhesion and invasion to Caco-2 cells in vitro and their antimicrobial effects in vivo. The former three strains inhibited Salmonella adhesion and invasion to Caco-2 cells in vitro, reduced the number of Salmonella in intestinal content, spleen and liver, reduced the levels of lipopolysaccharide-induced TNF-α factor (LITAF), IL-1ß, IL-6 and IL-12 in serum and increased the level of IL-10 in serum during a challenge study in vivo more efficiently than the latter three strains. These results suggest that in vitro immunomodulatory activities could be used as additional parameters to select more effective probiotics as feed supplements for poultry.

Evaluation of bacterial infiltration in teeth with apical barrier of MTA: an ex-vivo study

Objective: to evaluate the ability of different MTA thicknesses (3 and 5 mm), with or without conventional fillings, in preventing bacterial infiltration of Enterococcus faecalis in teeth with incompletely formed apices. Material and Methods: apical barriers were created which 3- and 5-mm-thick of MTA with and without conventional filling of the remaining root. 48 single-rooted human teeth were divided into four experimental groups (n=10) and two controls groups (n=8). After being mounted on a testing apparatus, Enterococcus faecalis was used as an indicator of bacterial infiltration. Inoculations were renewed every 3 days during a period of 60 days, and infiltration was assessed daily by checking the culture medium for turbidity. The results were analyzed using descriptive and analytical statistics (survival analysis). The level of significance was set at 5%. The tests were performed using the software GraphPad Prism 5.00. Results: the positive control group infiltrated within 24 hours, while the negative control group, no infiltrated. The Long-rank test did not demonstrate the existence of differences between groups. However, the Long-rank test for tendencies showed of a tendency to retard the infiltration. Conclusion: none of the MTA barriers were effective in preventing bacterial infiltration. The gutta-percha filling of the root segment in association with the 5-mm apical barrier of MTA showed a tendency to delay bacterial infiltration.

Immunomodulatory properties of Enterococcus faecium JWS 833 isolated from duck intestinal tract and suppression of Listeria monocytogenes infection.

The aim of this study was to evaluate the immunomodulatory properties of Enterococcus faecium JWS 833 (JWS 833) isolated from duck intestine and compare them to those of Lactobacillus rhamnosus GG (LGG), a proven immunity-enhancing probiotic. To investigate the immune-enhancing properties of JWS 833, production of nitric oxide (NO) and cytokines was measured in mouse peritoneal macrophages. In addition, a Listeria monocytogenes challenge model was used in the assessment. It was found that heat-killed JWS 833 stimulates mouse peritoneal macrophages to produce NO, interleukin-1 ß (IL-1ß) and tumor necrosis factor-α (TNF-α) and that oral administration of viable JWS833 enhances NO, IL-1ß and TNF-α synthesis upon L. monocytogenes challenge. Moreover, mice fed with JWS 833 were partially protected against lethal challenge with L. monocytogenes. JWS 833 strain has significantly greater immunostimulatory properties than LGG. Moreover, JWS 833 strain partially protects mice against lethal challenge with L. monocytogenes. JWS 833, a novel strain of E. faecium isolated from duck intestine, is potentially a useful feed supplement for controlling pathogens and enhancing host immune responses.

The acute-phase response impairs host defence against Enterococcus faecium peritonitis.

Enterococcus faecium is an emerging pathogen that causes infections in hospitalized patients with various co-morbid diseases. These underlying diseases are often associated with an acute-phase response that renders patients vulnerable to nosocomial infections. To study the influence of the acute-phase response induced by sterile tissue injury on host defence against E. faecium, mice were injected subcutaneously with either turpentine or casein 1 day before intraperitoneal infection with E. faecium. Control mice were subcutaneously injected with saline or sodium bicarbonate, respectively. Turpentine and casein induced an acute-phase response as reflected by increases in the plasma concentrations of interleukin-6, serum amyloid P and C3. A pre-existent acute-phase response in mice was associated with a strongly reduced capacity to clear E. faecium, resulting in prolonged bacteraemia for several days. The inflammatory response to E. faecium was impaired in mice with an acute-phase response, as shown by reduced capacity to mount a neutrophilic leucocytosis in peripheral blood and by decreased local cytokine concentrations. These data indicate that the acute-phase response impairs host defence against E. faecium, suggesting that this condition may contribute to the increased vulnerability of critically ill patients to enterococcal infections.

The immunomodulatory effect of lactulose on Enterococcus faecium fed preruminant calves.

Prebiotics and probiotics could represent an effective alternative to the use of synthetic antibiotics in nutrition. The mechanisms by which prebiotics affect the immune system have not yet been investigated in detail. Most effects have been attributed to increases in the innate and acquired immune responses. This study was conducted to elucidate the long-term effects of orally administered lactulose on the immune response in the intestinal tract of probiotic-fed calves. Preruminant calves were randomized to 3 feeding groups: milk replacer containing 1) no lactulose, 2) 1% lactulose, or 3) 3% lactulose. All 3 milk replacers contained 10(9) cfu Enterococcus faecium/kg. Messenger RNA expression of different cell activation markers, pro- and antiinflammatory cytokines, and IgA Fc receptor was investigated in the ileum, mesenterial lymph node, spleen, and white blood cells. A significantly greater number of blood lymphocytes were detected in the 3% lactulose group (P = 0.02) than in the control group. The expression results in male calves indicated that the transcription of IgA Fc receptor in the ileal mucosa of the 1% lactulose treatment group increased significantly (P = 0.04) and also tended to increase in the 3% lactulose group (P = 0.07). Furthermore, decreases in IL-10 and interferon-gamma mRNA expression were observed in the ileum (P = 0.04). The CD4-presenting lymphocytes were decreased significantly in the ileum (P = 0.04) and mesenteric lymph node (P = 0.01), whereas CD8-presenting lymphocytes were increased in the blood (P = 0.03) of females. Other proinflammatory cytokines (IL-1beta, IL-8, and tumor necrosis factor-alpha) and antiinflammatory cytokines (transforming growth factor-beta1) did not show significant differences in mRNA expression among treatment groups. The results indicate that additional lactulose feeding had an immunomodulatory effect on the composition of T-cell subsets in different immune compartments and had minor effects on pro- and antiinflammatory cytokine mRNA expression.

Neutrophils are essential for rapid clearance of Enterococcus faecium in mice.

A progressive increase in infections with multiresistant Enterococcus faecium has been reported, especially in cancer patients and neutropenic patients. Despite its increasing importance as a nosocomial pathogen, knowledge of the pathogenesis of E. faecium infections is highly limited. In this study, we investigated the role of neutrophils during peritonitis with subsequent bacteremia caused by E. faecium. Therefore, we depleted neutrophils by intraperitoneal injections of monoclonal antibody RB6-8C5. Mice were followed for 5 days, and the enterococcal outgrowth and inflammatory response were compared between neutropenic mice and immunoglobulin G-injected control mice. Neutropenic mice demonstrated a severe delay in enterococcal clearance from all cultured organs (peritoneal fluid, blood, and lung and liver tissue). In particular, neutropenic mice remained bacteremic for up to 3 days, whereas all nonneutropenic mice had cleared the bacteria from circulation by 2 days. Furthermore, neutropenic mice displayed elevated peritoneal cytokine and chemokine levels 1 day after the infection and attracted fewer macrophages into the peritoneal cavity. In the circulation, a prolonged elevation of tumor necrosis factor alpha, interleukin-6, and the acute-phase proteins serum amyloid A and complement 3 were measured in neutropenic mice. In conclusion, attraction of neutrophils to the primary site of E. faecium infection is important for a rapid clearance of this bacterium, thereby attenuating a systemic inflammatory response.

TLR2-dependent MyD88 signaling contributes to early host defense in murine Enterococcus faecium peritonitis.

The incidence of infections with Enterococcus faecium is increasing worldwide. TLRs have been implicated in the recognition of pathogens and the initiation of an adequate innate immune response. We here sought to determine the roles of MyD88, the common adaptor protein involved in TLR signaling, TLR2, TLR4, and CD14 in host defense against E. faecium peritonitis. MyD88 knockout (KO) mice demonstrated an impaired early response to E. faecium peritonitis, as reflected by higher bacterial loads in peritoneal fluid and liver accompanied by a markedly attenuated neutrophil influx into the abdominal cavity. In vitro, not only MyD88 KO macrophages but also TLR2 KO and CD14 KO macrophages displayed a reduced responsiveness to E. faecium. In accordance, transfection of TLR2 rendered human embryonic kidney 293 cells responsive to E. faecium, which was enhanced by cotransfection of CD14. TLR2 KO mice showed higher bacterial loads in peritoneal fluid after in vivo infection with E. faecium and a diminished influx of neutrophils, whereas CD14 KO mice had an unaltered host response. E. faecium phagocytosis and killing were not affected by MyD88, TLR2, or CD14 deficiency. TLR4 did not play a role in the immune response to E. faecium in vitro or in vivo. These data suggest that MyD88 contributes to the effective clearance of E. faecium during peritonitis at least in part via TLR2 and by facilitating neutrophil recruitment to the site of the infection.

Immune modulation and expression of cytokine genes in rainbow trout Oncorhynchus mykiss upon probiotic feeding.

This study elucidates the immune modulation including the expression of cytokine genes following dietary administration of three selected probiotic bacteria--Lactobacillus rhamnosus, Enterococcus faecium and Bacillus subtilis to fish, rainbow trout Oncorhynchus mykiss. They were fed for 45 days on either a basal control diet or one of the three probiotic diets containing the specific bacteria in freeze-dried form at a density of 10(9)CFUgfeed-1. The non-specific immune parameters examined--superoxide anion production by the head kidney leukocytes and the alternate complement activity of serum was improved by probiotic feeding. Besides this, the relative gene expressions of interleukin-1beta1, tumor necrosis factor 1 and 2 and transforming growth factor-beta were up regulated in the spleen and the head kidney. The comparatively better performance of E. faecium could possibly be linked to their suitable ambient temperature conditions. Thus, probiotic bacteria delivered in feed exerts its influence on the immune system of fish, both at cellular and molecular levels.

Identification of ABC transporters in vancomycin-resistant Enterococcus faecium as potential targets for antibody therapy.

The occurrence of an outbreak of septicaemias due to vancomycin-resistant Enterococcus faecium (VRE), in Manchester, UK, provided an opportunity to examine the antibody responses in patients infected by the same strain. Immunoblotting sera from 24 cases, six of whom died, showed an immunodominant cluster of antigens at 34, 54 and 97 kDa, with a statistically significant correlate between survival and immunoglobulin G to the 34 and 97 kDa bands (P<0.05). Screening a genomic expression library of VRE with seropositive serum and peritoneal dialysate from a survivor gave a recombinant clone with two contiguous open reading frames, the derived amino acid sequences of which both showed sequence homologue with ABC transporters, with a Walker A and Walker B motif and the signature sequence LSGGQ. The first open reading frame (putative VRE ABC1) showed 57% homologue with YbxA from Bacillus subtilis. A partial sequence (putative VRE ABC2) was also obtained, in the same recombinant clone, of a second ABC transporter with 72% homologue with ybaE from B. subtilis. Affinity selection with the seropositive serum and peritoneal dialysate used to screen the library showed that the eluted antibody bound to the 97, 54, 34 and 30 kDa bands. Direct amino acid sequencing identified this as a possible ABC transporter. Rabbit antiserum against peptides representing Walker A and an area adjacent to the Walker B site cross-reacted with bands at 34, 54, 97, 110 kDa and at 30, 34 and 54 kDa respectively. This therefore appeared to be an immunodominant complex of ABC transporters of which the smallest was the 30 kDa antigen. Epitope mapping of this antigen with seropositive patients' sera delineated three linear epitopes (KVGIV, FGPKNF and RVAI). The Walker A site represented by peptide 1 (GHNGSGKSTLAKTIN), epitope RVAI represented by peptides 2 (MRRVAIAGVLAMPRE) and 3 (ELSGGQMRRVAIAGV), epitope KVGIV represented by peptide 4 (LKPIRKKVGIVFQFP), and recombinant VRE ABC1 and VRE ABC2 expressed in Escherichia coli pBAD were then used to isolate human genetically recombinant antibodies from a phage antibody display library. An assessment of the protective potential of these antibodies was carried out in a mouse model of the infection. This study suggests that an ABC transporter homologue could be a target for antibody therapy against VRE infections.

Comparación de cuatro métodos para la detección de la resistencia a la vancomicina por el enterococo / Comparison of four methods for detection of resistance to vancomycin by enterococcus

Se evaluaron en forma prospectiva 108 cepas de enterococos (83 E. faecalis, 16 E. faecium, 5 E. avium y 4 E. gallinarum) aisladas de diversas muestras clínicas en la sección de Bacteriología del Laboratorio Metropolitano en el período comprendido entre enero y julio de 1999 con la finalidad de comparar cuatro de los métodos descritos para la detección de resistencia a vancomicina in vitro en este grupo de gérmenes: difusión en disco (DD), E test (AB Biodisk, Solina, Sweden), el sistema automatizado Vitek a través de la tarjeta GPS-102 (bioMérieux, Inc., Hazelwood, Mo.), tomando como referencia el agar cerebro-corazón con 6 µ/ml de vancomicina (VSA), observándose una correlación del 93,56 por ciento, 100 por ciento y 97,52 por ciento respectivamente. Se recomienda el uso en paralelo de cualquiera de los métodos en conjunto de la VSA para la segura detección de cepas de enterococcos con sensibilidad disminuida a la vancomicina.