Intranasal Application of S. epidermidis Prevents Colonization by Methicillin-Resistant Staphylococcus aureus in Mice

Methicillin-resistant S. aureus emerged in recent decades to become a leading cause of infection worldwide. Colonization with MRSA predisposes to infection and facilitates transmission of the pathogen; however, available regimens are ineffective at preventing MRSA colonization. Studies of human nasal flora suggest that resident bacteria play a critical role in limiting S. aureus growth, and prompted us to query whether application of commensal resident bacteria could prevent nasal colonization with MRSA. We established a murine model system to study this question, and showed that mice nasally pre-colonized with S. epidermidis became more resistant to colonization with MRSA. Our study suggests that application of commensal bacteria with antibiotics could represent a more effective strategy to prevent MRSA colonization

An experimental study of bacterial nitrogen fixation in the mammalian gut

学位の種別: 課程博士審査委員会委員 : (主査)東京大学教授 米田 穣, 東京大学教授 大矢 禎一, 東京大学教授 河村 正二, 東京大学教授 服部 正平, 理化学研究所チームリーダー 林 誠University of Tokyo(東京大学

Inhibition of Endothelial Interleukin-8 Production and Neutrophil Transmigration by Staphylococcus aureus Beta-Hemolysin▿

Neutrophils play a crucial role in the host response to infection with Staphylococcus aureus, which is a major human pathogen capable of causing life-threatening disease. Interleukin-8 (IL-8) is a potent chemoattractant and activator of neutrophils. We previously reported that S. aureus secretes a factor that suppresses IL-8 production by human endothelial cells. Here we isolated an inhibitor of IL-8 production from the supernatant and identified it as staphylococcal beta-hemolysin. Beta-hemolysin reduced IL-8 production without cytotoxicity to endothelial cells. Pretreatment with beta-hemolysin decreased the expression of both IL-8 mRNA and protein induced by tumor necrosis factor alpha (TNF-α). Migration of neutrophils across TNF-α-activated endothelium was also inhibited by beta-hemolysin. In contrast, beta-hemolysin had no effect on intercellular adhesive molecule 1 expression in activated endothelial cells. These results showed that beta-hemolysin produced by S. aureus interferes with inflammatory signaling in endothelial cells and may help S. aureus evade the host immune response

Hydrophobicity of Residue 128 of the Stress-Inducible Sigma Factor RpoS Is Critical for Its Activity

RpoS is a key stress-inducible sigma factor that regulates stress resistance genes in Escherichia coli, such as the katE gene encoding catalase HPII and the glg genes encoding glycogen synthesis proteins. Monitoring RpoS activity can provide information on the stress sensitivity of E. coli isolates in clinical settings because the RpoS in these isolates is often mutated. In the present study, we found a novel, missense point mutation at RpoS residue 128 in a clinical Shiga toxin-producing E. coli (STEC) isolate. This mutation caused RpoS dysfunction and increased stress sensitivity. A mutant rpoS was cloned from a clinical STEC that is vulnerable to cold temperature and oxidative stresses. Mutant RpoS protein expression was detected in the clinical isolate, and this RpoS was non-functional according to HPII activity and glycogen levels, which are positively regulated by RpoS and thus are used as indicators for RpoS function. A reporter assay with β-galactosidase indicated that the dysfunction occurred at the transcriptional level of genes regulated by RpoS. Furthermore, substitution analysis indicated that the hydrophobicity of the amino acid at residue 128 was critical for RpoS activity; the simulation analysis indicated that the amino acids of RNA polymerase (RNAP) that interact with RpoS residue 128 are hydrophobic, suggesting that this hydrophobic interaction is critical for RpoS activity. In addition, substitution of Ile128 to Pro128 abolished RpoS activity, possibly as a result of disruption of the secondsary structure around residue 128, indicating that the structure is also a crucial factor for RpoS activity. These results indicate that only one point mutation at a hydrophobic residue of the complex formed during transcription leads to a critical change in RpoS regulation. Moreover, we found that Ile128 is widely conserved among various bacteria: several bacterial strains have Met128 or Leu128, which are hydrophobic residues, and these strains had similar or higher RpoS activity than that observed with Ile128 in this study. These data indicate that the hydrophobicity of the amino acid at residue 128 is critical for RpoS activity and is consequently important for bacterial survival. Taken together, these findings may contribute to a deeper understanding of protein functional mechanisms and bacterial stress responses

The Composition and Structure of Biofilms Developed by Propionibacterium acnes Isolated from Cardiac Pacemaker Devices

The present study aimed to understand the biofilm formation mechanism of Propionibacterium acnes by analyzing the components and structure of the biofilms. P. acnes strains were isolated from the surface of explanted cardiac pacemaker devices that exhibited no clinical signs of infection. Culture tests using a simple stamp culture method (pressing pacemakers against the surface of agar plates) revealed frequent P. acnes colonization on the surface of cardiac pacemaker devices. P. acnes was isolated from 7/31 devices, and the isolates were categorized by multilocus sequence typing into five different sequence types (STs): ST4 (JK18.2), ST53 (JK17.1), ST69 (JK12.2 and JK13.1), ST124 (JK5.3), ST125 (JK6.2), and unknown ST (JK19.3). An in vitro biofilm formation assay using microtiter plates demonstrated that 5/7 isolates formed biofilms. Inhibitory effects of DNase I and proteinase K on biofilm formation varied among isolates. In contrast, dispersin B showed no inhibitory activity against all isolates. Three-dimensional live/dead imaging of P. acnes biofilms with different biochemical properties using confocal laser microscopy demonstrated different distributions and proportions of living and dead cells. Additionally, it was suggested that extracellular DNA (eDNA) plays a role in the formation of biofilms containing living cells. Ultrastructural analysis of P. acnes biofilms using a transmission electron microscope and atmospheric scanning electron microscope revealed leakage of cytoplasmic components along with cell lysis and fibrous structures of eDNA connecting cells. In conclusion, the biochemical properties and structures of the biofilms differed among P. acnes isolates. These findings may provide clues for establishing countermeasures against biofilm-associated infection by P. acnes

Isolation and Identification of ATP-Secreting Bacteria from Mice and Humans▿

In a recent report, ATP, which was possibly secreted by some intestinal bacteria, was shown to cause colitis in mice via Th17 cell differentiation. However, the ATP-secreting bacteria have not been isolated and identified. In the present study, we report that Enterococcus gallinarum, which is a vancomycin-resistant Gram-positive coccus isolated from mice and humans, secretes ATP