A novel vaccine modality is needed to generate both systemic and mucosal immunity in the respiratory tract, where pathogens are most likely to colonize and to initiate infection. Here, we demonstrated that intranasal immunization with probiotic Escherichia coli-derived membrane vesicles displaying serotype-14 pneumococcal capsule (CPS14+MVs) elicited potent IgG responses without adjuvant in mice, which were comparable and significantly superior to those of the injected CPS14+MVs and two licensed pneumococcal vaccines (conjugate and polysaccharide types), respectively. Notably, IgA class-switch recombination occurred only with intranasal CPS14+MVs immunization, resulting in robust secretory IgA (SIgA) production throughout a one-year-long-term study. Furthermore, the intranasal CPS14+MV vaccine induced both systemic and mucosal immunity regardless of mouse age at vaccination. Whereas opsonophagocytic activities were detected in sera of all immunization groups, only the intranasal CPS14+MV vaccine achieved dramatic pneumococcal clearance in the nasal cavity. Nevertheless, it failed to protect isogenic pIgR−/− mice, which are genetically impaired for SIgA translocation, from colonization of the respiratory tract. In conclusion, the present study could offer a novel vaccination strategy using chimeric probiotic E. coli MVs to provide antibody-mediated protection against pneumococcal colonization and infection.

Abstract Notch ligands present during interactions between T cells and dendritic cells (DCs) dictate cell phenotype through a myriad of effects including the induction of T cell regulation, survival, and cytokine response. The presence of Notch ligands on DCs varies with the context of the inflammatory response; Jagged-1 is constitutively expressed, whereas Delta-like 1 and Delta-like 4 are induced in response to pathogen exposure. Although Delta-like and Jagged ligands send different signals through the same Notch receptor, the role of these two ligands in peripheral T cell immunity is not clear. The goal of our studies was to determine the role of Jagged-1 in the pathogen-free inflammation induced by OVA during allergic airway disease in mice. Our studies show that a deletion in DC-expressed Jagged-1 causes a significant increase in cytokine production, resulting in increased mucus production and increased eosinophilia in the lungs of mice sensitized and challenged with OVA. We also observed that a reduction of Jagged-1 expression is correlated with increased expression of the Notch 1 receptor on the surface of CD4+ T cells in both the lung and lymph node. Through transfer studies using OT-II transgenic T cells, we demonstrate that Jagged-1 represses the expansion of CD44+CD62L+CCR7+ memory cells and promotes the expansion of CD44+CD62L− effector cells, but it has no effect on the expansion of naive cells during allergic airway disease. These data suggest that Jagged-1 may have different roles in Ag-specific T cell responses, depending on the maturity of the stimulated T cell.

Background: Intrawound vancomycin powder is effective in preventing surgical site infection after spine surgery. In a previous study, vancomycin-induced cytotoxicity in osteoblasts was investigated in vitro, and vitamin D3 was verified to be a candidate drug aiding recovery from vancomycin-induced cyto-toxicity. The treatment practices involving osteogenesis-promoting drugs vary widely. Teriparatide, an anabolic agent, highly promotes bone formation by inducing osteoblast activation, increasing bone for-mation and mineral density, and preventing vertebral fractures. Hence, teriparatide may be administered in combination with vancomycin. Methods: MC3T3-E1 cells were cultured in minimum essential medium supplemented with 10% fetal bovine serum at 37 degrees C in a humidified incubator containing 5% CO2. The experimental concentrations of vancomycin (2500, 5000, and 7500 mg/mL) were determined based on previous reports and our pre-liminary experiments. Teriparatide (100 ng/mL) was administered concomitantly to prevent cytotoxicity in osteoblasts, using pulsed vancomycin for 24 h (measured at 1, 3, and 7 days). Cell numbers and morphological changes in cells treated with vancomycin or vancomycin plus 100 ng/mL teriparatide were measured. Osteoblast differentiation was assessed using alkaline phosphatase staining, alkaline phos-phatase activity, and alizarin red S staining. Results: Teriparatide showed a recovery effect when vancomycin (7500 mg/mL) was administered only for 24 h. Microscopic examination revealed that teriparatide had a protective effect on osteoblasts exposed to 7500 mg/mL vancomycin. Addition of teriparatide led to the recovery of alkaline phosphatase staining and alizarin red staining. Conclusion: Vancomycin-induced cytotoxicity in osteoblasts could be inhibited by administering ter-iparatide concomitantly with vancomycin. (c) 2022 The Authors. Published by Elsevier B.V. on behalf of The Japanese Orthopaedic Association. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/ 4.0/).

BackgroundThe emergence of novel genomic-type clones, such as community-associated meticillin-resistant Staphylococcus aureus (MRSA) and livestock-associated MRSA, and their invasion into hospitals have become major concerns worldwide; however, little information is available regarding the prevalence of MRSA in Japan. Whole-genome sequencing (WGS) has been conducted to analyse various pathogens worldwide. Therefore, it is important to establish a genome database of clinical MRSA isolates available in Japan.AimA molecular epidemiological analysis of MRSA strains isolated from bloodstream-infected patients in a Japanese university hospital was conducted using WGS and single-nucleotide polymorphism (SNP) analysis. Additionally, through a review of patients' clinical characteristics, the effectiveness of SNP analysis as a tool for detecting silent nosocomial transmission that may be missed by other methods was evaluated in diverse settings and various time points of detection.MethodsPolymerase-chain-reaction-based staphylococcal cassette chromosome mec (SCCmec) typing was performed using 135 isolates obtained between 2014 and 2018, and WGS was performed using 88 isolates obtained between 2015 and 2017.FindingsSCCmec type II strains, prevalent in 2014, became rare in 2018, whereas the prevalence of SCCmec type IV strains increased from 18.75% to 83.87% of the population, and became the dominant clones. Clonal complex (CC) 5 CC8 and CC1 were detected between 2015 and 2017, with CC1 being dominant. In 88 cases, SNP analyses revealed nosocomial transmissions among 20 patients which involved highly homologous strains.ConclusionsRoutine monitoring of MRSA by whole-genome analysis is effective not only for gaining knowledge regarding molecular epidemiology, but also for detecting silent nosocomial transmission.

In Mycobacterium avium infections, macrophages play a critical role in the host defense response. Apoptosis inhibitor of macrophage (AIM), also known as CD5L, may represent a novel supportive therapy against various diseases, including metabolic syndrome and infectious diseases. The mechanisms of AIM include modulating lipid metabolism in macrophages and other host cells. We investigated the role of AIM in M. avium infections in vitro and in vivo. In a mouse model of M. avium pneumonia, foamy macrophages were induced 6 wk after infection. The bacteria localized in these macrophages. Flow cytometric analysis also confirmed that the percentage of CD11chighMHCclassIIhigh interstitial and alveolar macrophages, a cell surface marker defined as foamy macrophages, increased significantly after infection. AIM in alveolar lavage fluid and serum gradually increased after infection. Administration of recombinant AIM significantly increased the number of bacteria in the lungs of mice, accompanied by the induction of inflammatory cytokine and iNOS expression. In mouse bone marrow-derived macrophages, the mRNA expression of AIM after M. avium infection and the amount of AIM in the supernatant increased prior to the increase in intracellular bacteria. Infected cells treated with anti-AIM Abs had fewer bacteria and a higher percentage of apoptosis-positive cells than infected cells treated with isotype control Abs. Finally, AIM in the sera of patients with M. avium-pulmonary disease was measured and was significantly higher than in healthy volunteers. This suggests that AIM production is enhanced in M. avium-infected macrophages, increasing macrophage resistance to apoptosis and providing a possible site for bacterial growth.

In Japan, Staphylococcal cassette chromosome mec (SCCmec) type IV methicillin-resistant Staphylococcus aureus (MRSA) is an increasingly prominent cause of bacteremia, but the virulence of most of these strains is unclear. We aimed to investigate the relationship between the molecular characteristics and the ability to form biofilms in the presence of blood plasma (plasma-biofilms) of MRSA strains isolated from bloodstream infections. In this study, the molecular characteristics and biofilms of MRSA strains isolated from blood cultures between 2015 and 2017 were analyzed by PCR-based assays, crystal violet staining, and confocal reflection microscopy methods. Among the 90 MRSA isolates, the detection rate of SCCmec type II clones decreased from 60.7 to 20.6%. The SCCmec type IV clone replaced the SCCmec type II clone as the dominant clone, with a detection rate increasing from 32.1 to 73.5%. The plasma-biofilm formation ability of the SCCmec type IV clone was higher than the SCCmec type II clone and even higher in strains harboring the cna or arcA genes. Plasma-biofilms, mainly composed of proteins, were formed quickly and strongly. Our study demonstrated the increased plasma-biofilm formation ability of SCCmec type IV strains.

Pneumococcal conjugate vaccination (PCV) may prevent influenza-related pneumonia, including Streptococcus pneumoniae pneumonia. To investigate PCV efficacy against secondary pneumococcal pneumonia following influenza, PCV was administered intramuscularly 2 and 5 weeks before S. pneumoniae serotype-3 colonization of murine nasopharynges followed by intranasal challenge with a sublethal dose of influenza A virus. Bacterial and viral loads, including innate immune responses were compared across conditions. PCV vaccination improved the survival of mice with secondary pneumococcal pneumonia and significantly reduced the pulmonary bacterial burden. Increased monocyte/macrophage influx into the lungs, alleviated loss of alveolar macrophages and decreased neutrophil influx into the lungs occurred in PCV-treated mice irrespective of pneumococcal colonization. Higher monocyte chemoattractant protein 1 levels and lower levels of CXCL1, interferon-γ, interleukin-17A, and IL-10, were detected in PCV-treated mice. Additionally, PCV treatment activated the macrophage intracellular killing of S. pneumoniae. Collectively, PCV potentially modulates the host’s innate immunity and specific antibodies induction. Macrophage-related innate immunity should be further explored to elucidate the efficacy and mechanisms of PCV versus influenza-related life-threatening diseases.

Streptococcus pneumoniae may colonize the nasopharynx, and as pneumococcal colonization causes invasive diseases and the subsequent transmission, reducing bacterial burden in the nasal cavity is critical. Hochu-ekki-to (TJ-41) is a traditional Japanese herbal medicine that exerts immunomodulatory effects in host cells. In this study, we investigated the potency of TJ-41 in modulating pneumococcal colonization clearance by activating host immunity. Mice, intranasally inoculated with pneumococci, were treated orally with TJ-41. During colonization, TJ-41 treatment significantly reduced pneumococcal burden and increased macrophage population in the nasopharynx. Furthermore, interleukin 17A production was significantly enhanced after TJ-41 treatment. In vitro experiment using nasal-derived cells revealed that pneumococcal antigen exposure upregulated the transcription of interleukin 17A in the TJ-41-treated group compared with that in the control group. Macrophages activated by killed bacteria were significantly increased in the presence of TJ-41 in an interleukin 17A-dependent manner. Moreover, TJ-41 enhanced phagocytosis, inhibited bacterial growth, and improved the antigen-presenting capacity of macrophages. Our results demonstrate that TJ-41 accelerates the clearance of pneumococcal nasopharyngeal colonization via macrophage activation. Subsequent production of interleukin 17A provides an additional benefit to effector cells.

IL-17 plays a critical role in the immunological control of various infectious diseases; its function has been investigated in the removal of both extracellular and intracellular bacteria. Our group previously revealed the importance of IL-17 in neutrophil migration following Legionella infection by using IL-17AF knockout mice; however, aside from neutrophil infiltration, alternative causes for the reduced survival of these mice have not been characterized. In this study, we found that γδ T cells in IL-17AF knockout mice were markedly increased and produced the cytotoxic substances granzyme B and perforin. Moreover, the elimination of γδ T cells from these mice, via an anti-TCRδ Ab, caused a substantial reduction in the level of lactate dehydrogenase in bronchoalveolar lavage fluid, indicating that γδ T cells contribute to lung tissue damage. Moreover, although cells lysed by cytotoxic substances are typically eliminated by phagocytic cells, in IL-17AF knockout mice, lung homeostasis was not maintained because of a decrease in phagocytic cells that impaired the clearance of dead cells. Our results indicate that increased γδ T cells in IL-17AF knockout mice help eliminate Legionella by releasing cytotoxic substances and lysing infected cells; however, this results in tissue damage due to insufficient removal of dead cells by phagocytic cells. This study enhances our understanding of the protective response against Legionella and provides insights into γδ T cell–mediated protective immunity against various infectious diseases.

The AP-1 transcription factor JunB plays crucial roles in multiple biological processes, including placental formation and bone homeostasis. We recently reported that JunB is essential for development of Th17 cells, and thus Junb-deficient mice are resistant to experimental autoimmune encephalomyelitis. However, the role of JunB in CD4+ T cells under other inflammatory disease conditions is unknown. Here we show that mice lacking JunB in CD4+ T cells (Junbfl/flCd4-Cre mice) were more susceptible to dextran sulfate sodium (DSS)-induced colitis because of impaired development of regulatory T (Treg) cells. Production of interleukin (IL)-2 and expression of CD25, a high affinity IL-2 receptor component, were decreased in Junb-deficient CD4+ T cells in vitro and in vivo. Naive CD4+ T cells from Junbfl/flCd4-Cre mice failed to differentiate into Treg cells in the absence of exogenously added IL-2 in vitro. A mixed bone marrow transfer experiment revealed that defective Treg development of Junb-deficient CD4+ T cells was not rescued by co-transferred wild-type cells, indicating a significance of the cell-intrinsic defect. Injection of IL-2-anti-IL-2 antibody complexes induced expansion of Treg cells and alleviated DSS-induced colitis in Junbfl/flCd4-Cre mice. Thus JunB plays a crucial role in the development of Treg cells by facilitating IL-2 signaling.

In light of the increasing number of clinical cases resistant to traditional monotherapies and the lack of novel antimicrobial agents, combination therapy is an appealing solution for the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections. Here, we evaluated the efficacy of anti-MRSA agents, such as vancomycin (VAN), daptomycin (DAP), and linezolid (LZD), in conjunction with 13 beta-lactams and non-beta-lactams. We assessed the in vitro activities of the various combinations against 40 MRSA strains based on the maximum synergistic effect (MSE), an index calculated from the MIC change with a combination agent. Nearly all the anti-MRSA agents, which were combined with beta-lactams as well as VAN and DAP, showed a synergistic effect with arbekacin. VAN also exhibited varying degrees of synergy depending on the type of beta-lactam, whereas DAP and LZD showed similar synergy with different beta-lactams. These effects were confirmed by antibiotic kill curves, except for the apparent interaction between LZD and beta-lactams. The MSE results were analyzed according to strain characteristics including susceptibility to combination agents, staphylococcal cassette chromosome mec type, and presence of the blaZ gene; however, no obvious correlations were observed. In a fluorescence binding assay, the fluorescence intensity of boron-dipyrromethene (BODIPY)-VAN decreased, whereas that of BODIPY-DAP increased in combination with a beta-lactam agent. These findings suggest that beta-lactam combinations are promising treatment options for MRSA infections and that the type of beta-lactam combined with VAN is important for the synergistic effect.

Notch ligands are present during the interactions between T cells and dendritic cells (DC) and induce a myriad of effects that facilitate the activation of T cells, including the induction of T cell regulation, survival, and cytokine production. Although the ligands Delta-like 4 and Delta-like 1 are expressed as a function of DC activation, the notch ligand Jagged-1 is constitutively expressed on DC. We sought to determine the role of Jagged-1 in the interactions between CD4 + T cells and DC. We observed that Jagged-1 regulates Foxp3 expression, and Cd11c Cre+ Jagged ff mice have an altered expression of Foxp3 in effector cells that arise as a result of infection with the mycobacterium Bacille Calmette-Guerin . The observed changes in Foxp3 expression were correlated with an increase in cytokine production from cultures of antigen-stimulated draining lymph nodes.

Abstract Background The utility of vancomycin powder to prevent surgical site infection, mainly in spinal surgery, has been widely examined, and the local administration of vancomycin powder to wounds has been reported to be effective in preventing surgical site infections after spine surgery. However, in vitro studies have shown that high local concentrations of vancomycin may inhibit osteogenesis, although it remains unclear how these high concentrations influence osteoblasts. No candidate drug has been reported to recover cytotoxicity with high concentrations of vancomycin, but we suggest that vitamin D3, which induces osteoblast proliferation, may be administrated concomitantly with vancomycin in these situations. Questions/purposes (1) Does a high concentration of vancomycin reduce viable osteoblast numbers in cell culture compared with controls? (2) Does vitamin D3 administration confer a protective effect on osteoblasts when administered with continuous vancomycin? (3) Does vitamin D3 administration confer a protective effect on osteoblasts when administered with pulsed vancomycin (24 hours of administration)? (4) Does vitamin D3 administration confer alkaline phosphatase, mineralization, and gene expression when administered with pulsed vancomycin? Methods MC3T3-E1 cells were cultured at 37° C in an α-minimum essential medium supplemented with 10% fetal bovine serum in a humidified incubator containing 5% CO2. The experimental concentrations of vancomycin (2500 μg/mL, 5000 μg/mL, and 7500 μg/mL) were determined based on previous reports and preliminary experiments. We concomitantly administered vitamin D3 (0.01 nM) to prevent cytotoxicity in osteoblasts, using two different treatments: continuous vancomycin administration (measured at 6 hours, 12 hours, 24 hours, and 72 hours) and pulsed vancomycin for 24 hours (measured at 1 days, 3 days, and 7 days). We analyzed cell numbers and morphologic changes in cells treated with vancomycin or vancomycin plus 0.01 nM vitamin D3. Osteoblast differentiation was assessed with alkaline phosphatase staining, alkaline phosphatase activity, and Alizarin red S staining. Results The number of cells was reduced at 6 hours, 24 hours, 48 hours, and 72 hours in response to continuous vancomycin administration at 7500 µg/mL (at 72 hours, control 14.6 × 104 cells/mL ± 0.260 × 104 cells/mL, vancomycin at 0.917 × 104 cells/mL ± 0.288 × 104 cells/mL, mean difference -13.7 × 104 cells/mL ± 0.388 × 104 cells/mL [95% CI -14.5 to -12.9]; p < 0.001). Vitamin D3 did not have a protective effect when vancomycin was administered continuously at 7500 µg/mL (at 72 hours, vancomycin alone 0.917 × 104 cells/mL ± 0.288 × 104 cells/mL, vancomycin + vitamin D3 1.67 × 104 cells/mL ± 0.310 × 104 cells/mL, mean difference 0.75 × 104 cells/mL ± 0.423 × 104 cells/mL [95% CI -0.127 to 1.63]; p = 0.09). With pulsed administration for only the first 24 hours, the number of cells was reduced at 1 day, 3 days, and 7 days at 7500 μg/mL (at 7 days, control 18.6 × 104 cells/mL ± 1.29 × 104 cells/mL, vancomycin at 3.46 × 104 cells/mL ± 0.292 × 104 cells/mL, mean difference -15.1 × 104 cells/mL ±1.33 × 104 cells/mL [95% CI -17.9 to -12.4]; p < 0.001 for all). However, vitamin D3 had a recovery effect when vancomycin was administered only for 24 hours (cell number with 7500 μg/mL, day 7: vancomycin alone 3.46 × 104 cells/mL ± 0.292 × 104 cells/mL, vancomycin +vitamin D3 10.6 × 104 cells/mL ± 0.900 × 104 cells/mL, mean difference 7.13 × 104 cells/mL ± 0.946 × 104 cells/mL [95% CI 5.16 to 9.09]; p < 0.001). With the addition of vitamin D3, we observed recovery of alkaline phosphatase staining and Alizarin red staining (evidence of calcification) but no difference in the gene expression of Type I collagen (vancomycin alone 0.319 ± 0.0730, vancomycin + vitamin D3 0.511 ± 0.139, mean difference 0.192 ± 0.157 [95% CI -0.483 to 0.867]; p = 0.345), alkaline phosphatase (vancomycin alone 0.532 ± 0.0210, vancomycin + vitamin D3 0.785 ± 0.0590, mean difference 0.253 ± 0.0620 [95% CI -0.0150 to 0.521]; p = 0.0550), and cathelicidin antimicrobial peptide (vancomycin alone 0.885 ± 0.0520, vancomycin + vitamin D3 1.24 ± 0.125, mean difference 0.355 ± 0.135 [95% CI -0.0200 to 0.730]; p = 0.0580). Conclusion We found that 7500 µg/mL of vancomycin is cytotoxic to osteoblasts. Cytotoxicity could be prevented by administering vitamin D3 in combination with vancomycin. Clinical Relevance The high concentrations of vancomycin routinely used clinically raises concerns related to osteoblast cytotoxicity, which may contribute to pseudoarthrosis after spinal surgery. Thus, vitamin D3, which is frequently used to treat osteoporosis, may have efficacy as a concomitantly administered drug by inducing the proliferation of osteoblasts. These results indicate that a combination therapy of vancomycin and vitamin D3 may prevent adverse events such as osteoblast cytotoxicity.

In Legionella pneumophila infection, macrophages play a critical role in the host defense response. Metformin, an oral drug for type 2 diabetes, is attracting attention as a new supportive therapy against a variety of diseases, such as cancer and infectious diseases. The novel mechanisms for metformin actions include modulation of the effector functions of macrophages and other host immune cells. In this study, we have examined the effects of metformin on L. pneumophila infection in vitro and in vivo. Metformin treatment suppressed growth of L. pneumophila in a time- and concentration-dependent fashion in bone marrow–derived macrophages, RAW cells (mouse), and U937 cells (human). Metformin induced phosphorylation of AMP-activated protein kinase (AMPK) in L. pneumophila–infected bone marrow–derived macrophages, and the AMPK inhibitor Compound C negated metformin-mediated growth suppression. Also, metformin induced mitochondrial reactive oxygen species but not phagosomal NADPH oxidase–derived reactive oxygen species. Metformin-mediated growth suppression was mitigated in the presence of the reactive oxygen species scavenger glutathione. In a murine L. pneumophila pneumonia model, metformin treatment improved survival of mice, which was associated with a significant reduction in bacterial number in the lung. Similar to in vitro observations, induction of AMPK phosphorylation and mitochondrial ROS was demonstrated in the infected lungs of mice treated with metformin. Finally, glutathione treatment abolished metformin effects on lung bacterial clearance. Collectively, these data suggest that metformin promotes mitochondrial ROS production and AMPK signaling and enhances the bactericidal activity of macrophages, which may contribute to improved survival in L. pneumophila pneumonia.

Interleukin (IL)-17 is a key member of the Th17 cytokines and has been reported to be involved in the pathomechanisms underlying various diseases, including infectious diseases. Infections with community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) have garnered worldwide attention, and the representative USA300 strain is known to cause pneumonia in healthy people, which can be lethal. However, little is known about the role of IL-17 in CA-MRSA pneumonia. In this study, we investigated the role of IL-17 in a CA-MRSA pneumonia animal model. Mortality was higher and occurred at an earlier stage of infection in the IL-17A-knockout mice than in the wild-type (P < 0.01) and IL-17A/F-knockout mice (P < 0.05); however, no significant difference in the intrapulmonary bacterial counts was observed among the three groups of mice. Moreover, the IL-17A-knockout group showed significantly higher levels of IL-17F and granulocyte-colony stimulating factor (G-CSF) and a significantly higher neutrophil count in the bronchoalveolar lavage fluid than the other groups. These results confirmed that G-CSF expression significantly increased, and significant neutrophilic inflammation occurred under conditions of IL-17A deficiency in the murine CA-MRSA pneumonia model.

Introduction: The USA300 clone of community-associated MRSA is reported to be hypervirulent and epidemic in the United States. This clone causes a variety of diseases from lethal pneumonia to mild skin infections. We hypothesized that evolutionary diversity may exist among USA300 clones, which may link virulence traits with host responses and mortality rates. Methods: USA300 isolates from severe pneumonia (IP) and skin infection (IS) were characterized by pulsed-field gel electrophoresis (PFGE) and next-generation sequencing. Their virulence traits and host responses were compared in a lung infection model. Results: The two USA300 isolates were found to be identical in genomic analysis. Robust IL-6 production, aggregation of bacteria, and hemorrhaging were observed in IP-infected lungs, which were associated with a higher rate of mortality than that observed with strain IS. Few neutrophils were detected in the lungs infected with strain IP, even at high bacterial loads. Massive production of alpha-toxin and coagulase were evident during the early phase of IP infection, and robust gene expression of hla (alpha-toxin) and lukS-PV (Panton-Valentine leukocidin), but not coa, agrA, or rnaIII, was confirmed in vitro. Strain IP also induced strong hemolysis in red blood cells. Conclusions: The present data demonstrated latent diversity in the virulence of USA300 clones. Unknown regulatory mechanisms, probably involving a host factor(s) as a trigger, may govern the virulence expression and resultant high mortality in certain sub-clones of USA300.

OBJECTIVES:Mupirocin is a useful antibiotic against superficial skin infections. We compared the impact of mupirocin with a cephalosporin, a fluoroquinolone, an aminoglycoside, and a macrolide on planktonic cell growth and biofilm formation of methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA).MATERIALS AND METHODS:Minimum inhibitory concentration (MIC) of mupirocin was determined against S. aureus strains used in this study. Biofilm formation of S. aureus strains exposed to mupirocin was quantified by crystal violet staining assay. Moreover, biofilm structure and viability of the biofilm cells were visualized by Live/Dead staining assay. Biofilm-related gene expression was investigated by quantitative real-time PCR.RESULTS:MRSA USA300 clone was resistant to mupirocin with MIC of 1,024 mg/L, while MRSA ATCC-43300 and MSSA ATCC-29213 were susceptible with MICs of 0.03 mg/L. Planktonic cell growth of the S. aureus strains was inhibited by mupirocin in a dose-dependent manner. However, some of the low concentrations of mupirocin less than the MICs promoted biofilm formation. Confocal laser scanning microscopy of the biofilm structures and cell viabilities showed established biofilms of slightly higher cell density in the mupirocin treated groups, especially in the MRSA USA300 clone. Gene expression of RNAIII in planktonic cells and biofilms of MRSA USA300 clone showed the highest upregulation after initial exposure to sub-MIC of mupirocin followed by downregulation, whereas the other antibiotics showed various fluctuations.CONCLUSION:The results showed that subinhibitory concentrations of mupirocin promoted biofilm formation of S. aureus, in particular the MRSA USA300 clone.

Macrolides have been reported to exert a variety of effects on both host immunomodulation and repression of bacterial pathogenicity. In this study, we report that the 3',5'-cyclic diguanylic acid (c-di-GMP) signaling system, which regulates virulence in Pseudomonas aeruginosa, is affected by the macrolide azithromycin. Using DNA microarray analysis, we selected a gene encoding PA2567 related to c-di-GMP metabolism that was significantly affected by azithromycin treatment. Expression of the PA2567 gene was significantly repressed by azithromycin in a time- and dose-dependent manner, whereas no difference in PA2567 gene expression was observed in the absence of azithromycin. In-frame deletion of the PA2567 gene affected both virulence factors and the quorum-sensing system, and significantly decreased total bacteria in a mouse pneumonia model compared to the wild-type strain (P < 0.05). These results suggest that macrolides possess the ability to modulate c-di-GMP intracellular signaling in P. aeruginosa.

The Notch ligand delta-like 4 (DLL4) is known to fine-tune the CD4+ T cell cytokine response. DLL4 is expressed on the surface of antigen-presenting cells (APCs) in a MyD88-dependent manner. We found that DLL4 expression was upregulated on bone marrow progenitor cells and APCs in mice infected with BCG Mycobacterium. Transfer of DLL4+ progenitor cells from infected hosts resulted in an increase DLL4+ myeloid cells in the spleen, indicating that expression of the dll4 gene is propagated throughout hematopoiesis. We also found an increase in DLL4+ monocytes from individuals who were infected with Mycobacterium tuberculosis. In latent individuals, DLL4 expression correlated with increased cytokine production from T cells in response to PPD stimulation. Finally, antibody blockade of DLL4 reduced T cell cytokine production from naïve T cells stimulated with antigen. These results demonstrate that the Notch ligand DLL4 can influence T cell cytokine production in both humans and mice, and further reveal that expression of DLL4 is upregulated on early hematopoietic progenitors in response to chronic mycobacterial infection. These data suggest that widespread DLL4 expression may occur as a result of mycobacterial infection, and that this expression may alter CD4+ T cell responses to both previously encountered and novel antigens.

Clostridium difficile infection (CDI) is a toxin-mediated intestinal disease. Toxin A, toxin B and binary toxin are believed to be responsible for the pathogenesis of CDI, which is characterized by massive infiltration of neutrophils at the infected intestinal mucosa. IL-17 is one of the cytokines that play critical roles in several inflammatory and immunological diseases through various actions, including promoting neutrophil recruitment. The aim of this study was to examine the role of this cytokine in CDI by employing IL-17 A and F double knockout (IL-17 KO) mice for the CDI model. We demonstrated that IL-17 KO mice were more resistant to CDI than WT mice using several factors, such as diarrhoea score, weight change and survival rate. Although the bacterial numbers of C. difficile in faeces were not different, the inflammatory mediator levels at the large intestine on day 3 post-infection were attenuated in IL-17 KO mice. Finally, we showed that infiltration of neutrophils, but not macrophages, in the large intestine was significantly decreased in IL-17 KO mice compared to WT mice. In conclusion, the data demonstrate that endogenous IL-17 may be a factor determining the severity of CDI in mice. Although the mechanism is totally unknown, IL-17-mediated inflammatory responses, such as cytokine/chemokine production and neutrophil accumulation, may be plausible targets for future investigations.