Toshiaki Komori

Estimation of Sulphoconjugated Catecholamine Concentrations in Plasma by High-Performance Liquid Chromatography

Free and sulphoconjugated catecholamine (CA) concentrations were measured in plasma using a fully automated and sensitive analyser equipped with a three-column system of high-performance liquid chromatography (HPLC). Although the free dopamine (DA) concentration has been below the detection limit of HPLC analysers thus far available, this new CA analyser can measure as little as 0·03 nmol/L. For the estimation of sulphoconjugated CA, we performed enzymatic deconjugation with arylsulphatase prior to HPLC analysis. The difference between free and total concentrations represents that of sulphoconjugated CA. The intraassay coefficient variation was less than 2·2% for free noradrenaline (NA) and adrenaline (A), 12·62% for free DA, and less than 4·3% for total A, and DA. These assays of free and sulphoconjugated CA are simple and can be performed routinely by a suitably equipped laboratory.

CTX-M-27- and CTX-M-14-producing, ciprofloxacin-resistant Escherichia coli of the H30 subclonal group within ST131 drive a Japanese regional ESBL epidemic

OBJECTIVES The global increase in ESBL-producing Escherichia coli is associated with the ST131 clonal group, especially its CTX-M-15-producing H30Rx subset. To understand the rapid spread of ESBL-producing E. coli in Japan, we investigated the molecular epidemiology and ESBL-associated genetic environments of Japanese ST131 isolates. METHODS Between 2001 and 2012, 1079 ESBL-producing E. coli isolates were collected at 10 Japanese acute-care hospitals. ESBL types, ST131 status, fimH allele, H30Rx-defining sequences and ESBL-associated genetic environments were defined using PCR and sequencing. Subclonal groups were defined based on fimH allele and H30Rx status. RESULTS Overall, 461 (43%) of the 1079 ESBL-producing E. coli isolates represented ST131. According to fimH-based subclonal typing, the ST131 isolates included 398 fimH allele 30 (H30) isolates, 49 H41 isolates, 10 H22 isolates and 4 other fimH-type isolates. The 398 H30 isolates included 396 ciprofloxacin-resistant H30R isolates, of which 64 (16%) represented the H30Rx subset. Between 2001 and 2007, the CTX-M-14-producing H30R subgroup predominated, accounting for 46% of ST131 isolates, whereas the CTX-M-27-producing H30R and CTX-M-15-producing H30Rx subgroups were rarely detected. In contrast, from 2008 onward the latter two subgroups rose to dominance, accounting for 45% and 24% of ST131 isolates, respectively, versus only 15% for the (formerly dominant) CTX-M-14-producing H30R subgroup. The emergent CTX-M-27-H30R subgroup frequently had an IS26-ΔISEcp1-blaCTX-M-27-ΔIS903D-IS26-like structure, whereas the older CTX-M-14-H30R subgroup frequently had an ISEcp1-blaCTX-M-14-IS903D-like structure. CONCLUSIONS This Japanese regional ESBL-producing E. coli epidemic is closely associated with newly identified CTX-M-27- and CTX-M-14-producing ST131 H30R subclonal groups and with mobile elements IS26, ISEcp1 and IS903D.

Eicosapentaenoic Acid Stimulates Nitric Oxide Production And Decreases Cardiac Noradrenaline In Diabetic Rats

1. The aim of the present study was to investigate whether long‐term oral administration of eicosapentaenoic acid increases nitric oxide (NO) production and affects cardiac sympathetic activity in rats with diabetes mellitus.

Prevalence of plasmid-mediated AmpC β-lactamase-producing Escherichia coli and spread of the ST131 clone among extended-spectrum β-lactamase-producing E. coli in Japan

In 2010, a total of 1327 clinical Escherichia coli isolates from five hospitals in the Kyoto and Shiga regions of Japan were analysed by PCR. The prevalences of plasmid-mediated AmpC β-lactamase (pAmpC)-producers, extended-spectrum β-lactamase (ESBL)-producers and co-producers of pAmpC and ESBL were 1.7%, 9.7% and 0.3%, respectively. Less than one-half of the pAmpC-producers were reported to be resistant to third-generation cephalosporins, cephamycins and β-lactam/β-lactam inhibitors using the old 2009 Clinical and Laboratory Standards Institute (CLSI) breakpoints. CMY-2 was the most prevalent pAmpC type (95%), and CTX-M-14 (38%), CTX-M-15 (26%) and CTX-M-27 (19%) were the most prevalent ESBL types. The worldwide O25b-ST131-B2 clone accounted for 11% of pAmpC-producers and 41% of ESBL-producers. The O25b-ST131-B2 clone was characterised by a CTX-M-27- or CTX-M-15-type ESBL and ciprofloxacin-non-susceptibility with quadruple mutations in the quinolone resistance-determining regions (S83L and D87N in GyrA and S80I and E84V in ParC). A significant proportion of pAmpC-producers and the O25b-ST131-B2 clone were found in Japan by a recent regional surveillance programme.

Multicenter Retrospective Study of Cefmetazole and Flomoxef for Treatment of Extended-Spectrum-β-Lactamase-Producing Escherichia coli Bacteremia

ABSTRACT The efficacy of cefmetazole and flomoxef (CF) for the treatment of patients with extended-spectrum β-lactamase-producing Escherichia coli (ESBL-EC) bacteremia (ESBL-CF group) was compared with that of carbapenem treatment for ESBL-EC patients (ESBL-carbapenem group) and with that of CF treatment in patients with non-ESBL-EC bacteremia (non-ESBL-CF group). Adult patients treated for E. coli bacteremia in four hospitals were retrospectively evaluated. The 30-day mortality rates in patients belonging to the ESBL-CF, ESBL-carbapenem, and non-ESBL-CF groups were compared as 2 (empirical and definitive therapy) cohorts. The adjusted hazard ratios (aHRs) for mortality were calculated using Cox regression models with weighting according to the inverse probability of propensity scores for receiving CF or carbapenem treatment. The empirical-therapy cohort included 104 patients (ESBL-CF, 26; ESBL-carbapenem, 45; non-ESBL-CF, 33), and the definitive-therapy cohort included 133 patients (ESBL-CF, 59; ESBL-carbapenem, 54; non-ESBL-CF, 20). The crude 30-day mortality rates for patients in the ESBL-CF, ESBL-carbapenem, and non-ESBL-CF groups were, respectively, 7.7%, 8.9%, and 3.0% in the empirical-therapy cohort and 5.1%, 9.3%, and 5.0% in the definitve-therapy cohort. In patients without hematological malignancy and neutropenia, CF treatment for ESBL-EC patients was not associated with mortality compared with carbapenem treatment (empirical-therapy cohort: aHR, 0.87; 95% confidence interval [CI], 0.11 to 6.52; definitive therapy cohort: aHR, 1.04; CI, 0.24 to 4.49). CF therapy may represent an effective alternative to carbapenem treatment for patients with ESBL-EC bacteremia for empirical and definitive therapy in adult patients who do not have hematological malignancy and neutropenia.

Anaerobic bacteremia: the yield of positive anaerobic blood cultures: patient characteristics and potential risk factors.

Abstract The anaerobic blood culture (AN) bottle is routinely used in Japan with little discussion as to its justification or validity. We retrospectively studied the AN bottle yield of obligate anaerobes and the characteristics of, and potential risk factors in, patients with anaerobic bacteremia during a 2-year period (1999–2000) at four university hospitals and one community hospital. Thirty-four of 18310 aerobic and anaerobic blood culture sets from 6215 patients taken at the university hospitals, and 35 of 2464 samples taken from 838 patients at the community hospital, yielded obligate anaerobes. Bacteroides species and Clostridium species accounted for 60% of the isolates. Fifty-seven patients from 69 blood culture sets containing anaerobes had clinically significant anaerobic bacteremia. Among these 57 patients, 24 (49%) were oncology patients, 40 (70%) had an obvious source of anaerobic infection, 15 (26%) had recent surgery and/or were in an immunosuppressed state. We concluded that the recovery rate of obligate anaerobes isolated from AN bottles was low, and the patients with anaerobic bacteremia had limited number of underlying diseases or potential risk factors for anaerobic infections. Therefore, anaerobic blood cultures may be selectively used according to the potential risk for anaerobic infections.

Dopamine stimulation of cardiac β‐adrenoceptors: the involvement of sympathetic amine transporters and the effect of SKF38393

1 Mechanisms underlying β‐adrenoceptor stimulation by dopamine were examined on guinea‐pig Langendorff‐perfused hearts and isolated cells from the right atrium, by using the chronotropic effects and the enhancement of L‐type Ca2+ current (ICa,L) in the presence of prazosin as indicators of β‐adrenoceptor stimulation. Dopamine‐induced overflow of noradrenaline (NA) concentrations was measured by high‐performance liquid chromatography. 2 Dopamine caused positive chronotropic effects with an EC50 of 2.5 μm and induced NA overflow with a similar EC50 (1.3 μm). The chronotropic effect of dopamine was abolished by bisoprolol (1 μm). 3 The effects of dopamine were maintained during prolonged application, whereas the effects of tyramine faded with time. Dopamine (3 μm) restored the chronotropic effects and the NA release suppressed by pretreatment with tyramine, suggesting a de novo synthesis of NA during the exposure to dopamine. 4 Dopamine (3 μm)‐induced NA release was not affected by tetrodotoxin, ω‐conotoxin, rauwolscine, ICI118551 or sulpiride, but was inhibited by desipramine, a NA uptake inhibitor (IC50 ∼1 μm). It was also not affected by GBR12909 and bupropion, dopamine uptake inhibitors in the central nervous system. 5 SKF38393, a D1 receptor partial agonist, potently inhibited the 3 μm dopamine‐induced release of NA (IC50 ∼0.1 μm). D1 receptors are not involved in the DA‐induced release of NA, since SCH23390 (3 μm), a potent D1 antagonist, inhibited the NA release only slightly, and dihydrexidine (1 μm) and chloro‐APB (1 μm), full D1 agonists, caused no significant NA release. 6 SKF38393 inhibited tyramine‐induced overflow of NA, and potentiated the field stimulation‐induced NA release. SKF38393 and desipramine retarded the decay of the stimulation‐induced tachycardia in a similar manner. These results indicate that SKF38393 is a potent monoamine transport inhibitor and a useful tool for the functional evaluation of indirectly‐acting sympathomimetic agonists in the heart. In the presence of SKF38393 (10 μm), dopamine at 1 μm showed no chronotropic effect. 7 Voltage clamp experiments with isolated atrial cells revealed that dopamine is a weak partial agonist. The EC50 for ICa,L stimulation by dopamine was high (13 μm). As a result, dopamine at 1 μm did not affect ICa,L. Bisoprolol abolished the stimulation of ICa,L by dopamine (30 μm), and dihydrexidine (1 μm) did not affect ICa,L. 8 It was concluded that the cardiac effects of dopamine at clinically relevant concentrations (<1 μm) result almost exclusively from the indirect effect of β adrenoceptor stimulation, involving the release of NA from sympathetic nerve terminals. The roles of the direct stimulation of β adrenoceptors by dopamine at these concentrations and the stimulation of postjunctional D1 receptors seem negligible. The desipramine‐ and SKF38393‐sensitive monoamine transporter mediates the release of NA.

Molecular and clinical characterization of plasmid-mediated AmpC β-lactamase-producing Escherichia coli bacteraemia: a comparison with extended-spectrum β-lactamase-producing and non-resistant E. coli bacteraemia

Plasmid-mediated AmpC β-lactamase-producing Escherichia coli (AmpC-E) bacteraemia was characterized by comparison with bacteraemia caused by extended-spectrum β-lactamase (ESBL)-producing E. coli (ESBL-E) and non-resistant E. coli (NR-E) in the era of the worldwide spread of the CTX-M-15-producing O25b-ST131-B2 clone. Of 706 bloodstream E. coli isolates collected between 2005 and 2010 in three Japanese university hospitals, 111 ESBL screening-positive isolates were analysed for AmpC and ESBL genes by PCR. A case-control study was performed in which the cases consisted of all of the patients with AmpC-E bacteraemia. Phylogenetic groups, sequence types and O25b serotype were determined. Twenty-seven AmpC-E isolates (26 of which were of the CMY-2 type) were identified, and 54 ESBL-E and 54 NR-E isolates were selected for the controls. Nineteen AmpC-E isolates were also positive for ESBL. CTX-M-14 was the most prevalent ESBL type among both the AmpC-E and ESBL-E isolates. The O25b-ST131-B2 clone was the most prevalent among the ESBL-E isolates (26%) and the second most prevalent among the NR-E isolates (13%), but only one O25b-ST131-B2 clone was found among the AmpC-E isolates. Twenty-three different sequence types were identified among the AmpC-E isolates. When compared with bacteraemia with ESBL-E, previous isolation of multidrug-resistant bacteria and intravascular catheterization were independently associated with a lower risk for AmpC-E. When compared with NR-E bacteraemia, prior use of antibiotics was the only significant risk factor for AmpC-E. Unlike the spread of the O25b-ST131-B2 clone between ESBL-E and NR-E, the AmpC-E isolates were not dominated by any specific clone.

Optimized dosage and frequency of cefozopran for patients with febrile neutropenia based on population pharmacokinetic and pharmacodynamic analysis

OBJECTIVES To establish a cefozopran (a fourth-generation cephem) population pharmacokinetic model using patient data and use it to explore alternative dosage regimens that could optimize the currently used dosing regimen to achieve higher likelihood of pharmacodynamic exposure against pathogenic bacteria. METHODS We conducted a prospective clinical trial of cefozopran for haematological patients with febrile neutropenia (FN). Twenty-two patients (30 episodes) were selected to receive intravenous cefozopran every 8 h on a daily basis. We gathered concentration data and performed the NONMEM program. The Monte Carlo simulation was performed to assess the pharmacodynamic exposure based on the population pharmacokinetics and MIC. RESULTS The NONMEM program demonstrated that a two-compartment model provided a best fit for the data, that is, CL of 4.62 (L/h), V1 of 10.3 (L), Q of 4.47 (L/h), and V2 of 4.48 (L). On the basis of the Japanese national surveillance findings for Pseudomonas aeruginosa, methicillin-sensitive Staphylococcus aureus, coagulase-negative Staphylococcus, viridans group streptococci, Escherichia coli and Klebsiella pneumoniae, Monte Carlo simulation data showed that probability of target attainment(T>MIC = 70%) is 67% to 97% for dosing every 8 h, and 48% to 88% for dosing every 12 h. For the patients in whom the efficacy of cefozopran could be evaluated, 17 of 22 patients (77.2%) survived the episode of FN without requiring further antibacterial treatment. CONCLUSIONS Our study proved that Monte Carlo simulation based on population pharmacokinetics can determine optimized dosage and method. The optimal regimen for this cephem was found to be three times daily.

The mechanisms underlying heart stimulation by dopamine, with special reference to direct and indirect β adrenoceptor stimulation

1. The positive chronotropic and norepinephrine-releasing effects of dopamine were examined in the isolated guinea pig heart, using the Langendorff model. 2. The released norepinephrine was estimated from the norepinephrine concentration measured in the post-perfusion solution using HPLC. 3. The dose-response curve for dopamine to stimulate the heart rate (HR) closely resembled that for the norepinephrine release. A selective beta 1 antagonist bisoprolol completely abolished the positive chronotropic effect, but did not affect the norepinephrine release. 4. The HR increase in response to 3 mumol/L dopamine was 54 +/- 15% (n = 14) of the control in normal hearts. The response was decreased to 15 +/- 7% (n = 6) by pretreatment with reserpine. 5. A D1 antagonist, SKF83742, (3 mumol/L) shifted the dose-response curve for the dopamine-induced norepinephrine release toward the right, indicating the involvement of D1-like dopamine receptors. 6. Voltage clamp experiments using single cells isolated from the right atrium revealed that dopamine is a weak partial agonist for beta adrenoceptors. Dopamine stimulated the L-type Ca2+ current with a threshold concentration of 3 mumol/L. 7. These findings indicate the important role of the norepinephrine release in the stimulation of beta adrenoceptors by dopamine at clinically relevant concentrations.