2-methacryloyloxyethyl phosphorylcholine polymer treatment prevents Candida albicans biofilm formation on acrylic resin

We aimed to evaluate the effectiveness of photoreactive 2-methacryloyloxyethyl phosphorylcholine (MPC) in inhibiting Candida albicans biofilm formation on polymethyl methacrylate (PMMA) and assess its mechanism and need for re-application by evaluating its interaction with salivary mucin and durability during temperature changes. PMMA discs were used as specimens. The MPC coating was applied using the spray and cure technique for the treatment groups, whereas no coating was applied to the control. The MPC treatment (MT) groups were further differentiated based on the number of thermal cycles involved (0, 1000, 2500, and 5000). The optical density was measured to assess mucin adsorption (MA). Contact angle (CA) was calculated to evaluate surface hydrophilicity. The presence of MPC components on the PMMA surface was assessed using X-ray photoelectron spectroscopy (XPS). C. albicans biofilms were evaluated qualitatively (scanning electron microscope images) and quantitatively (colony-forming units (CFUs)). Statistical analysis was conducted using two-way analysis of variance and Tukey's multiple comparison test. MA rate and CA increased significantly in the MT groups, which exhibited significantly fewer CFUs and thinner biofilms than those of the control group. Based on the XPS, MA, and CFU evaluations, the durability and efficacy of the MPC coating were considered stable up to 2500 thermal cycles. Additionally, a significant interaction was observed between mucin concentration and MPC efficacy. The photoreactive MPC coating, which was resistant to temperature changes for approximately 3 months, effectively prevented C. albicans biofilm formation by modifying surface hydrophilicity and increasing mucin adsorption.


Porphyromonas gingivalis Outer Membrane Vesicles Stimulate Gingival Epithelial Cells to Induce Pro-Inflammatory Cytokines via the MAPK and STING Pathways

() is a keystone pathogen associated with chronic periodontitis and produces outer membrane vesicles (OMVs) that contain lipopolysaccharide (LPS), gingipains, and pathogen-derived DNA and RNA. -OMVs are involved in the pathogenesis of periodontitis. -OMV-activated pathways that induce the production of the pro-inflammatory cytokines, interleukin (IL)-6, and IL-8 in the human gingival epithelial cell line, OBA-9, were investigated. The role of mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-κB in levels of -OMV-induced pro-inflammatory cytokines was investigated using Western blot analysis and specific pathway inhibitors. -OMVs induced IL-6 and IL-8 production via the extracellular signal-regulated kinase (Erk) 1/2, c-Jun N-terminal kinase (JNK), p38 MAPK, and NF-κB signaling pathways in OBA-9 cells. In addition, the stimulator of interferon genes (STING), an essential innate immune signaling molecule, was triggered by a cytosolic pathogen DNA. -OMV-induced IL-6 and IL-8 mRNA expression and production were significantly suppressed by STING-specific small interfering RNA. Taken together, these results demonstrated that -OMV-activated Erk1/2, JNK, p38 MAPK, STING, and NF-κB signaling pathways resulting in increased IL-6 and IL-8 expression in human gingival epithelial cells. These results suggest that -OMVs may play important roles in periodontitis exacerbation by stimulating various pathways.


pruR and PA0065 Genes Are Responsible for Decreasing Antibiotic Tolerance by Autoinducer Analog-1 (AIA-1) in Pseudomonas aeruginosa


Anti-Inflammatory and Protective Effects of Juncus effusus L. Water Extract on Oral Keratinocytes

Periodontitis is a chronic inflammatory disease caused by periodontopathogenic bacteria that form biofilms in periodontal pockets. The gingival epithelium acts as the first physical barrier in fighting attacks by periodontopathogenic pathogens, such as the primary etiological agent , and various exogenous chemicals, as well as regulates the local innate immune responses. Therefore, the development of novel oral care products to inhibit inflammatory reactions caused by bacterial infection and protect the gingival epithelium is necessary. L. has generally been used as an indigenous medicine, such as a diuretic, an antipyretic, and an analgesic, in ancient practice. In this study, we examined the effects of a water extract from L. on the inhibition of the inflammatory reaction elicited by bacterial infection and protection of the oral epithelium by chemical irritation. Pretreatment of oral epithelial cells with the water extract from L. significantly reduced or its lipopolysaccharide- (LPS-) mediated production of chemokines (interleukin-8 and C-C-chemokine ligand20) in a concentration-dependent manner with comparable to or greater effects than epigallocatechin gallate and protected oral epithelial cells from injury by chemical irritants, cetylpyridinium chloride, and benzethonium chloride. Moreover, the water extract from . L. in the presence of antimicrobial agents or antifibrinolytics already used as ingredients in mouthwash could significantly reduce the production of chemokines from LPS-stimulated oral epithelial cells in a concentration-dependent manner. These findings suggest that the water extract from . L. is potentially useful for oral care to prevent oral infections, such as periodontal infections, and maintain oral epithelial function.


Autoinducer Analogs Can Provide Bactericidal Activity to Macrolides in Pseudomonas aeruginosa through Antibiotic Tolerance Reduction

Macrolide antibiotics are used in treating chronic biofilm infections despite their unsatisfactory antibacterial activity, because they display several special activities, such as modulation of the bacterial quorum sensing and immunomodulatory effects on the host. In this study, we investigated the effects of the newly synthesized quorum-sensing autoinducer analogs (AIA-1, -2) on the activity of azithromycin and clarithromycin against . In the killing assay of planktonic cells, AIA-1 and -2 enhanced the bactericidal ability of macrolides against PAO1; however, they did not affect the minimum inhibitory concentrations of macrolides. In addition, AIA-1 and -2 considerably improved the killing activity of azithromycin and clarithromycin in biofilm cells. The results indicated that AIA-1 and -2 could affect antibiotic tolerance. Moreover, the results of hydrocarbon adherence and cell membrane permeability assays suggested that AIA-1 and -2 changed bacterial cell surface hydrophobicity and accelerated the outer membrane permeability of the hydrophobic antibiotics such as azithromycin and clarithromycin. Our study demonstrated that the new combination therapy of macrolides and AIA-1 and -2 may improve the therapeutic efficacy of macrolides in the treatment of chronic biofilm infections.


Clinical Significance of Carbapenem-Tolerant Pseudomonas aeruginosa Isolated in the Respiratory Tract


Suppresive effects of 2-methacryloyloxyethyl phosphorylcholine (MPC)-polymer on the adherence of Candida species and MRSA to acrylic denture resin.


2-Methacryloyloxyethyl phosphorylcholine (MPC)-polymer suppresses an increase of oral bacteria: a single-blind, crossover clinical trial

The biocompatible 2-methacryloyloxyethyl phosphorylcholine (MPC)-polymers, which mimic a biomembrane, reduce protein adsorption and bacterial adhesion and inhibit cell attachment. The aim of this study is to clarify whether MPC-polymer can suppress the bacterial adherence in oral cavity by a crossover design. We also investigated the number of Fusobacterium nucleatum, which is the key bacterium forming dental plaque, in clinical samples. This study was a randomized, placebo-controlled, single-blind, crossover study, with two treatment periods separated by a 2-week washout period. We conducted clinical trial with 20 healthy subjects to evaluate the effect of 5% MPC-polymer mouthwash after 5 h on oral microflora. PBS was used as a control. The bacterial number in the gargling sample before and after intervention was counted by an electronic bacterial counter and a culture method. DNA amounts of total bacteria and F. nucleatum were examined by q-PCR. The numbers of total bacteria and oral streptcocci after 5 h of 5% MPC-polymer treatment significantly decreased, compared to the control group. Moreover, the DNA amounts of total bacteria and F. nucleatum significantly decreased by 5% MPC-polymer mouthwash. We suggest that MPC-polymer coating in the oral cavity may suppress the oral bacterial adherence. MPC-polymer can be a potent compound for the control of oral microflora to prevent oral infection.


The pathogenic factors from oral Streptococci for systemic diseases

International Journal of Molecular Sciences


Royal jelly inhibits Pseudomonas aeruginosa adherence and reduces excessive inflammatory responses in human epithelial cells

is a Gram-negative bacterium and causes respiratory infection especially in elderly patients. Royal jelly has been used worldwide as a traditional remedy and as a nutrient; however, the effect against is unclear. The aim of this study was to analyze antibacterial, antiadherent, and anti-inflammatory effects of royal jelly against . Wild-type strain PAO1 and clinical isolates of were used for antibacterial assay and antiadherent assay to abiotic surface and epithelial cells, which are pharynx (Detroit 562) and lung (NCI-H292) epithelial cells. In anti-inflammatory assay, epithelial cells were pretreated with royal jelly before bacterial exposure to investigate its inhibitory effect on interleukin (IL-8) and macrophage inflammatory protein-3/CCL20 overproduction. Although royal jelly did not have antibacterial activity at concentration of 50% w/v, antiadherent activity was confirmed on the abiotic surface and epithelial cells under concentration of 25%. Pretreatment with royal jelly significantly inhibited overproduction of IL-8 and CCL20 from both cells. These results demonstrated that royal jelly inhibits adherence and protects epithelial cells from excessive inflammatory responses against infection. Our findings suggested that royal jelly may be a useful supplement as complementary and alternative medicine for preventing respiratory infection caused by .


Antibiofilm and Anti-Inflammatory Activities of+ Houttuynia cordata Decoction for Oral Care

Dental biofilms that form in the oral cavity play a critical role in the pathogenesis of several infectious oral diseases, including dental caries, periodontal disease, and oral candidiasis. (HC, Saururaceae) is a widely used traditional medicine, for both internal and external application. A decoction of dried HC leaves (dHC) has long been consumed as a health-promoting herbal tea in Japan. We have recently reported that a water solution of HC poultice ethanol extract (wHCP) exerts antimicrobial and antibiofilm effects against several important oral pathogens. It also exhibits anti-inflammatory effects on human keratinocytes. In our current study, we examined the effects of dHC on infectious oral pathogens and inflammation. Our results demonstrated that dHC exerts moderate antimicrobial effects against methicillin-resistant (MRSA) and other oral microorganisms. dHC also exhibited antibiofilm effects against MRSA, (involved in dental plaque formation), and and inhibitory effects on interleukin-8, CCL20, IP-10, and GRO productions by human oral keratinocytes stimulated by lipopolysaccharide (a cause of periodontal disease), without cytotoxic effects. This suggests that dHC exhibits multiple activities in microorganisms and host cells. dHC can be easily prepared and may be effective in preventing infectious oral diseases.

Evidence-Based Complementary and Alternative Medicine : eCAM


Effects of an autoinducer analogue on antibiotic tolerance in Pseudomonas aeruginosa

Antibiotic tolerance causes chronic, refractory and persistent infections. In order to advance the development of a new type of drug for the treatment of infectious diseases, we herein investigated the effects of a newly synthesized analogue of the Pseudomonas aeruginosa quorum-sensing autoinducer named AIA-1 ( a uto i nducer a nalogue) on antibiotic tolerance in P. aeruginosa . A P. aeruginosa luminescent strain derived from PAO1 was injected into neutropenic ICR mice and bioluminescence images were acquired for a period of time after treatments with antibiotics and AIA-1. In vitro susceptibility testing and killing assays for the planktonic and biofilm cells of PAO1 were performed using antibiotics and AIA-1. The expression of quorum-sensing-related genes was examined using real-time PCR. In vivo and in vitro assays showed that AIA-1 alone did not exert any bactericidal effects and also did not affect the MICs of antibiotics. However, the combined use of AIA-1 and antibiotics exerted markedly stronger therapeutic effects against experimental infection than antibiotics alone. The presence of AIA-1 also enhanced the killing effects of antibiotics in planktonic and biofilm cells. Although AIA-1 did not inhibit the expression of lasB and rhlA genes, which are directly regulated by quorum sensing, it clearly suppressed expression of the rpoS gene. The new compound, AIA-1, did not alter the antibiotic susceptibility of P. aeruginosa by itself; however, its addition enhanced the antibacterial activity of antibiotics. AIA-1 did not inhibit quorum sensing, but reduced the antibiotic tolerance of P. aeruginosa by suppressing rpoS gene expression.

The Journal of Antimicrobial Chemotherapy


Antibacterial and antibiofilm effects of Reuterin-Related Compounds to periodontopathic bacteria


Role of psl Genes in Antibiotic Tolerance of Adherent Pseudomonas aeruginosa.

Bacteria attached to a surface are generally more tolerant to antibiotics than their planktonic counterparts, even without the formation of a biofilm. The mechanism of antibiotic tolerance in biofilm communities is multifactorial, and the genetic background underlying this antibiotic tolerance has not yet been fully elucidated. Using transposon mutagenesis, we isolated a mutant with reduced tolerance to biapenem (relative to that of the wild type) from adherent cells. Sequencing analysis revealed a mutation in the pslL gene, which is part of the polysaccharide biosynthesis operon. The Pseudomonas aeruginosa PAO1 slBCD mutant demonstrated a 100-fold-lower survival rate during the exposure of planktonic and biofilm cells to biapenem; a similar phenotype was observed in a mouse infection model and in clinical strains. Transcriptional analysis of adherent cells revealed increased expression of both pslA and pelA, which are directly regulated by bis-(3',5')-cyclic dimeric GMP (c-di-GMP). Inactivation of wspF resulted in significantly increased tolerance to biapenem due to increased production of c-di-GMP. The loss of pslBCD in the spF mutant background abolished the biapenem-tolerant phenotype of the spF mutant, underscoring the importance of psl in biapenem tolerance. Overexpression of PA2133, which can catalyze the degradation of c-di-GMP, led to a significant reduction in biapenem tolerance in adherent cells, indicating that c-di-GMP is essential in mediating the tolerance effect. The effect of pslBCD on antibiotic tolerance was evident, with 50- and 200-fold-lower survival in the presence of ofloxacin and tobramycin, respectively. We speculate that the psl genes, which are activated by surface adherence through elevated intracellular c-di-GMP levels, confer tolerance to antimicrobials.


Synthesis and evaluation of c-di-4'-thioAMP as an artificial ligand for c-di-AMP riboswitch

Cyclic-di-adenosine monophosphate (c-di-AMP) is a bacterial second messenger that binds to an RNA receptor called riboswitch and regulates its downstream genes involving cell wall metabolism, ion transport, and spore germination. Therefore, the c-di-AMP riboswitch can be a novel target of antibiotics. In this study, we synthesized c-di-4'-thioAMP (1), which possesses a sulfur atom instead of an oxygen atom in the furanose ring, as a candidate of a bioisoster for natural c-di-AMP. The resulting 1 bound to the c-di-AMP riboswitch with a micromolar affinity (34.8M), and the phosphodiesterase resistance of 1 was >12-times higher than that of c-di-AMP. Thus, 1 can be considered to be a stable ligand against a c-di-AMP riboswitch.


RpoN Promotes Pseudomonas aeruginosa Survival in the Presence of Tobramycin.

Pseudomonas aeruginosa has developed diverse strategies to respond and adapt to antibiotic stress. Among the factors that modulate survival in the presence of antibiotics, alternative sigma factors play an important role. Here, we demonstrate that the alternative sigma factor RpoN ( (54)) promotes survival in the presence of tobramycin. The tobramycin-sensitive phenotype of logarithmic phase poN mutant cells is suppressed by the loss of the alternative sigma factor RpoS. Transcriptional analysis indicated that RpoN positively regulates the expression of RsmA, an RNA-binding protein, in the P. aeruginosa stationary growth phase in a nutrient-rich medium. The loss of RpoS led to the upregulation of gacA expression in the nutrient-limited medium-grown stationary phase cells. Conversely, in the logarithmic growth phase, the poS mutant demonstrated lower expression of gacA, underscoring a regulatory role of RpoS for GacA. Supplementation of tobramycin to stationary phase poN mutant cells grown in nutrient-rich medium resulted in decreased expression of gacA, relA, and rpoS without altering the expression of rsmA relative to wild-type PAO1. The observed downregulation of gacA and relA in the poN mutant in the presence of tobramycin could be reversed through the mutation of rpoS in the poN mutant background. The tobramycin-tolerant phenotype of the poN poS mutant logarithmic phase cells may be associated with the expression of relA, which remained unresponsive upon addition of tobramycin. The logarithmic phase poS and poN poS mutant cells demonstrated increased expression of gacA in response to tobramycin. Together, these results suggest that a complex regulatory interaction between RpoN, RpoS, the Gac/Rsm pathway, and RelA modulates the P. aeruginosa response to tobramycin.


Novel reuterin-related compounds suppress odour by periodontopathic bacteria

Halitosis is caused by volatile sulphur compounds including methyl mercaptan (CH3 SH) in the oral cavity and is a serious problem that limits interpersonal social communication. The aim of study was to evaluate the effects of reuterin-related compounds (RRCs) on halitosis-related periodontopathic bacteria in vitro. RRC-01, RRC-02 and RRC-03 (32 and 64 g ml(-1) ) in culture media containing Fusobacterium nucleatum JCM8523 and Porphyromonas gingivalis ATCC33277 were used. The effects of RRCs on CH3 SH production and detectable odour by F. nucleatum and P. gingivalis were examined by CH3 SH production assay and organoleptic test, respectively. The number of bacterial cells was also measured using an ATP assay. In P. gingivalis treated with RRCs, the expression of mgl gene, which is responsible for CH3 SH production, was examined by qRT-PCR. CH3 SH production and the score of detectable odour from F. nucleatum and P. gingivalis culture media containing RRCs were significantly lower than that without RRCs (P < 0.05). The expression of mgl gene in P. gingivalis was significantly downregulated by RRC-01 (P < 0.01), but not by RRC-02 or RRC-03. RRCs are potent oral care products for preventing halitosis via reducing CH3 SH production.


Explorative gene analysis of antibiotic tolerance-related genes in adherent and biofilm cells of Pseudomonas aeruginosa.

Antibiotic tolerance has attracted worldwide attention, as it leads to chronic, refractory, and persistent infections that are difficult to control. Bacterial biofilms are well known to be more tolerant to antibiotics compared to planktonic bacteria. We previously revealed that adherent bacteria on a solid surface also exhibited tolerance to antibiotics before forming a biofilm. However, little is known about the mechanisms of antibiotic tolerance for adherent or biofilm cells. We investigated the mechanisms of antibiotic tolerance in the biofilm life cycle using adherent and biofilm cells, and evaluated the possibility that common mechanisms operate at each stage. We constructed transposon mutants of Pseudomonas aeruginosa PAO1 and screened for low-tolerant mutants with two different methods, using adherent cells and biofilm cells. Fourteen and nine mutants exhibiting low antibiotic tolerance were detected in the adherent cells and biofilm cells, and 14 and 7 candidate genes linked to this tolerance were identified by sequencing, respectively. Eight of the 14 genes related to the antibiotic tolerance of the adherent cells were involved in biofilm formation. Two of the seven genes related to the antibiotic tolerance of biofilm cells participated in the antibiotic tolerance of adherent cells. The antibiotic tolerance of adherent cells and biofilm formation appear to be under the same regulation mechanism to promote survival in the presence of antibiotics. Antibiotic tolerance shows a complex regulation mechanism at each stage of biofilm formation.


Role of the interplay between quorum sensing regulator VqsR and the Pseudomonas quinolone signal in mediating carbapenem tolerance in Pseudomonas aeruginosa.

Pseudomonas aeruginosa coordinates its response to environmental conditions through activation of a quorum sensing (QS) system. In this study, we investigated the regulatory interaction between the QS transcriptional regulator VqsR and the Pseudomonas quinolone signal (PQS) through integration of sigma factor RpoS, and we addressed whether one of the pathways controlling carbapenem tolerance can be attributed to VqsR. We demonstrate that vqsR expression at the transcriptional level is regulated by pqsA, pqsR, and pqsE. Assessment of the transcriptional expression of vqsR, lasI, rhlI, and qscR in qsA and qsA poS mutants provided insight into pqsA- and rpoS-dependent regulation of vqsR and vqsR-controlled genes. Exogenously supplemented PQS reversed expression of vqsR and vqsR-controlled genes in the qsA mutant to wild-type levels, but failed to increase expression levels of lasI and qscR in the qsA poS mutant to levels observed in wild-type PAO1. The qsR mutant showed reduced survival when challenged with carbapenems compared to wild-type PAO1. Introduction of a pqsA mutation into the qsR mutant completely abolished its carbapenem-sensitive phenotype. We conclude that a regulatory link between PQS and vqsR exists, and that RpoS is important in their interaction. We also demonstrate that VqsR affects carbapenem tolerance.


Evaluation of the effectiveness of the potent bis-quaternary ammonium compound, 4,4'-( , -hexametylenedithio) bis (1-octylpyridinium bromide) (4DTBP-6,8) on Pseudomonas aeruginosa.

Quaternary ammonium compounds (QACs), including benzalkonium chloride (BAC) and cetylpyridinium chloride (CPC) are cationic surfactants and have been used widely as general disinfectants in the medical field due to their strong antibacterial effects and low cytotoxicity to human cells. 4,4'-( , -hexametylenedithio) bis (1-octylpyridinium bromide) (4DTBP-6,8) is one of the potent bis-QACs synthesized to improve the antimicrobial activities of mono-QACs such as BAC. This study aimed to assess the effectiveness of 4DTBP-6,8 against Pseudomonas aeruginosa, a prevalent hospital pathogen. The minimum inhibitory concentrations of 4DTBP-6,8, CPC and BAC against P. aeruginosa were measured. 4DTBP-6,8 exhibited strong antibacterial activity. We assessed the bactericidal effects of QACs against P. aeruginosa under certain conditions and their cytotoxicities in human epithelial cells using lactate dehydrogenase (LDH) release. 4DTBP-6,8 exerted excellent bactericidal effects against high concentrations of bacteria, biofilm cells and even in the presence of contaminated proteins. Cellular LDH was not released by the treatment with 4DTBP-6,8. 4DTBP-6,8 exhibited the strongest bactericidal activity against P. aeruginosa among the three QACs tested without any cytotoxicity. The potent bis-QAC, 4DTBP-6,8 has the potential to be an effective disinfectant in preventing hospital infections caused by P. aeruginosa.


A Pseudomonas aeruginosa Quorum-Sensing autoinducer analog enhances the activity of antibiotics against resistant strains.

In this study, we have investigated the effects of the newly synthesized analog of Pseudomonas aeruginosa quorum-sensing autoinducer named AIA-1 (autoinducer analog) against antibiotic-resistant bacteria. In vitro susceptibility and killing assays for P. aeruginosa PAO1 prD mutant and clinical isolates were performed by using antibiotics and AIA-1. In an in vivo assay, a luminescent carbapenem-resistant strain derived from PAO1 prD was injected into neutropenic ICR mice and bioluminescence images were acquired after the treatment with antibiotics and AIA-1. Additionally, we investigated the effects of the combination use against carbapenem-resistant Enterobacteriaceae (CRE). Using killing assays in P. aeruginosa, the survival rates in the presence of antibiotics and AIA-1 significantly decreased in comparison with those with antibiotics alone. Furthermore, dual treatment of biapenem and AIA-1 was more effective than biapenem alone in a mouse infection model. AIA-1 did not change the MICs in P. aeruginosa, suggesting that AIA-1 acts on the mechanism of antibiotic tolerance. Conversely, the MICs of antibiotics decreased in the presence of AIA-1 in some CRE strains, indicating that AIA-1 may require additional mechanism to act on CRE. In conclusion, AIA-1 may be a potent drug for clinical treatment of infections caused by antibiotic-resistant bacteria. J. Med. Invest. 64: 101-109, February, 2017.

The Journal of Medical Investigation : JMI


RpoN Modulates Carbapenem Tolerance in Pseudomonas aeruginosa through Pseudomonas Quinolone Signal and PqsE.

The ability of Pseudomonas aeruginosa to rapidly modulate its response to antibiotic stress and persist in the presence of antibiotics is closely associated with the process of cell-to-cell signaling. The alternative sigma factor RpoN ( (54)) is involved in the regulation of quorum sensing (QS) and plays an important role in the survival of stationary-phase cells in the presence of carbapenems. Here, we demonstrate that a poN mutant grown in nutrient-rich medium has increased expression of pqsA, pqsH, and pqsR throughout growth, resulting in the increased production of the Pseudomonas quinolone signal (PQS). The link between pqsA and its role in carbapenem tolerance was studied using a poN qsA mutant, in which the carbapenem-tolerant phenotype of the poN mutant was abolished. In addition, we demonstrate that another mechanism leading to carbapenem tolerance in the poN mutant is mediated through pqsE Exogenously supplied PQS abolished the biapenem-sensitive phenotype of the poN qsA mutant, and overexpression of pqsE failed to alter the susceptibility of the poN qsA mutant to biapenem. The mutations in the poN hlR mutant and the poN qsH mutant led to susceptibility to biapenem. Comparison of the changes in the expression of the genes involved in QS in wild-type PAO1 with their expression in the poN mutant and the poN mutant-derived strains demonstrated the regulatory effect of RpoN on the transcript levels of rhlR, vqsR, and rpoS The findings of this study demonstrate that RpoN negatively regulates the expression of PQS in nutrient-rich medium and provide evidence that RpoN interacts with pqsA, pqsE, pqsH, and rhlR in response to antibiotic stress.


Preventive Effects of Houttuynia cordata Extract for Oral Infectious Diseases.

Houttuynia cordata (HC) (Saururaceae) has been used internally and externally as a traditional medicine and as an herbal tea for healthcare in Japan. Our recent survey showed that HC poultice (HCP) prepared from smothering fresh leaves of HC had been frequently used for the treatment of purulent skin diseases with high effectiveness. Our experimental study also demonstrated that ethanol extract of HCP (eHCP) has antibacterial, antibiofilm, and anti-inflammatory effects against S. aureus which caused purulent skin diseases. In this study, we focused on novel effects of HCP against oral infectious diseases, such as periodontal disease and dental caries. We determined the antimicrobial and antibiofilm effects of water solution of HCP ethanol extract (wHCP) against important oral pathogens and investigated its cytotoxicity and anti-inflammatory effects on human oral epithelial cells. wHCP had moderate antimicrobial effects against some oral microorganisms and profound antibiofilm effects against Fusobacterium nucleatum, Streptococcus mutans, and Candida albicans. In addition, wHCP had no cytotoxic effects and could inhibit interleukin-8 and CCL20 productions by Porphyromonas gingivalis lipopolysaccharide-stimulated human oral keratinocytes. Our findings suggested that wHCP may be clinically useful for preventing oral infectious diseases as a mouthwash for oral care.


Anti-bacterial and anti-inflammatory effects of ethanol extract from Houttuynia cordata poultice.

Houttuynia cordata (HC) has been commonly used as many traditional remedies in local areas of Japan. Although many pharmacological activities of HC have been reported, the mechanism underlying the effect of HC remains unknown. We conducted the interview survey in Japan to verify how HC was actually used. The interview survey revealed that HC poultice (HCP) prepared from smothering fresh leaves of HC was most frequently used for the treatment of purulent skin diseases including furuncle and carbuncle with high effectiveness. Ethanol extract of HCP (eHCP) showed anti-bacterial effects against methicillin-resistant Staphylococcus aureus (MRSA), and showed an anti-biofilm activity against MRSA. eHCP showed dose-dependent inhibition of S. aureus lipoteichoic acid (LTA)-induced interleukin-8 and CCL20 production in human keratinocyte without any cytotoxicity. These results suggest that HCP is effective for skin abscess and its underlying mechanism might be the complicated multiple activities for both bacteria and host cells.

Bioscience, Biotechnology, and Biochemistry


Anti-inflammatory and protective effects of 2-methacryloyloxyethyl phosphorylcholine polymer on oral epithelial cells.

Periodontitis is a chronic inflammatory disease initiated by a microbial biofilm formed in the periodontal pocket. Gingival epithelium plays important roles as the first physical barrier to bacterial invasion and in orchestrating the innate immune reaction via toll-like receptors (TLRs), which recognize various bacterial products, and maintaining its function. Newly developed oral care products to inhibit bacterial adherence, subsequent inflammatory reaction and protect the gingival epithelium are expected. We previously reported that 2-methacryloyloxyethyl phosphorylcholine (MPC)-polymer coating decreased bacterial adhesion to human oral keratinocytes, RT-7, and mouth-rinsing with MPC-polymer inhibited the increase of oral bacteria. In this study, regarding the possibility of MPC-polymer application for preventing the adherence of periodontal pathogen, subsequent inflammatory reaction and protection of gingival epithelium, we examined the effects of MPC-polymer on the adherence of Porphyromonas gingivalis, major periodontitis-related pathogen, and TLR2 ligand to RT-7 and subsequent interleukin (IL)-8 production. MPC-polymer treatment significantly reduced P. gingivalis adherence by 44% and TLR2-mediated IL-8 production by blocking the binding of its specific-ligand in a concentration-dependent manner. Furthermore, MPC-polymer pretreatment protected RT-7 from injury by chemical irritants, cetylpyridinium chloride. These findings suggest that MPC-polymer is potentially useful for oral care to prevent oral infection and to maintain oral epithelial function. © 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2014.

Journal of Biomedical Materials Research. Part A


Effect of extracellular DNA on pyocyanin production from Pseudomonas aeruginosa


マウスを用いた緑膿菌性尿路バイオフィルム感染症モデルの作製

マウスを用いた緑膿菌性尿路バイオフィルム感染症モデルの作製


Effects of extracellular DNA from Candida albicans and pneumonia-related pathogens on Candida biofilm formation and hyphal transformation.

The aim of this study was to investigate the effects of genomic DNA purified from Candida albicans and pneumonia-related pathogens, Pseudomonas aeruginosa and Staphylococcus aureus, on in vitro biofilm formation and morphological change of 3 Candida species (C. albicans, C. glabrata, and C. tropicalis). Biofilm formation was evaluated by the crystal violet assay and colony-forming unit counts. Morphological characteristics of biofilms were evaluated by scanning electron microscopy and fluorescence microscopy. Addition of DNA at a low concentration (<1·0 μg ml(-1)) significantly increased biofilm mass of all three Candida species. In contrast, the addition of DNA at a high concentration (10 μg ml(-1)) decreased the biofilm mass. Interestingly, the formation of hyphae in a dense network of yeast cells was observed in C. albicans biofilms exposed to a low concentration of DNA (<1·0 μg ml(-1)). These findings demonstrated that extracellular DNA (eDNA) plays a crucial role in Candida biofilm formation and suggested that eDNA may induce the morphological transition from yeast to hyphal growth form during C. albicans biofilm development. A novel therapy targeting eDNA may be applicable for Candida infection to decrease biofilm formation and hyphal formation.


付着及びバイオフィルム形成緑膿菌の抗菌薬抵抗性関連遺伝子の探索

付着及びバイオフィルム形成緑膿菌の抗菌薬抵抗性関連遺伝子の探索


Role of Extracellular DNA and DNA-binding protein in biofilm formation of Streptococcus intermeius


Effects of extracellular DNA and DNA-binding protein on the development of a Streptococcus intermedius biofilm.

The aim of this study was to clarify the effects of homologous and heterologous extracellular DNAs (eDNAs) and histone-like DNA-binding protein (HLP) on Streptococcus intermedius biofilm development and rigidity. Formed biofilm mass was measured with 0·1% crystal violet staining method and observed with a scanning electron microscope. The localizations of eDNA and extracellular HLP (eHLP) in formed biofilm were detected by staining with 7-hydoxyl-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) and anti-HLP antibody without fixation, respectively. DNase I treatment (200 U ml(-1) ) markedly decreased biofilm formation and cell density in biofilms. Colocalization of eHLP and eDNA in biofilm was confirmed. The addition of eDNA (up to 1 μg ml(-1) ) purified from Strep. intermedius, other Gram-positive bacteria, Gram-negative bacteria, or human KB cells into the Strep. intermedius culture increased the biofilm mass of all tested strains of Strep. intermedius, wild-type, HLP-downregulated strain and control strains. In contrast, the addition of eDNA (>1 μg ml(-1) ) decreased the biofilm mass of all Strep. intermedius strains. These findings demonstrated that eDNA and eHLP play crucial roles in biofilm development and its rigidity. eDNA- and HLP-targeting strategies may be applicable to novel treatments for bacterial biofilm-related infectious diseases.


Fluorescence-based reporter for gauging cyclic di-GMP levels in Pseudomonas aeruginosa.

The increased tolerance toward the host immune system and antibiotics displayed by biofilm-forming Pseudomonas aeruginosa and other bacteria in chronic infections such as cystic fibrosis bronchopneumonia is of major concern. Targeting of biofilm formation is believed to be a key aspect in the development of novel antipathogenic drugs that can augment the effect of classic antibiotics by decreasing antimicrobial tolerance. The second messenger cyclic di-GMP is a positive regulator of biofilm formation, and cyclic di-GMP signaling is now regarded as a potential target for the development of antipathogenic compounds. Here we describe the development of fluorescent monitors that can gauge the cellular level of cyclic di-GMP in P. aeruginosa. We have created cyclic di-GMP level reporters by transcriptionally fusing the cyclic di-GMP-responsive cdrA promoter to genes encoding green fluorescent protein. We show that the reporter constructs give a fluorescent readout of the intracellular level of cyclic di-GMP in P. aeruginosa strains with different levels of cyclic di-GMP. Furthermore, we show that the reporters are able to detect increased turnover of cyclic di-GMP mediated by treatment of P. aeruginosa with the phosphodiesterase inducer nitric oxide. Considering that biofilm formation is a necessity for the subsequent development of a chronic infection and therefore a pathogenicity trait, the reporters display a significant potential for use in the identification of novel antipathogenic compounds targeting cyclic di-GMP signaling, as well as for use in research aiming at understanding the biofilm biology of P. aeruginosa.


The FleQ protein from Pseudomonas aeruginosa functions as both a repressor and an activator to control gene expression from the pel operon promoter in response to c-di-GMP.

Bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) modulates the transition between planktonic and biofilm life styles. In response to c-di-GMP, the enhancer binding protein FleQ from Pseudomonas aeruginosa derepresses the expression of Pel exopolysaccharide genes required for biofilm formation when a second protein, FleN is present. A model is that binding of c-di-GMP to FleQ induces its dissociation from the pelA promoter allowing RNA polymerase to access this site. To test this, we analyzed pelA DNA footprinting patterns with various combinations of FleQ, FleN and c-di-GMP, coupled to in vivo promoter activities. FleQ binds to two sites called box 1 and 2. FleN binds to FleQ bound at these sites causing the intervening DNA to bend. Binding of c-di-GMP to FleQ relieves the DNA distortion but FleQ remains bound to the two sites. Analysis of wild type and mutated versions of pelA-lacZ transcriptional fusions suggests that FleQ represses gene expression from box 2 and activates gene expression in response to c-di-GMP from box 1. The role of c-di-GMP is thus to convert FleQ from a repressor to an activator. The mechanism of action of FleQ is distinct from that of other bacterial transcription factors that both activate and repress gene expression from a single promoter.


MPC-Polymer Reduces Adherence and Biofilm Formation by Oral Bacteria.

Oral biofilms such as dental plaque cause dental caries and periodontitis, as well as aspiration pneumonia and infectious endocarditis by translocation. Hence, the suppression of oral biofilm formation is an issue of considerable importance. Mechanical removal, disinfectants, inhibition of polysaccharide formation, and artificial sugar have been used for the reduction of oral biofilm. From the viewpoint of the inhibition of bacterial adherence, we investigated whether aqueous biocompatible 2-methacryloyloxyethyl phosphorylcholine (MPC)-polymer can reduce streptococcal colonization and biofilm formation. We examined the effects of MPC-polymer on streptococcal adherence to saliva-coated hydroxyapatite and oral epithelial cells, and the adherence of Fusobacterium nucleatum to streptococcal biofilm. MPC-polymer application markedly inhibited both the adherence and biofilm formation of Streptococcus mutans on saliva-coated hydroxyapatite and streptococcal adherence to oral epithelial cells, and reduced the adherence of F. nucleatum to streptococcal biofilms. A small-scale clinical trial revealed that mouthrinsing with MPC-polymer inhibited the increase of oral bacterial numbers, especially of S. mutans. These findings suggest that MPC-polymer is a potent inhibitor of bacterial adherence and biofilm development, and may be useful to prevent dental-plaque-related diseases. (UMIN Clinical Trial Registry UMIN000003471). Abbreviations: QOL, quality of life; MPC, 2-methacryloyloxyethyl phosphorylcholine; BHI, brain-heart infusion broth; PBS, phosphate-buffered saline; SEM, scanning electron microscopy; CFU, colony-forming unit.


The pel polysaccharide can serve a structural and protective role in the biofilm matrix of Pseudomonas aeruginosa.

Bacterial extracellular polysaccharides are a key constituent of the extracellular matrix material of biofilms. Pseudomonas aeruginosa is a model organism for biofilm studies and produces three extracellular polysaccharides that have been implicated in biofilm development, alginate, Psl and Pel. Significant work has been conducted on the roles of alginate and Psl in biofilm development, however we know little regarding Pel. In this study, we demonstrate that Pel can serve two functions in biofilms. Using a novel assay involving optical tweezers, we demonstrate that Pel is crucial for maintaining cell-to-cell interactions in a PA14 biofilm, serving as a primary structural scaffold for the community. Deletion of pelB resulted in a severe biofilm deficiency. Interestingly, this effect is strain-specific. Loss of Pel production in the laboratory strain PAO1 resulted in no difference in attachment or biofilm development; instead Psl proved to be the primary structural polysaccharide for biofilm maturity. Furthermore, we demonstrate that Pel plays a second role by enhancing resistance to aminoglycoside antibiotics. This protection occurs only in biofilm populations. We show that expression of the pel gene cluster and PelF protein levels are enhanced during biofilm growth compared to liquid cultures. Thus, we propose that Pel is capable of playing both a structural and a protective role in P. aeruginosa biofilms.


Intermedilysin induces EGR-1 expression through calcineurin/NFAT pathway in human cholangiocellular carcinoma cells.

(Intermedilysin (ILY) is a cholesterol-dependent cytolysin produced by Streptococcus intermedius, which is associated with human brain and liver abscesses. Although intrahepatic bile duct cells play a valuable role in the pathogenesis of liver abscess, the molecular mechanism of ILY-treated intrahepatic bile duct cells remains unknown. In this study, we report that ILY induced a nuclear accumulation of intracellular calcium ([Ca(2+)]i) in human cholangiocellular cells HuCCT1. We also demonstrate that 10 ng/ml ILY induced NFAT1 dephosphorylation and its nuclear translocation in HuCCT1 cells. In contrast to the result that ILY induced NF-B translocation in human hepatic HepG2 cells, ILY did not affect NF-B localization in HuCCT1 cells. Dephosphorylation and nuclear translocation of NFAT1 caused by ILY were prevented by [Ca(2+)]i calcium chelator, BAPTA/AM, and calcineurin inhibitors, cyclosporine A and tacrolimus. ILY induced early growth response-1 (EGR-1) expression and it was inhibited by the pre-treatment with cyclosporine A, indicating that the calcineurin/NFAT pathway was involved in EGR-1 expression in response to ILY. ILY-induced calcineurin/NFAT1 activation and sequential EGR-1 expression might be related to the pathogenesis of S. intermedius in human bile duct cells.)

Biochemical and Biophysical Research Communications


Pseudomonas aeruginosa uses a cyclic-di-GMP-regulated adhesin to reinforce the biofilm extracellular matrix.

Pseudomonas aeruginosa, the principal pathogen of cystic fibrosis patients, forms antibiotic-resistant biofilms promoting chronic colonization of the airways. The extracellular (EPS) matrix is a crucial component of biofilms that provides the community multiple benefits. Recent work suggests that the secondary messenger, cyclic-di-GMP, promotes biofilm formation. An analysis of factors specifically expressed in P. aeruginosa under conditions of elevated c-di-GMP, revealed functions involved in the production and maintenance of the biofilm extracellular matrix. We have characterized one of these components, encoded by the PA4625 gene, as a putative adhesin and designated it cdrA. CdrA shares structural similarities to extracellular adhesins that belong to two-partner secretion systems. The cdrA gene is in a two gene operon that also encodes a putative outer membrane transporter, CdrB. The cdrA gene encodes a 220 KDa protein that is predicted to be rod-shaped protein harbouring a beta-helix structural motif. Western analysis indicates that the CdrA is produced as a 220 kDa proprotein and processed to 150 kDa before secretion into the extracellular medium. We demonstrated that cdrAB expression is minimal in liquid culture, but is elevated in biofilm cultures. CdrAB expression was found to promote biofilm formation and auto-aggregation in liquid culture. Aggregation mediated by CdrA is dependent on the Psl polysaccharide and can be disrupted by adding mannose, a key structural component of Psl. Immunoprecipitation of Psl present in culture supernatants resulted in co-immunoprecipitation of CdrA, providing additional evidence that CdrA directly binds to Psl. A mutation in cdrA caused a decrease in biofilm biomass and resulted in the formation of biofilms exhibiting decreased structural integrity. Psl-specific lectin staining suggests that CdrA either cross-links Psl polysaccharide polymers and/or tethers Psl to the cells, resulting in increased biofilm structural stability. Thus, this study identifies a key protein structural component of the P. aeruginosa EPS matrix.


Effect of substrate surface hydrophobicity on the adherence of yeast and hyphal Candida

A biofilm composed of various microorganisms including Candida is found on denture surfaces and is likely to be involved in the etiology of denture-induced stomatitis. The purpose of this study was to examine the role of hydrophobic interactions in candidal adherence to acrylic surfaces, particularly that of the hyphal form of Candida albicans. Candida clinical isolates were used. Acrylic plates coated with carrageenan and hydrocolloid (Hitachi chemical, Tokyo, Japan) were used as a hydrophilic substratum. A microbial suspension was placed on each acrylic plate and incubated. All plates were washed in phosphate-buffered saline containing CaCl(2) and MgCl(2) [PBS (+)] and cells still adhering to the acrylic surface were collected by 0.25% trypsin treatment. Cell-surface hydrophobicity was estimated using a modification of the technique used to measure adherence to hydrocarbons. When the acrylic plates were coated with hydrophilic materials, the adherence of hydrophobic clinical isolates of Candida and the hydrophobic hyphal C. albicans decreased, whereas the adherence of non-hydrophobic Candida was not affected or increased. We suggest that hydrophilic coating of denture surfaces could be a potent method for reduction of the adherence of relatively hydrophobic fungal cells, particularly hyphal C. albicans, which causes denture stomatitis and related infections.


The role of rpoS gene and quorum-sensing system in ofloxacin tolerance in Pseudomonas aeruginosa

The basis of the bactericidal action of antibiotics and the mechanisms of antibiotic tolerance are largely unknown. To elucidate one of the mechanisms of antibiotic tolerance, the present study investigated the role of Pseudomonas aeruginosa quorum sensing (QS) and the rpoS gene in antibiotic tolerance. The survival rates of the lasR and lasI mutants were observed to be lower than that of the parental strain in time-dependent killing studies with 8 microg mL(-1) ofloxacin, but the survival rates of the rhlR and rhlI mutants were not different from that of the parental strain. Moreover, a lasR-overexpressing strain was more tolerant to ofloxacin than the parental strain, but this was not the case for an rhlR-overexpressing strain. The mRNA expression levels of lasR, lasI, and rpoS in the wild-type strain in the presence of bactericidal concentration of ofloxacin were lower than that in the absence of ofloxacin. In addition, the significant loss of antibiotic tolerance in the lasR mutant was recovered by the overexpression of rpoS. These results suggest that the Las QS system in P. aeruginosa is involved in the development of ofloxacin tolerance, and the tolerance induced by the Las-system is regulated by rpoS gene.


Heterologous expression of a histone-like protein from Streptococcus intermedius in Escherichia coli alters the nucleoid structure and inhibits the growth of E. coli

Escherichia coli failed to survive after transformation with a Streptococcus intermedius histone-like protein gene (Si-hlp) and its promoter-harbored plasmid. The promoter function of Si-hlp in E. coli was determined using enhanced green fluorescence protein (egfp) gene as a reporter. The inhibitory effect of Si-HLP on E. coli viability was verified by a tetracycline-inducible gene expression system. Further study suggested that Si-HLP may alter the bacterial nucleoid structure, leading to the growth inhibition of E. coli.


The essentiality and involvement of Streptococcus intermedius histone-like DNA-binding protein in bacterial viability and normal growth

Streptococcus intermedius histone-like DNA-binding protein (Si-HLP) is a homodimeric protein and, conserved with Escherichia coli HU, a well-documented nucleoid-associated protein (NAP). In E. coli, HU plays important roles as both structural and regulatory factors, but it is not essential for E. coli viability. Streptococcal HLP has been found to bind host cells and induce cytokine production, but its physiological role remains poorly defined. In the present study, using gene insertion knockout and tetracycline-regulated antisense RNA expression techniques, we determined whether Si-HLP is essential for bacterial viability and normal growth in S. intermedius. The Si-HLP-downregulated S. intermedius strain showed alterations in its morphology and surface properties. Downregulation of Si-HLP led to an expanded nucleoid to fill the intracellular space. Transcription levels of several genes, including virulence-associated factors, were found to be activated or repressed in the antisense Si-hlp RNA-expressing strain by real-time PCR and reverse-transcription PCR. Collectively, these data suggest that Si-HLP serves as an essential NAP governing the nucleoid architecture and controlling the gene transcription profile in S. intermedius.


Histone-like DNA binding protein of Streptococcus intermedius induces the expression of pro-inflammatory cytokines in human monocytes via action of ERK1/2 and JNK pathways

Streptococcus intermedius is a commensal associated with serious, deep-seated purulent infections in major organs, such as the brain and liver. Histone-like DNA binding protein (HLP) is an accessory architectural protein in a variety of bacterial cellular processes. In this study, we investigated the mechanisms of pro-inflammatory cytokine inductions in THP-1 cells by stimulation with recombinant HLP of S. intermedius (rSi-HLP). rSi-HLP stimulation-induced production of pro-inflammatory cytokines (IL-8, IL-1 beta and TNF-alpha) occurred in a time- and dose-dependent manner. In contrast with the heat-stable activity of DNA binding, the induction activity of rSi-HLP was heat-unstable. In subsequent studies, rSi-HLP acted cooperatively with lipoteichoic acid, the synthetic Toll-like receptor 2 agonist, Pam3CSK4, and the cytosolic nucleotide binding oligomerization domain 2 receptor agonist, muramyldipeptide. Furthermore, Western blot and blocking assays with specific inhibitors showed that rSi-HLP stimulation induced the activation of cell signal transduction pathways, extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK). In addition to its physiological role in bacterial growth through DNA binding, these results indicate that Si-HLP can trigger a cascade of events that induce pro-inflammatory responses via ERK1/2 and JNK signal pathways, and suggest that bacterial HLP may contribute to the activation of host innate immunity during bacterial infection.


rpoN gene of Pseudomonas aeruginosa alters Its susceptibility to quinolones and cabapenems

The alternative sigma factor sigma54 has been implicated in diverse functions within the cells. In this study, we have constructed an rpoN mutant of Pseudomonas aeruginosa and investigated its importance as a target for antimicrobial agents, such as quinolones and carbapenems. The stationary-phase cells of the rpoN mutant displayed a survival rate approximately 15 times higher than that of the wild-type cells in the presence of quinolones and carbapenems. The stationary phase led to substantial production of pyoverdine by the P. aeruginosa rpoN mutant. Pyoverdine synthesis correlated with decreased susceptibility to antimicrobial agents. Quantitative real-time PCR revealed that stationary-phase cells of the rpoN mutant grown without an antimicrobial agent had approximately 4- to 140- and 2- to 14-fold-higher levels of transcripts of the pvdS and vqsR genes, respectively, than the wild-type strain. In the presence of an antimicrobial agent, levels of pvdS and vqsR transcripts were elevated 400- and 5-fold, respectively, in comparison to the wild-type levels. Flow cytometry assays using a green fluorescent protein reporter demonstrated increased expression of the vqsR gene in the rpoN mutant throughout growth. A pvdS mutant of P. aeruginosa, deficient in pyoverdine production, was shown to be susceptible to biapenem. These findings suggest that rpoN is involved in tolerance to antimicrobial agents in P. aeruginosa and that its tolerant effect is partly dependent on increased pyoverdine production and vqsR gene expression.


Functional Analysis of spoT, relA and dksA Genes on Quinolone Tolerance in Pseudomonas aeruginosa under Nongrowing Condition

To assess the contribution of ppGpp in antibiotic tolerance to quinolone in Pseudomonas aeruginosa, knockout mutants of the genes involved or linked with the stringent response, such as relA, spoT and dksA, were constructed and investigated for their antibiotic susceptibility to quinolones. The survival of the dksA and spoT mutants in the presence of 8 microg/ml of ofloxacin and 1 microg/ml of ciprofloxacin were shown to be approximately 20-180 and 10-40 times respectively, higher than the same for the wild type strain. The intracellular levels of ppGpp determined with high performance liquid chromatography (HPLC) demonstrated that spoT and dksA mutants possess higher basal levels of ppGpp. The data suggest that elevated basal levels of ppGpp may be responsible for rendering these mutants tolerant to quinolones and expand the importance of ppGpp as an antimicrobial target in P. aeruginosa.


CXCL12 and CXCR4 expression by human gingival fibroblasts in periodontal disease

CXCL12 is a CXC chemokine that is related to lymphocyte infiltration and angiogenesis in inflammatory sites such as arthritis. However, the expression and roles of CXCL12 in periodontal disease are uncertain. The aim of this study was to assess the expression of CXCL12 and its receptor, CXCR4, in periodontal tissue and to investigate the properties of CXCL12 and CXCR4 expression by human gingival fibroblasts (HGF). RT-PCR analysis revealed that CXCL12 and CXCR4 mRNA were expressed in both normal gingival tissues and periodontal diseased tissues. Immunohistochemistry disclosed that CXCL12 was expressed and CXCR4 positive cells were found in both normal and periodontal diseased gingival tissues. Our in vitro experiments elucidated that HGF constitutively produced CXCL12, and the levels were enhanced by stimulation with tumour necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), transforming growth factor-beta (TGF-beta), regulated upon activation normal T cell expressed and secreted (RANTES) and macrophage inflammatory protein 3(alpha) (MIP-3(alpha)). On the other hand, heat killed Porphyromonas gingivalis (P. gingivalis) and P. gingivalis LPS reduced the CXCL12 production by HGF. Flow cytometry analysis clarified that CXCR4 was highly expressed on HGF, and CXCR4 expression was abrogated by TNF-alpha, IFN-gamma and P. gingivalis LPS. Moreover, CXCL12 induced vascular endothelial growth factor (VEGF) production by HGF. Our results demonstrated that CXCL12 might be related to CXCR4+ cells infiltration and angiogenesis both in normal periodontal tissues and periodontal diseased tissue. P. gingivalis, a known periodontal pathogen, inhibits the production of CXCL12 and the expression of CXCR4 by HGF. This fact means that P. gingivalis may inhibit CXCR4+ cells infiltration and neovascularization in periodontal tissue and escape from the immune response.


Coating of a surface with 2-methacryloyloxyethyl phosphorylcholine (MPC) co-polymer significantly reduces retention of human pathogenic microorganisms

The present study compares the retention of four species that are often isolated in association with biomedical device-related infections - Staphylococcus aureus, Streptococcus mutans, Pseudomonas aeruginosa, and Candida albicans - to three different surfaces. All four bacterial species were found to bind significantly less well to MPC-coated surfaces than to non-coated surfaces. We attribute this effect to the "superhydrophilicity" of MPC-coated surfaces, whereas hydrophobic surfaces are well known to reduce bacterial retention and thus to inhibit a crucial step in the formation of bacterial biofilms that lead to biomedical device-related infections and complications.


Role for rpoS gene of Pseudomonas aeruginosa in antibiotic tolerance

The alternative sigma factor, RpoS has been described as a central regulator of many stationary phase-inducible genes and a master stress-response regulator under various stress conditions. We constructed an rpoS mutant in Pseudomonas aeruginosa and investigated the role of rpoS gene in antibiotic tolerance. The survival of the rpoS mutant cells in stationary phase was approximately 70 times lower when compared with that of the parental strain at 37 degrees C for 2 h after the addition of biapenem. For imipenem, the survival was approximately 40 times lower. Heat stress promoted an increase in the survival of the parental strain to biapenem, but the same was not found to be the case for the rpoS mutant. Our results indicate that rpoS gene is involved in tolerance to antibiotics in P. aeruginosa during the stationary phase and heat stress. However, under osmotic stress, tolerance to biapenem was not dependent on the rpoS gene.


Novel Pseudomonas aeruginosa gene that suppresses tolerance to carbapenems

A biapenem-tolerant mutant of Pseudomonas aeruginosa was isolated by Tn1737KH insertion. The survival of the mutant 3 h after the addition of biapenem was about 1000 times greater than that of the wild type. The mutant was also tolerant to other biapenems, such as imipenem, panipenem, and meropenem.


Correlation between resistance of Pseudomonas aeruginosa to quaternary ammonium compounds and expression of outer membrane protein OprR

論文題目:緑膿菌の細胞膜タンパク質OprRの発現と第四アンモニウム塩に対する耐性化の相関 第四アンモニウム塩系消毒剤に耐性化した緑膿菌において顕著に発現量が増加する約25.6kDaの蛋白質を2次元電気泳動で分離し，そのN末端アミノ酸配列の解析によって，それが外膜蛋白質のOprRであることを明らかとした．またその耐性化株でOprRのノックアウト株を作成したところ，第四アンモニウム塩系消毒剤に対する感受性が野生株と同程度まで上昇することから，OprRがこの消毒薬耐性に重要な役割を果たしていることを示した．


Effect of varidase (streptodornase) on biofilm formed by Pseudomonas aeruginosa

The biofilm of Pseudomonas aeruginosa could be removed by Varidase (streptodornase) that was used as defibrinating drug. After cultivating the biofilm on a silicone chip in a modified alginate-producing medium in vitro, it was treated with Varidase or DNase I. In both treatments, the biofilm was removed depending on the concentration of the reagents. Varidase was apparently effective under the condition that the biofilm was exposed to more than 625 U/ml (a quarter of the concentration of the medical use) for 3 h, twice, at 37 degrees C. The result of this experiment indicates that (a) the DNases, DNase I and Varidase, were effective in destroying the biofilm of P. aeruginosa in vitro, and (b) in a clinical field, Varidase could be useful for P. aeruginosa focal infection, such as urinary tract infection, by removing the biofilm.


緑膿菌におけるストレス反応と抗菌薬抵抗性

緑膿菌におけるストレス反応と抗菌薬抵抗性

感染症の治療において，起炎菌に対するMICを基準にした適切な抗菌薬を選択したにも関わらず，十分な効果が得られないことも少なくない．この原因には，殺菌的抗菌薬を用いても，薬剤に対する抵抗性が上昇しているために，殺菌的効果が得られていない可能性が考えられる．緑膿菌PAOI株では，カルパペネム系抗菌薬であるbiapenemに対し，浮遊菌でのMBCに比べ付着菌では128倍高いMBCADを示した．また浮遊菌においても，定常期の細菌は，対数増殖期のそれよりも抵抗性を示し，heat shockにおいても抗菌薬抵抗性が上昇することが明らかになった．


Effect of varidase (streptokinase) on biofilm formed by Staphylococcus aureus

Staphylococcus aureus forms a fibrin-rich biofilm in the presence of plasma which is highly resistant to attack by the human immune system and to chemotherapy. Varidase, composed mainly of streptokinase, is used for hydrolyzing clots. In this study, we attempted to destroy the biofilm of S. aureus with Varidase and to apply this drug in the treatment of staphylococcal infections. Four clinical isolates were used in the experiments. These organisms formed a several-millimeter-thick biofilm on type IV collagen coated coverslips in trypticase soy broth containing 50% human plasma. The biofilm was composed of bacterial cell which adhered to fibrillar fibers and of sediment derived from plasma. 10,000 U/ml of Varidase, the dose which is used clinically, removed the sediment and reduced the number of live bacteria in biofilms to less than 20% of control. 200 U/ml of Varidase was also effective against biofilms of the organisms. An equal combination of Varidase and ofloxacin had an additive effect on the bacteria. The results of this study demonstrate that Varidase is highly effective in destroying biofilms of S. aureus in vitro and suggest that this drug would be useful for treating staphylococcal infections.


緑膿菌の抗菌薬抵抗性に対する新規化合物の作用機序

緑膿菌の抗菌薬抵抗性に対する新規化合物の作用機序


新規化合物によるレボフロキサシン及びトブラマイシン殺菌作用の増強効果

新規化合物によるレボフロキサシン及びトブラマイシン殺菌作用の増強効果


Streptococcus intermedius biofilm componentsの糖鎖結合能と炎症反応誘導能に関する解析


新規化合物によるビアペネム殺菌作用の増強効果について

新規化合物によるビアペネム殺菌作用の増強効果について


クラリスロマイシンによる緑膿菌c-di-GMPの誘導

クラリスロマイシンによる緑膿菌c-di-GMPの誘導

第50回緑膿菌感染症研究会講演記録 /緑膿菌感染症研究会事務局編

第50回緑膿菌感染症研究会講演記録 /緑膿菌感染症研究会事務局編


水溶性MPCポリマーの口腔細菌付着抑制効果

水溶性MPCポリマーの口腔細菌付着抑制効果

口腔細菌によるbiofilm，すなわちdental plaqueの形成にはいくつかのkey stepがあり，dental plaque形成の抑制のためにはそれらのkey stepに効果的に働く方法を選択する必要が有る．我々は，超親水性表面を得ることができる2-methacryloloxethyl phosphorylcholine(MPC)co-polymerを用いてプラスチック表面をコートした界面は微生物の付着抑制に関与することを報告してきた．本実験では5%水溶性MPCポリマーで処理したHydroxyapatite(HY)プレートと培養歯肉上皮細胞(RT-7)へのStreptococcus mutansの付着状況を顕微鏡下にて観察した．その結果，5%水溶性MPCポリマー処理でHYプレート及び培養歯肉上皮細胞上へのS.mutansの付着は抑制されることが示された．以上よりHYプレートや培養歯肉上皮細胞に吸着した水溶性MPCポリマーは，プラスチックへのコーティングと同様にS.mutansの付着抑制効果を発揮することが示唆された．


緑膿菌臨床分離株におけるRSCVsの分離とその性状について

緑膿菌臨床分離株におけるRSCVsの分離とその性状について

緑膿菌感染症において，寒天培地上で小さな皺上のコロニー性状を示す，rugose small colony variants(RSCVs)の存在が報告されている．このRSCVsは，細胞内のセカンドメッセンジャーであるc-di-GMPの細胞内濃度が上昇しているためであると考えられている．今回我々は，徳島大学病院で分離された緑膿菌30株についてRSCVsの有無を検討したところ，2株が小さな皺状のコロニー性状を示し，バイオフィルム形性能が高く，運動性も低下していたことから，RSCVsであると考えられる．そのうちの1株は，c-di-GMP合成に関与する遺伝子オペロンの1つであるwspF遺伝子の変異の結果である事が明らかとなった．もう1株では，wspF遺伝子には大きな変異は見られなかった．これらの結果から，臨床分離株30株中2株のRSCVsが確認されたものの，その原因となる遺伝子は異なっていることが明らかとなった．


付着緑膿菌の抗菌薬抵抗性とQuorum sensing機構について

付着緑膿菌の抗菌薬抵抗性とQuorum sensing機構について


Biological Roles of Extracellular DNA in Bacterial Infection

Asean Plus and Tokushima Joint International Conference, Program and Proceeding Book


High prevalence of Pseudomonas aeruginosa from oropharyngeal biofilm in patients with cerebrovascular infarction and dysphagia.