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1.  Association of Fluoroquinolone Resistance, Virulence Genes, and IncF Plasmids with Extended-Spectrum-β-Lactamase-Producing Escherichia coli Sequence Type 131 (ST131) and ST405 Clonal Groups 
Antimicrobial Agents and Chemotherapy  2013;57(10):4736-4742.
The global increase of extended-spectrum-β-lactamase (ESBL)-producing Escherichia coli is associated with the specific clonal group sequence type 131 (ST131). In order to understand the successful spread of ESBL-producing E. coli clonal groups, we characterized fluoroquinolone resistance determinants, virulence genotypes, and plasmid replicons of ST131 and another global clonal group, ST405. We investigated 41 ST131-O25b, 26 ST131-O16, 41 ST405, and 41 other ST (OST) ESBL-producing isolates, which were collected at seven acute care hospitals in Japan. The detection of ESBL types, fluoroquinolone resistance-associated mutations (including quinolone resistance-determining regions [QRDRs]), virulence genotypes, plasmid replicon types, and IncF replicon sequence types was performed using PCR and sequencing. blaCTX-M, specifically blaCTX-M-14, was the most common ESBL gene type among the four groups. Ciprofloxacin resistance was found in 90% of ST131-O25b, 19% of ST131-O16, 100% of ST405, and 54% of OST isolates. Multidrug resistance was more common in the ST405 group than in the ST131-O25 group (56% versus 32%; P = 0.045). All ST131-O25b isolates except one had four characteristic mutations in QRDRs, but most of the isolates from the other three groups had three mutations in common. The ST131-O25b and ST405 groups had larger numbers of virulence genes than the OST group. All of the ST131-O25b and ST405 isolates and most of the ST131-O16 and OST isolates carried IncF replicons. The most prevalent IncF replicon sequence types differed between the four clonal groups. Both the ST131-O25b and ST405 clonal groups had a fluoroquinolone resistance mechanism in QRDRs, multidrug resistance, high virulence, and IncF plasmids, suggesting the potential for further global expansion and a need for measures against these clonal groups.
PMCID: PMC3811414  PMID: 23856781
2.  Clinical characteristics and risk factors of non-Candida fungaemia 
BMC Infectious Diseases  2013;13:247.
The incidence of fungaemia has been increasing worldwide. It is important to distinguish non-Candida fungaemia from candidaemia because of their different antifungal susceptibilities. The aims of this study were to investigate the clinical characteristics of non-Candida fungaemia and identify the clinical factors that differentiate it from candidaemia.
We investigated the clinical manifestations and mortality of non-Candida fungaemia in Kyoto University Hospital from 2004 to 2009.
There were 110 episodes of fungaemia during the study period. There were 11 renal replacement therapy episodes of fungaemia due to non-Candida yeasts (10.0%), including 6 episodes with Cryptococcus neoformans, 4 with Trichosporon asahii, and 1 with Kodamaea ohmeri, in addition to 99 episodes of candidaemia (90.0%). The presence of collagen disease [odds ratio (OR) 9.00; 95% confidence interval (CI) 1.58-51.4; P = 0.01] or renal replacement therapy (OR 15.0; 95% CI 3.06-73.4; P < 0.01) was significantly more common in non-Candida fungaemia patients than in candidaemia patients. Prior colonisation by the species may be a predictor of non-Candida fungaemia. Non-Candida fungaemia had a higher mortality than candidaemia (54.5% versus 21.2%, P = 0.03).
Although Candida species frequently cause fungaemia, we should also be aware of non-Candida yeasts because of their high mortality, particularly among high-risk patients, such as those with collagen disease and those under renal replacement therapy. Prior colonisation by the causative organisms may be an important predictor of non-Candida fungaemia.
PMCID: PMC3668224  PMID: 23714136
Fungaemia; Non-Candida yeast; Risk factor; Mortality; Colonisation
3.  Pneumocystis jiroveci pneumonia and colonization in patients with advanced lung cancer 
Oncology Letters  2012;5(2):601-604.
Pneumocystis jiroveci pneumonia (PCP) has long been recognized as a cause of mortality in immuno-compromised populations, including those with advanced lung cancer. Although Pneumocystis colonization has only recently been described due to the development of more sensitive molecular techniques, including polymerase chain reaction (PCR), it is unknown whether Pneumocystis colonization leads to the development of PCP. In the present study, we aimed to determine the prevalence of Pneumocystis colonization in advanced lung cancer patients. Furthermore, the association between PCP and Pneumocystis colonization was also investigated. Advanced lung cancer patients with no indication of PCP were evaluated to determine the prevalence of Pneumocystis colonization. We analyzed their oral wash (OW) samples and retrospectively evaluated advanced lung cancer patients with PCP by analyzing their sections of formalin-fixed, paraffin-embedded lung tissues obtained following a diagnosis of lung cancer. Pneumocystis colonization was determined by a PCR test for Pneumocystis jiroveci (P. jiroveci). No P. jiroveci was detected by PCR in the OW samples of 47 advanced lung cancer patients with no indication of PCP, or in the lung tissues of four advanced lung cancer patients with PCP. These results indicate that PCP is not associated with Pneumocystis colonization in advanced lung cancer patients, although this study is limited since this was a cross-sectional and retrospective study.
PMCID: PMC3572999  PMID: 23420670
Pneumocystis jiroveci pneumonia; colonization; lung cancer; immunocompromised host
4.  Validation of the japanese version of the sarcoidosis health questionnaire: A cross-sectional study 
Although impaired health-related quality of life (HRQOL) has been reported in patients with sarcoidosis, there is currently no sarcoidosis-specific questionnaire in Japan. The 29-item Sarcoidosis Health Questionnaire (SHQ), originally developed in the United States, is the only sarcoidosis-specific HRQOL questionnaire currently available. The primary aim of this study was to develop and validate a Japanese version of the SHQ.
The SHQ was translated into Japanese following the forward-backward procedure. The reliability and validity of the Japanese version of the SHQ were examined. One hundred twenty-two Japanese patients with biopsy-proven sarcoidosis were evaluated by the SHQ, the Medical Outcomes Study 36-item short form (SF-36), the St. George's Respiratory Questionnaire (SGRQ), chest radiography, an electrocardiogram, laboratory blood tests, pulmonary function tests, an echocardiogram, and assessments of dyspnea and depressive symptoms. The SHQ was found to have acceptable levels of internal consistency (Cronbach's coefficient α values = 0.68 to 0.91). SHQ scores correlated significantly with scores on the SF-36 and SGRQ. The domain or total scores on the SHQ also significantly correlated with serum levels of the soluble interleukin-2 receptor, the percentage of the predicted forced vital capacity, pulmonary arterial systolic pressure, dyspnea, and depressive symptoms. Also, the SHQ scores of patients who had one or two organ systems affected by sarcoidosis were significantly different from those of patients who had three or more organ systems involvement.
The Japanese version of the SHQ can be used to assess the HRQOL of patients with sarcoidosis.
PMCID: PMC3118310  PMID: 21569587
5.  Clinical characteristics of Pneumocystis pneumonia in non-HIV patients and prognostic factors including microbiological genotypes 
The number of patients with non-HIV Pneumocystis pneumonia (PCP) is increasing with widespread immunosuppressive treatment. We investigated the clinical characteristics of non-HIV PCP and its association with microbiological genotypes.
Between January 2005 and March 2010, all patients in 2 university hospitals who had been diagnosed with PCP by PCR were enrolled in this study. Retrospective chart review of patients, microbiological genotypes, and association with 30-day mortality were examined.
Of the 82 adult patients investigated, 50 patients (61%) had inflammatory diseases, 17 (21%) had solid malignancies, 12 (15%) had hematological malignancies, and 6 (7%) had received transplantations. All patients received immunosuppressive agents or antitumor chemotherapeutic drugs. Plasma (1→3) β-D-glucan levels were elevated in 80% of patients, and were significantly reduced after treatment in both survivors and non-survivors. However, β-D-glucan increased in 18% of survivors and was normal in only 33% after treatment. Concomitant invasive pulmonary aspergillosis was detected in 5 patients. Fifty-six respiratory samples were stored for genotyping. A dihydropteroate synthase mutation associated with trimethoprim-sulfamethoxazole resistance was found in only 1 of the 53 patients. The most prevalent genotype of mitochondrial large-subunit rRNA was genotype 1, followed by genotype 4. The most prevalent genotype of internal transcribed spacers of the nuclear rRNA operon was Eb, followed by Eg and Bi. Thirty-day mortality was 24%, in which logistic regression analysis revealed association with serum albumin and mechanical ventilation, but no association with genotypes.
In non-HIV PCP, poorer general and respiratory conditions at diagnosis were independent predictors of mortality. β-D-glucan may not be useful for monitoring the response to treatment, and genotypes were not associated with mortality.
PMCID: PMC3073915  PMID: 21439061
6.  Infectious background of febrile advanced lung cancer patients who received chemotherapy 
Oncology Letters  2010;1(5):849-853.
The study aimed to determine the diagnostic utility of procalcitonin (PCT) in order to discriminate between infective fever and fever due to inflammation in febrile advanced lung cancer patients treated with cytotoxic chemotherapy. A total of 121 patients with advanced lung cancer, treated with a cytotoxic chemotherapy regimen between September 2007 and September 2008 at Kyoto University Hospital, were recruited. Blood samples were obtained on the first day of the fever. Serum c-reactive protein (CRP) and PCT levels were measured. At least two blood cultures were performed, and sputum was taken for Gram staining and culture. There were 71 episodes in 61 patients in the 12 months of the study, representing 50.4% of our study population. A total of 41 patients (57.7%) were diagnosed with pneumonia using imaging modalities, 6 (8.5%) with bacteremia using blood culture and 4 (5.6%) with urinary tract infections using urine culture. Among the 41 pneumonia cases, culture from sputum revealed pathologic bacteria in 21 (51.2%) and fungal disease in 14 (34.1%) cases. Among the 71 febrile episodes, serum procalcitonin and CRP were measured in 50 episodes. Serum procalcitonin-positive patients showed poor outcomes on antibiotics therapy (Fisher's exact test, p=0.042). Furthermore, serum procalcitonin positivity was able to discriminate infective fever from fever due to inflammation (Chi-square test, p=0.001). We showed the causative organisms of febrile advanced lung cancer patients who received cytotoxic chemotherapy, as well as the possibility of PCT to discriminate infective fever from fever due to inflammation.
PMCID: PMC3436404  PMID: 22966393
pneumonia; advanced lung cancer; procalcitonin; immunocompromised host; c-reactive protein; prognosis
7.  Genotypes and Related Factors Reflecting Macrolide Resistance in Pneumococcal Pneumonia Infections in Japan▿  
Journal of Clinical Microbiology  2007;45(5):1440-1446.
Although macrolide-resistant Streptococcus pneumoniae strains possessing either the ermB or mefA gene are very common in Japan, clinical and microbial factors in community-acquired pneumonia (CAP) caused by different macrolide resistance genotypes have not been evaluated. A multicenter study of CAP caused by S. pneumoniae was performed in Japan from 2003 to 2005. A total of 156 isolates were tested for susceptibility to antibiotics correlated with ermB and mefA genotyping. Independent relationships between tested variables and possession of either the ermB or the mefA gene were identified. Of 156 isolates, 127 (81.4%) were resistant to erythromycin, with the following distribution of resistance genotypes: ermB alone (50.0%), mefA alone (23.7%), and both ermB and mefA (7.1%). All isolates were susceptible to telithromycin. By multivariate analysis, oxygen saturation of <90% on admission increased the risk for ermB-positive pneumococcal pneumonia (odds ratio [OR] = 11.1; 95% confidence interval [CI] = 1.30 to 95.0; P = 0.03), but there were no associations with mefA or with ermB mefA positivity. Penicillin nonsusceptibility was associated with mefA-positive and with ermB- and mefA-positive isolates (OR = 14.2; 95% CI = 4.27 to 46.9; P < 0.0001 and P < 0.0001, respectively) but not with ermB-positive isolates. The overall patient mortality was 5.1%. Mortality, the duration of hospitalization, and the resolution of several clinical markers were not associated with the different erythromycin resistance genotypes. In Japan, S. pneumoniae with erythromycin resistance or possession of ermB, mefA, or both genes was highly prevalent in patients with CAP. The risk factors for ermB-positive, mefA-positive, and double ermB-mefA-positive pneumococcal pneumonia were different, but the clinical outcomes did not differ.
PMCID: PMC1865875  PMID: 17344362
8.  Differences in Gamma Interferon Production Induced by Listeriolysin O and Ivanolysin O Result in Different Levels of Protective Immunity in Mice Infected with Listeria monocytogenes and Listeria ivanovii  
Infection and Immunity  2003;71(5):2447-2454.
Two pathogenic species in the genus Listeria, Listeria monocytogenes and Listeria ivanovii, are characterized by the production of hemolysins belonging to cholesterol-dependent cytolysins, listeriolysin O (LLO) and ivanolysin O (ILO), respectively. LLO, produced by L. monocytogenes, is able to induce gamma interferon (IFN-γ) production and contributes to the generation of Th1-dependent protective immunity. On the other hand, nothing is known about the role of ILO, produced by L. ivanovii, in this regard. In this study, we immunized mice with 0.1 50% lethal dose (LD50) of L. monocytogenes and L. ivanovii. Protective immunity against a challenge with 10 LD50 was generated in mice infected with L. monocytogenes, whereas L. ivanovii infection did not induce protection. After immunization, the level of IFN-γ in serum samples was increased in mice given L. monocytogenes but not in those given L. ivanovii. To determine the IFN-γ-inducing activity of cytolysins, recombinant protein was constructed. Recombinant ILO exhibited significantly lower IFN-γ-inducing activity than LLO. By comparing the IFN-γ-inducing activity of a chimera incorporating LLO and ILO, it was found that domains 1 to 3 of LLO were critical for IFN-γ-inducing activity while the counterpart in ILO was unable to induce cytokine production. These results suggested that the weak ability of ILO to induce IFN-γ production is responsible for the failure of L. ivanovii to generate effective protective immunity.
PMCID: PMC153848  PMID: 12704115
9.  An NMR study on the interaction of Escherichia coli DinI with RecA–ssDNA complexes 
Nucleic Acids Research  2003;31(6):1735-1743.
The SOS response, a set of cellular phenomena exhibited by eubacteria, is initiated by various causes that include DNA damage-induced replication arrest, and is positively regulated by the co- protease activity of RecA. Escherichia coli DinI, a LexA-regulated SOS gene product, shuts off the initiation of the SOS response when overexpressed in vivo. Biochemical and genetic studies indicated that DinI physically interacts with RecA to inhibit its co-protease activity. Using nuclear magnetic resonance (NMR) spectroscopy, we show that DinI tightly binds to the central region of RecA (between the N- and C-terminal domains) and that this interaction is enhanced upon the oligomerisation of RecA. On the other hand, DinI did not inhibit the interaction between 4mer single-stranded (ss)DNA and RecA– ATPγS, but had a slight effect on the structure of ssDNA–RecA–ATPγS complexes involving 8mer and 12mer ssDNA. We hypothesise that prevention of repressor binding to the intermolecular cleft region of RecA protomers by DinI, with the possibility of a slight conformational change induced in the DinI-bound ssDNA–RecA–ATPγS complex, together function to inhibit the co-protease activity of RecA.
PMCID: PMC152859  PMID: 12626715
10.  Seeligeriolysin O, a Cholesterol-Dependent Cytolysin of Listeria seeligeri, Induces Gamma Interferon from Spleen Cells of Mice  
Infection and Immunity  2003;71(1):234-241.
Seeligeriolysin O (LSO), one of the cholesterol-dependent cytolysins produced by Listeria seeligeri, shows 80% homology to listeriolysin O (LLO) produced by Listeria monocytogenes at the amino acid sequence level. In addition to cytolytic activity, LLO has been shown to exhibit cytokine-inducing activity. In order to determine whether LSO is also capable of exhibiting these two different activities, we constructed a recombinant full-length LSO (rLSO530) and a noncytolytic truncated derivative with a C-terminal deletion (rLSO483) and compared these molecules with recombinant LLO. The cytolytic rLSO530 molecule could induce gamma interferon (IFN-γ) production in spleen cells when the cytolytic activity was blocked by treatment with cholesterol. The noncytolytic truncated rLSO483 molecule also induced IFN-γ production. Anti-LLO polyclonal antibody inhibited not only LLO-induced IFN-γ production but also LSO-induced IFN-γ production. Both NK cells and CD11b+ cells were required for LSO-induced IFN-γ production. Among the various cytokines expressed in CD11b+ cells, interleukin-12 (IL-12) and IL-18 appeared to be essential. We concluded that LSO exhibits the same biological activity as LLO.
PMCID: PMC143279  PMID: 12496171
11.  Dissociated Linkage of Cytokine-Inducing Activity and Cytotoxicity to Different Domains of Listeriolysin O from Listeria monocytogenes 
Infection and Immunity  2002;70(3):1334-1341.
Listeriolysin O (LLO), a cholesterol-binding cytolysin of Listeria monocytogenes, exhibits cytokine-inducing and cytolytic activities. Because the cytolytic activity was abolished by cholesterol treatment but the cytokine-inducing activity was not, these activities appeared to be linked to different domains of the LLO molecule. In this study, we constructed recombinant full-length LLO (rLLO529) and various truncated derivatives and examined their cytolytic, cholesterol-binding, and gamma interferon (IFN-γ)-inducing activities. rLLO529 exhibited both IFN-γ-inducing and cytolytic activities. Four truncated rLLOs possessing different C termini, which did not exert either cytolytic or cholesterol-binding activity, stimulated IFN-γ production in normal spleen cells. However, a truncated rLLO corresponding to domain 4 (rLLO416-529) did not exhibit IFN-γ-inducing activity, whereas it did bind to immobilized cholesterol. In addition, though the hemolysis induced by rLLO529 was inhibited by rLLO416-529, such inhibition was not detected upon rLLO529-induced IFN-γ production. These data indicated that domain 4 was responsible for binding of LLO to membrane cholesterol followed by oligomerization and pore formation by the entire LLO molecule. In contrast, the other part of LLO, corresponding to domain 1-3, was essential for IFN-γ-inducing activity. These findings implied a novel aspect of the function of LLO as a bacterial modulin.
PMCID: PMC127785  PMID: 11854218
12.  Essential Role of Interleukin-12 (IL-12) and IL-18 for Gamma Interferon Production Induced by Listeriolysin O in Mouse Spleen Cells 
Infection and Immunity  2002;70(3):1049-1055.
The mechanism of gamma interferon (IFN-γ) production induced by listeriolysin O (LLO), a cytolytic virulence factor of Listeria monocytogenes, was analyzed with special reference to the involvement of macrophage-derived cytokines in spleen cells of mice. LLO purified from the culture supernatant of L. monocytogenes was capable of inducing a high level of IFN-γ when its cytolytic activity was blocked by cholesterol treatment. The IFN-γ-inducing ability of LLO was not dependent on possibly contaminating lipopolysaccharide. Depletion of CD11b+ cells resulted in a profound decrease in IFN-γ production in response to LLO stimulation. Negative selection also suggested the contribution of DX5+ cells in IFN-γ production. Reverse transcription-PCR revealed that expression of interleukin-12 (IL-12) p35 and p40 was induced by LLO but that the IL-18 mRNA level in the CD11b+ fraction of spleen cells was unchanged. There was no change in the expression of the IFN-γ-inducing cytokine genes in the CD11b− fraction. Neutralization of IL-12 and IL-18 in culture abolished the IFN-γ production almost completely. Spleen cells from IL-12- or IL-18-deficient mice never produced IFN-γ after stimulation with LLO. These results clearly indicated that LLO, a well-known virulence factor of L. monocytogenes, is capable of inducing IFN-γ from NK cells through induction of IL-12 and IL-18 from macrophages. LLO appeared to play essential roles, not only as a bacterial virulence factor but also as a bacterial modulin in the immune response of the host.
PMCID: PMC127750  PMID: 11854182
13.  Induction of Gamma Interferon and Nitric Oxide by Truncated Pneumolysin That Lacks Pore-Forming Activity  
Infection and Immunity  2002;70(1):107-113.
Pneumolysin (PLY), an important virulence factor of Streptococcus pneumoniae, is known to exert various effects on the host immune cells, including cytokine induction, in addition to its known cytolytic activity as a member of the thiol-activated cytolysins. It is of interest to determine whether cytolytic activity is involved in triggering the cytokine production. In this study, we constructed full-length recombinant PLY and noncytolytic truncated PLYs with C-terminal deletions to examine the response of spleen cells to these PLY preparations. When cytolytic activity was blocked by treatment with cholesterol, full-length PLY was capable of inducing gamma interferon (IFN-γ) production. Truncated PLYs that originally exhibited no cytolytic activity were also active in IFN-γ induction. Therefore, the IFN-γ-inducing ability of PLY appeared to be independent of the cytolytic activity. Furthermore, IFN-γ-inducing preparations were also capable of inducing nitric oxide synthase expression and nitric oxide (NO) production, and the addition of neutralizing antibody to IFN-γ abolished the NO production. These results clearly demonstrated that PLY is capable of inducing IFN-γ production in spleen cells by a mechanism different from pore formation and that the induced IFN-γ stimulates NO production. These findings were discussed with reference to the contribution of PLY to the virulence of S. pneumoniae in vivo.
PMCID: PMC127632  PMID: 11748170

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