Kazuhiro Tateda

Azithromycin Inhibits Quorum Sensing in Pseudomonas aeruginosa

ABSTRACT We report that 2 μg of azithromycin/ml inhibits the quorum-sensing circuitry of Pseudomonas aeruginosa strain PAO1. Addition of synthetic autoinducers partially restored the expression of the trancriptional activator-encoding geneslasR and rhlR but not that of the autoinducer synthase-encoding gene lasI. We propose that azithromycin interferes with the synthesis of autoinducers, by an unknown mechanism, leading to a reduction of virulence factor production.

Chemokine-dependent neutrophil recruitment in a murine model of Legionella pneumonia: Potential role of neutrophils as immunoregulatory cells

ABSTRACT The roles of CXC chemokine-mediated host responses were examined with an A/J mouse model of Legionella pneumophilapneumonia. After intratracheal inoculation of 106 CFU ofL. pneumophila, the bacterial numbers in the lungs increased 10-fold by day 2; this increase was accompanied by the massive accumulation of neutrophils. Reverse transcription-PCR data demonstrated the up-regulation of CXC chemokines, such as keratinocyte-derived chemokine, macrophage inflammatory protein 2 (MIP-2), and lipopolysaccharide-induced CXC chemokine (LIX). Consistent with these data, increased levels of KC, MIP-2, and LIX proteins were observed in the lungs and peaked at days 1, 2, and 2, respectively. Although the administration of anti-KC or anti–MIP-2 antibody resulted in an approximately 20% decrease in neutrophil recruitment on day 2, no increase in mortality was observed. In contrast, the blockade of CXC chemokine receptor 2 (CXCR2), a receptor for CXC chemokines, including KC and MIP-2, strikingly enhanced mortality; this effect coincided with a 67% decrease in neutrophil recruitment. Interestingly, anti-CXCR2 antibody did not affect bacterial burden by day 2, even in the presence of a lethal challenge of bacteria. Moreover, a significant decrease in interleukin-12 (IL-12) levels, in contrast to the increases in KC, MIP-2, and LIX levels, was demonstrated for CXCR2-blocked mice. These data indicated that CXCR2-mediated neutrophil accumulation may play a crucial role in host defense against L. pneumophilapneumonia in mice. The increase in lethality without a change in early bacterial clearance suggested that neutrophils may exert their protective effect not through direct killing but through more immunomodulatory actions in L. pneumophila pneumonia. We speculate that a decrease in the levels of the protective cytokine IL-12 may explain, at least in part, the high mortality in the setting of reduced neutrophil recruitment.

Alveolar macrophage deactivation in murine septic peritonitis: Role of interleukin 10

ABSTRACT Sepsis predisposes the host to a number of infectious sequelae, particularly the development of nosocomial pneumonia. Mechanisms by which sepsis results in impairment of lung antibacterial host defense have not been well defined. Alveolar macrophages (AM) represent important immune effector cells of the lung airspace. In this study, we examined the effects of cecal ligation and puncture (CLP) on murine AM function ex vivo, including the expression of proinflammatory cytokines and AM phagocytic activity. AM were harvested from mice subjected to a sham operation and CLP 24 h after laparotomy, adherence purified, and challenged with lipopolysaccharide (LPS) or left unstimulated. Both unstimulated and LPS-stimulated AM from mice subjected to CLP (CLP mice) produced significantly smaller amounts of proinflammatory cytokines tumor necrosis factor alpha and interleukin (IL-12) and C-X-C chemokines KC and macrophage inflammatory protein 2 than similarly treated AM from animals subjected to a sham operation. Furthermore, AM isolated from CLP mice displayed a marked impairment in phagocytic activity, as determined by flow cytometry, with this defect persisting to 48 h post-CLP. Induction of peritoneal sepsis syndrome resulted in a time-dependent increase in IL-10 in plasma and peritoneal fluid. Interestingly, the impairment in AM proinflammatory-cytokine production and phagocytic activity observed in AM from CLP mice was partially reversed by the in vivo neutralization of IL-10 prior to AM harvest. These observations suggest that abdominal sepsis syndrome results in significant impairment in AM effector cell function, which is mediated, in part, by sepsis-induced expression of IL-10.

Hyperoxia Mediates Acute Lung Injury and Increased Lethality in Murine Legionella Pneumonia: The Role of Apoptosis

Legionella pneumophila is a major cause of life-threatening pneumonia, which is characterized by a high incidence of acute lung injury and resultant severe hypoxemia. Mechanical ventilation using high oxygen concentrations is often required in the treatment of patients with L. pneumophila pneumonia. Unfortunately, oxygen itself may propagate various forms of tissue damage, including acute lung injury. The effect of hyperoxia as a cofactor in the course of L. pneumophila pneumonia is poorly understood. In this study, we show that exposure to hyperoxic conditions during the evolution of pneumonia results in a marked increase in lethality in mice with Legionella pneumonia. The enhanced lethality was associated with an increase in lung permeability, but not changes in either lung bacterial burden or leukocyte accumulation. Interestingly, accelerated apoptosis as evidenced by assessment of histone-DNA fragments and caspase-3 activity were noted in the infected lungs of mice exposed to hyperoxia. TUNEL staining of infected lung sections demonstrated increased apoptosis in hyperoxic mice, predominantly in macrophages and alveolar epithelial cells. In vitro exposure of primary murine alveolar epithelial cells to Legionella in conjunction with hyperoxia accelerated apoptosis and loss of barrier function. Fas-deficient mice demonstrated partial resistance to the lethal effects of Legionella infection induced by hyperoxia, which was associated with attenuated apoptosis in the lung. These results demonstrate that hyperoxia serves as an important cofactor for the development of acute lung injury and lethality in L. pneumophila pneumonia. Exaggerated apoptosis, in part through Fas-mediated signaling, may accelerate hyperoxia-induced acute lung injury in Legionella pneumonia.

Transient transgenic expression of gamma interferon promotes legionella pneumophila clearance in immunocompetent hosts

ABSTRACT Gamma interferon (IFN-γ) and T1-phenotype immune responses are important components of host defense against a variety of intracellular pathogens, including Legionella pneumophila. The benefit of intrapulmonary adenovirus-mediated IFN-γ gene therapy was investigated in a nonlethal murine model of experimental L. pneumophilapneumonia. Intratracheal (i.t.) administration of 106 CFU of L. pneumophila induced the expression of T1 phenotype cytokines, such as IFN-γ and interleukin-12 (IL-12). Natural killer cells were identified as the major cellular source of IFN-γ. To determine if enhanced expression of IFN-γ in the lung could promote pulmonary clearance of L. pneumophila, we i.t. administered 5 × 108 PFU of a recombinant adenovirus vector containing the murine IFN-γ cDNA (AdmIFN-γ) concomitant with L. pneumophila. We observed a 10-fold decrease in lung bacterial CFU at day 2 in the AdmIFN-γ-treated group compared to controls (P < 0.01). Alveolar macrophages isolated from AdmIFN-γ-treated animals displayed enhanced killing of intracellular L. pneumophila organisms ex vivo. Similar improvements in bacterial clearance were observed with i.t. recombinant IFN-γ treatment. The transient transgenic expression of IL-12, a known inducer of IFN-γ and promoter of T1-type immune responses, resulted in more modest improvement in bacterial clearance (sixfold reduction;P < 0.05). These results demonstrate that, even in immunocompetent hosts, exogenous administration or transient transgenic expression of IFN-γ, and to a lesser extent IL-12, may be of potential therapeutic benefit in the treatment of patients withLegionella pneumonia.

Induction of interleukin-10 and down-regulation of cytokine production by Klebsiella pneumoniae capsule in mice with pulmonary infection.

The role of the capsule of Klebsiella pneumoniae in inducing cytokine production was investigated by comparing the responses of mice with experimentally induced pneumonia caused by capsulate (strain DT-S) or non-capsulate (mutant strain DT-X) K. pneumoniae. Anaesthetised ICR mice were inoculated intranasally. Whereas all DT-S-infected mice died within 3 days, no deaths were observed in DT-X-infected mice by 14 days after infection. During the early stage of infection, interferon-gamma (IFN-gamma) levels in bronchoalveolar lavage fluid (BALF) of DT-X-infected mice were significantly higher than those in DT-S-infected mice. In contrast, in the late stage of infection, serum levels of granulocyte macrophage-colony stimulating factor (GM-CSF) and IFN-gamma in DT-S-infected mice were significantly higher than those in DT-X-infected mice. Levels of interleukin10 (IL-10) in BALF and serum of DT-S-infected mice were significantly and persistently higher than those of DT-X-infected mice. The IL-10/TNF-alpha (tumour necrosis factor-a) ratios in BALF and serum indicated that higher levels of IL-10 production were induced in mice infected with strain DT-S than in those infected with strain DT-X. The results suggest that the capsule of K. pneumoniae may induce IL-10 production at the site of infection and, thereafter, these high IL-10 levels may serve to down-regulate the expression of pro-inflammatory cytokines.

In vitro and in vivo antibacterial activities of L-084, a novel oral carbapenem, against causative organisms of respiratory tract infections

ABSTRACT L-084 (a prodrug of LJC 11,036 [L-036]) is a new oral carbapenem. Here we compared the in vitro and in vivo antibacterial activities of L-036 with those of imipenem, faropenem, ceditoren-pivoxil, cefdinir, amoxicillin, and levofloxacin. The MICs at which 90% of the isolates were inhibited of L-036 against methicillin-susceptible staphylococci,Streptococcus pneumoniae including penicillin-resistant organisms, Escherichia coli, Klebsiella pneumoniae, Haemophilus influenzae including ampicillin-resistant organisms, Legionella pneumophila, andMoraxella catarrhalis were equal to or less than 1 μg/ml. In pharmacokinetics studies of L-084 in lungs of mice, the maximum concentration in serum, half-life, and area under the concentration-time curve of this drug were 9.09 μg/g of tissue, 6.18 h, and 31.0 μg · h/ml, respectively. In murine respiratory infection models of penicillin-susceptible and -resistantS. pneumoniae and H. influenzae, the efficacies of L-084 were better than those of reference drugs. Our results indicate that the in vitro high potency and good distribution in the lungs might be the underlying mechanisms of its efficacy in the murine model of pneumonia.

Development of systemic bacteraemia after oral inoculation of vancomycin-resistant enterococci in mice

Bacteraemia caused by vancomycin-resistant enterococci (VRE) is an important clinical problem because there are only a few potent antimicrobial agents against such bacteria. Therefore, understanding the pathogenic mechanisms of VRE bacteraemia is important for prophylaxis. This study shows that treatment of mice with cyclophosphamide and a combination of metronidazole, kanamycin and vancomycin reduced normal intestinal flora and induced systemic VRE bacteraemia. Translocation of VRE and the normal intestinal flora to the mesenteric lymph nodes, liver, spleen and blood, and mortality rate were dependent on treatment with cyclophosphamide and each of the three antimicrobial drugs. Among the different strains studied, C57BL/6 mice were the most susceptible to VRE. The virulence of vancomycin-resistant Enterococcus faecalis was greater than that of vancomycin-resistant Ent. faecium. On the day after inoculation of VRE, Escherichia coli was also detected in many VRE-positive specimens including blood, liver and the mesenteric lymph nodes. Moreover, both VRE and E. coli were detected simultaneously in almost all blood samples obtained from dead and dying mice, and VRE organisms outnumbered E. coli in those samples by 100:1 or more. These results indicate that changes in normal intestinal flora by administration of antimicrobial drugs and severity of neutropenia induced by cyclophosphamide are important factors that contribute to the development of systemic VRE bacteraemia. E. coli may be intimately associated with the establishment of VRE translocation.

In Vivo Efficacy of Telithromycin (HMR3647) against Streptococcus pneumoniae and Haemophilus influenzae

ABSTRACT The in vivo activity of telithromycin against erythromycin A- and penicillin G-resistant Streptococcus pneumoniae was superior to that of azithromycin, clarithromycin, cefdinir, and levofloxacin. In respiratory tract infections caused by erythromycin A-susceptible S. pneumoniae or Haemophilus influenzae in mice, telithromycin was more effective than clarithromycin and comparable to azithromycin.