Suppressed distribution of protein A on the surface of Staphylococcus aureus as a morphological characteristic of erythromycin-resistant strain.

To identify a new morphological phenotype of erythromycin (EM)-resistant Staphylococcus aureus (S. aureus) were isolated in vitro from EM-sensitive parent strain, and the distribution of staphylococcus specific protein A (SpA) on the surface of these strains was examined morphologically by using applied immunoelectron microscopy. The isolated EM-resistant strains had thickened cell walls, and the distribution of SpA on the surfaces of these strains was demonstrated to be lower than that of the parent strain. The SpA suppression was confirmed by enzyme-linked immunosorbent assay (ELISA) using fixed EM-resistant cells. Moreover, the spa gene of EM-resistant cells was detected by polymerase chain reaction (PCR) and confirmed by quantitative real-time PCR assay, showing that the expression of SpA was repressed at the transcriptional level in these strains. Furthermore, ELISA assay showed that whole EM-resistant cell SpA content was significantly decreased. Therefore, it was considered that the suppression of surface SpA on the EM-resistant strain was due to regulated SpA production, and not dependent on the conformational change in SpA molecule expression through cell wall thickening. These results strongly suggest that suppressed SpA distribution on the EM-resistant S. aureus is a phenotypical characteristic in these strains.

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

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Porphyromonas gingivalis (Pg) 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. Pg-OMVs are involved in the pathogenesis of periodontitis. Pg-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 Pg-OMV-induced pro-inflammatory cytokines was investigated using Western blot analysis and specific pathway inhibitors. Pg-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. Pg-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 Pg-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 Pg-OMVs may play important roles in periodontitis exacerbation by stimulating various pathways.

Characteristics of Antibiotic Resistance and Tolerance of Environmentally Endemic Pseudomonas aeruginosa.

Antibiotic-resistant bacteria remain a serious public health threat. In order to determine the percentage of antibiotic-resistant and -tolerant Pseudomonas aeruginosa cells present and to provide a more detailed infection risk of bacteria present in the environment, an isolation method using a combination of 41 °C culture and specific primers was established to evaluate P. aeruginosa in the environment. The 50 strains were randomly selected among 110 isolated from the river. The results of antibiotic susceptibility evaluation showed that only 4% of environmental strains were classified as antibiotic-resistant, while 35.7% of clinical strains isolated in the same area were antibiotic-resistant, indicating a clear difference between environmental and clinical strains. However, the percentage of antibiotic-tolerance, an indicator of potential resistance risk for strains that have not become resistant, was 78.8% for clinical strains and 90% for environmental strains, suggesting that P. aeruginosa, a known cause of nosocomial infections, has a high rate of antibiotic-tolerance even in environmentally derived strains. It suggested that the rate of antibiotic-tolerance is not elicited by the presence or absence of antimicrobial exposure. The combination of established isolation and risk analysis methods presented in this study should provide accurate and efficient information on the risk level of P. aeruginosa in various regions and samples.

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

Pseudomonas aeruginosa infection is considered a high-risk nosocomial infection and is very difficult to eradicate because of its tolerance to antibiotic treatment. A new compound, autoinducer analog-1 (AIA-1), has been demonstrated to reduce antibiotic tolerance, but its mechanisms remain unknown. This study aimed to investigate the mechanisms of AIA-1 in the antibiotic tolerance of P. aeruginosa. A transposon mutant library was constructed using miniTn5pro, and screening was performed to isolate high tolerant mutants upon exposure to biapenem and AIA-1. We constructed a deletion mutant and complementation strain of the genes detected in transposon insertion site determination, pruR and PA0066-65-64, and performed killing assays with antibiotics and AIA-1. Gene expression upon exposure to biapenem and AIA-1 and their relationship to stress response genes were analyzed. High antibiotic tolerance was observed in Tn5-pruR and Tn5-PA0065 transposon mutants and their deletion mutants, ΔpruR and ΔPA0066-65-64. Complemented strains of pruR and PA0066-65-64 with their respective deletion mutants exhibited suppressed antibiotic tolerance. It was determined that deletion of PA0066-65-64 increased rpoS expression, and PA0066-65-64 affects antibiotic tolerance via the rpoS pathway. Additionally, antibiotics and AIA-1 were found to inhibit pruR and PA0066-65-64. This study proposed that pruR and PA0066-65-64 are members of the antibiotic tolerance suppressors.

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 Porphyromonas gingivalis, 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. Juncus effusus 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 J. effusus 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 J. effusus L. significantly reduced P. gingivalis 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 J. effusus L. in the presence of antimicrobial agents or antifibrinolytics already used as ingredients in mouthwash could significantly reduce the production of chemokines from P. gingivalis LPS-stimulated oral epithelial cells in a concentration-dependent manner. These findings suggest that the water extract from J. effusus L. is potentially useful for oral care to prevent oral infections, such as periodontal infections, and maintain oral epithelial function.

Infective Endocarditis from Furuncle with Meningitis Complication Caused by Methicillin-resistant Staphylococcus aureus.

Infective endocarditis (IE) may be acquired in the community as community-acquired (CA) IE or in the healthcare setting. In Japan, cases of CA-methicillin-resistant Staphylococcus aureus (MRSA) infection as skin infection have been increasing. CA-MRSA strains, including the USA300 clone, have higher pathogenicity and are more destructive to tissue than healthcare-associated MRSA strains because of the toxins they produce, including arginine-catabolic mobile element (ACME) and Panton-Valentine leukocidin (PVL). However, only a few IE cases induced by USA300 have been reported. We herein report a 64-year-old man who developed CA-IE from a furuncle caused by USA300 MRSA producing PVL and ACME, which resulted in complications of meningitis.

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

Macrolide antibiotics are used in treating Pseudomonas aeruginosa 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 P. aeruginosa quorum-sensing autoinducer analogs (AIA-1, -2) on the activity of azithromycin and clarithromycin against P. aeruginosa. In the killing assay of planktonic cells, AIA-1 and -2 enhanced the bactericidal ability of macrolides against P. aeruginosa 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 P. aeruginosa biofilm infections.

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

We often come across difficult to treat infections-even after administering appropriate antibiotics according to the minimal inhibitory concentration of the causative bacteria. Antibiotic tolerance has recently started to garner attention as a crucial mechanism of refractory infections. However, few studies have reported the correlation between clinical outcomes and antibiotic tolerance. This study aims to clarify the effect of antibiotic tolerance on clinical outcomes of respiratory tract infection caused by Pseudomonas aeuginosa (P. aeruginosa). We examined a total of 63 strains isolated from sputum samples of different patients and conducted a retrospective survey with the medical records of 37 patients with imipenem-sensitive P. aeruginosa infections. Among them, we selected 15 patients with respiratory infections, and they were divided into high-tolerance minimal bactericidal concentration for adherent bacteria (MBCAD)/minimal inhibitory concentration for adherent bacteria (MICAD) ≥ 32 (n = 9) group and low-tolerance MBCAD/MICAD ≤ 16 (n = 6) group for further investigations. The findings indicated that the high-tolerance group consisted of many cases requiring hospitalization. Chest computed tomography findings showed that the disease was more extensive in the high-tolerance group compared to the low-tolerance group. Regarding the bacterial phenotypic characterization, the high-tolerance group significantly upregulated the production of the virulence factors compared to the low-tolerance group. Our study provided evidence that carbapenem tolerance level is a potent prognostic marker of P. aeruginosa infections, and carbapenem tolerance could be a potential target for new antimicrobial agents to inhibit the progression of persistent P. aeruginosa infections.

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

OBJECTIVES: The effects of 2-methacryloyloxyethyl phosphorylcholine (MPC)-polymer on the adherence of microorganisms such as non-Candida albicans Candida (NCAC) and methicillin-resistant Staphylococcus aureus (MRSA), frequently detected in oral infections in immunocompromised and/or elderly people, to denture resin material, are still unclear. Here, we report the effects of MPC-polymer on the adherence of C. albicans, NCAC, and MRSA to acrylic denture resin. METHODS: Sixteen strains of C. albicans, seven strains of C. glabrata, two strains of C. tropicalis, one strain of C. parapsilosis, and six strains of MRSA were used. We cultured the fungal/bacterial strains and examined the cell growth and adherence of fungi/bacteria to mucin-coated acrylic denture resin plates (ADRP) with or without MPC-polymer coating, by scanning electron microscopy. The cell surface hydrophobicity of the fungal/bacterial strains was measured by the adsorption to hydrocarbons. RESULTS: MPC-polymer did not affect the growth of all strains of Candida species and MRSA, but significantly suppressed adherence to ADRP in most strains of C. albicans and all strains of NCAC and MRSA. A significant positive correlation was found between cell hydrophobicity and the reduction rates of microbial adherence to ADRP treated with 5% of MPC-polymer. CONCLUSIONS: MPC-polymer treatment for acrylic resin material suppresses the adherence of C. albicans, NCAC and MRSA via their hydrophilicity interaction. CLINICAL SIGNIFICANCE: The application of MPC-polymer for denture hygiene is potent to prevent oral candidiasis, denture stomatitis and opportunistic infection, caused by Candida species and MRSA, via suppressing the adherence of those fungus/bacteria.

The Pathogenic Factors from Oral Streptococci for Systemic Diseases.

The oral cavity is suggested as the reservoir of bacterial infection, and the oral and pharyngeal biofilms formed by oral bacterial flora, which is comprised of over 700 microbial species, have been found to be associated with systemic conditions. Almost all oral microorganisms are non-pathogenic opportunistic commensals to maintain oral health condition and defend against pathogenic microorganisms. However, oral Streptococci, the first microorganisms to colonize oral surfaces and the dominant microorganisms in the human mouth, has recently gained attention as the pathogens of various systemic diseases, such as infective endocarditis, purulent infections, brain hemorrhage, intestinal inflammation, and autoimmune diseases, as well as bacteremia. As pathogenic factors from oral Streptococci, extracellular polymeric substances, toxins, proteins and nucleic acids as well as vesicles, which secrete these components outside of bacterial cells in biofilm, have been reported. Therefore, it is necessary to consider that the relevance of these pathogenic factors to systemic diseases and also vaccine candidates to protect infectious diseases caused by Streptococci. This review article focuses on the mechanistic links among pathogenic factors from oral Streptococci, inflammation, and systemic diseases to provide the current understanding of oral biofilm infections based on biofilm and widespread systemic diseases.

Heterogeneity in surface sensing suggests a division of labor in Pseudomonas aeruginosa populations.

The second messenger signaling molecule cyclic diguanylate monophosphate (c-di-GMP) drives the transition between planktonic and biofilm growth in many bacterial species. Pseudomonas aeruginosa has two surface sensing systems that produce c-di-GMP in response to surface adherence. Current thinking in the field is that once cells attach to a surface, they uniformly respond by producing c-di-GMP. Here, we describe how the Wsp system generates heterogeneity in surface sensing, resulting in two physiologically distinct subpopulations of cells. One subpopulation has elevated c-di-GMP and produces biofilm matrix, serving as the founders of initial microcolonies. The other subpopulation has low c-di-GMP and engages in surface motility, allowing for exploration of the surface. We also show that this heterogeneity strongly correlates to surface behavior for descendent cells. Together, our results suggest that after surface attachment, P. aeruginosa engages in a division of labor that persists across generations, accelerating early biofilm formation and surface exploration.

Bacteria can adopt different lifestyles, depending on the environment in which they grow. They can exist as single cells that are free to explore their environment or group together to form 'biofilms'. The bacteria in biofilms stick to a surface, and produce a slimy 'matrix' that covers and thereby protects them. Biofilms have been found in lung infections that affect people with the genetic disorder cystic fibrosis, and can also form on the surface of medical implants. Because the biofilm lifestyle protects bacteria from the immune system and antimicrobial drugs, learning about how biofilms form could help researchers to discover ways to prevent and treat such infections. Many bacteria switch between the free-living and biofilm lifestyles by altering their levels of a signaling molecule called cyclic diguanylate monophosphate (called c-di-GMP for short). Bacteria living in biofilms have much higher levels of c-di-GMP than their free-living counterparts, and bacteria that have high levels of c-di-GMP produce more biofilm matrix. Bacteria called Pseudomonas aeruginosa use a protein signaling complex called the Wsp system to sense that they are on a surface and increase c-di-GMP production. Questions remained about how quickly this change in production occurs, and whether bacteria pass on their c-di-GMP levels to the new descendant cells when they divide. Armbruster et al. monitored individual cells of P. aeruginosa producing c-di-GMP as they began to form biofilms. Unexpectedly, not all cells increased their c-di-GMP levels when they first attached to a surface. Instead, Armbruster et al. found that there are two populations ­ high and low c-di-GMP cells ­ that each perform complementary and important tasks in the early stages of biofilm formation. The high c-di-GMP cells represent 'biofilm founders' that start to produce the biofilm matrix, whereas the low c-di-GMP cells represent 'surface explorers' that spend more time traveling along the surface. Armbruster et al. found that the Wsp surface sensing system generates these two populations of cells. Moreover, the c-di-GMP levels in a bacterial cell even affect the behavior of the descendant cells that form when it divides. This effect can persist for several cell generations. More work is needed to examine exactly how the biofilm founders and surface explorers interact and influence how biofilms form, and to discover if blocking c-di-GMP signaling prevents biofilm formation. This could ultimately lead to new strategies to prevent and treat infections in humans.

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

OBJECTIVES: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSIONS: We suggest that MPC-polymer coating in the oral cavity may suppress the oral bacterial adherence. CLINICAL RELEVANCE: MPC-polymer can be a potent compound for the control of oral microflora to prevent oral infection.

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. Houttuynia cordata (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 Staphylococcus aureus (MRSA) and other oral microorganisms. dHC also exhibited antibiofilm effects against MRSA, Fusobacterium nucleatum (involved in dental plaque formation), and Candida albicans and inhibitory effects on interleukin-8, CCL20, IP-10, and GROα productions by human oral keratinocytes stimulated by Porphyromonas gingivalis 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.

Royal Jelly Inhibits Pseudomonas aeruginosa Adherence and Reduces Excessive Inflammatory Responses in Human Epithelial Cells.

Pseudomonas aeruginosa 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 P. aeruginosa is unclear. The aim of this study was to analyze antibacterial, antiadherent, and anti-inflammatory effects of royal jelly against P. aeruginosa. Wild-type strain PAO1 and clinical isolates of P. aeruginosa 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-3α/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 P. aeruginosa adherence and protects epithelial cells from excessive inflammatory responses against P. aeruginosa infection. Our findings suggested that royal jelly may be a useful supplement as complementary and alternative medicine for preventing respiratory infection caused by P. aeruginosa.

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 (34.8µM), 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.

Effects of an autoinducer analogue on antibiotic tolerance in Pseudomonas aeruginosa.

Objectives: 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 . Methods: 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. Results: 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. Conclusions: 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.

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 (σ54) promotes survival in the presence of tobramycin. The tobramycin-sensitive phenotype of logarithmic phase ΔrpoN 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 ΔrpoS mutant demonstrated lower expression of gacA, underscoring a regulatory role of RpoS for GacA. Supplementation of tobramycin to stationary phase ΔrpoN 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 ΔrpoN mutant in the presence of tobramycin could be reversed through the mutation of rpoS in the ΔrpoN mutant background. The tobramycin-tolerant phenotype of the ΔrpoNΔrpoS mutant logarithmic phase cells may be associated with the expression of relA, which remained unresponsive upon addition of tobramycin. The logarithmic phase ΔrpoS and ΔrpoNΔrpoS 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.

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 PAO1ΔpslBCD 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 ΔwspF mutant background abolished the biapenem-tolerant phenotype of the ΔwspF 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.