In vitro activity of tedizolid against 43 species of Nocardia species.

The purpose of the present study was to evaluate the in vitro activity of tedizolid against several clinically significant species of Nocardia by comparing with that of linezolid. A total of 286 isolates of Nocardia species, including 236 clinical isolates recovered from patients in Japan and 50 strains (43 species) purchased from NITE Biological Resource Center, were studied. Antimicrobial susceptibility testing was performed using the broth microdilution method. For the 286 Nocardia isolates, the minimal inhibitory concentration (MIC)50 and MIC90 values of tedizolid were 0.25 and 0.5 µg/ml, and those of linezolid were 2 and 2 µg/ml, respectively. The distribution of the linezolid/tedizolid ratios (MICs of linezolid/MICs of tedizolid) showed that tedizolid had four- to eight-fold higher activity than linezolid in 96.1% (275/286) of Nocardia isolates. Both the tedizolid and linezolid MIC90 values for Nocardia brasiliensis were two-fold higher than those for the other Nocardia species. Both tedizolid and linezolid had low MIC values, 0.25-1 µg/ml and 0.5-4 µg/ml, respectively, even against nine isolates (five species) that were resistant to trimethoprim/sulfamethoxazole. One Nocardia sputorum isolate showed reduced susceptibility to tedizolid (4 µg/ml). Bioinformatics analysis suggests different resistance mechanisms than the oxazolidinone resistance seen in enterococci and staphylococci.

Nocardia sputorum sp. nov., an actinobacterium isolated from clinical specimens in Japan.

Two novel actinobacteria, designated IFM 12276T and IFM 12275, were isolated from clinical specimens in Japan, and their taxonomic positions were investigated using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequence comparisons revealed that strains IFM 12276 T and IFM 12275 have completely identical 16S rRNA gene sequences and were closely related to members of the genus Nocardia. The highest 16S rRNA gene sequence similarity was observed to Nocardia beijingensis (99.6 %) and Nocarida sputi (99.6 %), followed by Nocardia niwae (99.3 %) and Nocardia araoensis (99.3 %). The whole-cell hydrolysates of strains IFM 12276T and IFM 12275 contained meso-diaminopimelic acid, arabinose and galactose. The acyl type of muramic acid was N-glycolyl. The predominant isoprenoid quinone was MK-8(H4, ω-cycl.) and the principal polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannosides. Strains IFM 12276T and IFM 12275 contained mycolic acids that co-migrated with those from the type strain of N. niwae. These chemotaxonomic features corresponded to those of the genus Nocardia. Meanwhile, the differences in some phenotypic characteristics, along with the results of average nucleotide identity and digital DNA-DNA hybridization analyses, indicated that strains IFM 12276 T and IFM 12275 should be distinguished from the recognized species of the genus Nocardia. Therefore, these strains represent a novel species of the genus Nocardia, for which the name Nocardia sputorum sp. nov. is proposed. The type strain is IFM 12276T (=NBRC 115477T=TBRC 17096T).

Phase I dose-escalation study of milademetan in patients with relapsed or refractory acute myeloid leukemia.

Long-term survival in patients with acute myeloid leukemia (AML) remains low, and current treatment modalities are inadequate. Milademetan (DS-3032, RAIN-32), a small-molecule specific murine double minute 2 inhibitor, has shown a p53 status-dependent antitumor effect in vitro studies. This is the first phase I study report of milademetan monotherapy in relapsed/refractory (R/R) AML patients evaluating the safety, tolerability, pharmacokinetics, and preliminary tumor response for further clinical development. Fourteen patients received 90 (starting dose, n = 4), 120 (n = 6), or 160 mg (n = 4) of oral milademetan once daily in a 14/28 treatment cycle. The median total treatment duration was 1.5 cycles. Dose-limiting toxicity did not occur, and the maximum tolerated dose was not reached. Thus, the recommended dose was defined as 160 mg. The most common adverse events (AEs) were decreased appetite (64.3%), febrile neutropenia (50%), nausea (42.9%), and anemia (35.7%). No deaths or AEs leading to treatment discontinuation occurred. Five serious treatment-emergent AEs occurred in 4 patients. Plasma concentration increased linearly with milademetan dose. However, trends in the safety and efficacy of oral milademetan in patients with R/R AML warrant further clinical investigation. This study can inform future milademetan studies in hematologic malignancies.

Safety and pharmacokinetics of milademetan, a MDM2 inhibitor, in Japanese patients with solid tumors: A phase I study.

Milademetan (DS-3032, RAIN-32) is an orally available mouse double minute 2 (MDM2) antagonist with potential antineoplastic activity owing to increase in p53 activity through interruption of the MDM2-p53 interaction. This phase I, dose-escalating study assessed the safety, tolerability, efficacy, and pharmacokinetics of milademetan in 18 Japanese patients with solid tumors who relapsed after or were refractory to standard therapy. Patients aged ≥ 20 years received oral milademetan once daily (60 mg, n = 3; 90 mg, n = 11; or 120 mg, n = 4) on days 1 to 21 in a 28-day cycle. Dose-limiting toxicities, safety, tolerability, maximum tolerated dose, pharmacokinetics, and recommended dose for phase II were determined. The most frequent treatment-emergent adverse events included nausea (72.2%), decreased appetite (61.1%), platelet count decreased (61.1%), white blood cell count decreased (50.0%), fatigue (50.0%), and anemia (50.0%). Dose-limiting toxicities (three events of platelet count decreased and one nausea) were observed in the 120-mg cohort. The plasma concentrations of milademetan increased in a dose-dependent manner. Stable disease was observed in seven out of 16 patients (43.8%). Milademetan was well tolerated and showed modest antitumor activity in Japanese patients with solid tumors. The recommended dose for phase II was considered to be 90 mg in the once-daily 21/28-day schedule. Future studies would be needed to further evaluate the potential safety, tolerability, and clinical activity of milademetan in patients with solid tumors and lymphomas. The trial was registered with Clinicaltrials.jp: JapicCTI-142693.

Patritumab plus trastuzumab and paclitaxel in human epidermal growth factor receptor 2-overexpressing metastatic breast cancer.

Human epidermal growth factor receptor 3 (HER3) expression in lung and breast cancers has a negative impact on survival. Patritumab, a human anti-HER3 mAb, has shown anticancer activity in preclinical models. This study examined the safety and pharmacokinetics of patritumab in combination with trastuzumab and paclitaxel in patients with HER2-overexpressing metastatic breast cancer. In this open-label, multicenter, dose-escalation, phase Ib study, patients received patritumab 9 or 18 mg/kg plus trastuzumab and paclitaxel at known tolerated doses. Safety and tolerability were assessed based on dose-limiting toxicities and other non-life threatening adverse events. The pharmacokinetic profile for patritumab was determined based on the target trough level. Clinical efficacy was evaluated based on the overall response rate and progression-free survival. Six patients received patritumab 9 mg/kg and 12 received 18 mg/kg. The most common adverse events were diarrhea, alopecia, leukopenia, neutropenia, and maculopapular rash. No dose-limiting toxicities were observed. The target trough serum concentration was achieved in all patients at a dose of 18 mg/kg. Overall response rate was 38.9% and median progression-free survival was 274 days. In conclusion, patritumab plus trastuzumab and paclitaxel was tolerable and efficacious at both doses. We recommend the dose level of 18 mg/kg for future phase II studies. (Clinical trial registration: JapicCTI-121772.).

Blood pressure-lowering effects of angiotensin receptor antagonist monotherapy and in combination with other anti-hypertensive drugs in primary care settings in Japan.

Combination therapy with multiple anti-hypertensives is required to achieve target blood pressure (BP) control and is recommended as the first-line therapy in hypertension. Although angiotensin receptor blockers (ARBs) may be combined with other anti-hypertensives, it is unclear how the effects of ARBs are influenced by co-administered anti-hypertensives. We investigated the effect of olmesartan medoxomil (OLM) when it is given alone (monotherapy) or concomitantly with other anti-hypertensives in 6507 OLM-naive Japanese in "real world" clinical practice. After a 12-week treatment, BP was significantly reduced from baseline in both the monotherapy group and the combination therapy group (P < 0.0001). The BP-lowering efficacy after treatment and achievement rates of target BP were similar in both groups. In the combination therapy group, no significant difference of achieved BP level was detected between patients taking Calcium channel blockers and any other class of anti-hypertensive drugs. This study suggests that ARBs such as OLM-elicits BP-lowering efficacy as either a first- or second-line agent and its effects are minimally influenced by co-administered anti-hypertensives.

[Evaluation of three selective enrichment media for detection of Group B Streptococcus in vaginal swabs].

As the most common cause of neonatal sepsis, Lancefield Group B Streptococcus (GBS) must be diagnosed as early as possible in pregnant women is prevent neonatal infection. A selective enrichment broth medium has been widely recommended to optimally recover GBS from genital and anorectal samples. To establish a culture suitable for screening vaginal swab specimens, we compared subcultures of three selective enrichment media to direct culture on agar medium. Vaginal swab samples were inoculated directly onto 5% sheep blood agar and into New Granada medium (Eiken), Lim broth (Becton, Dickinson, and Company), and Todd Hewitt broth with gentamicin and nalidixic acid (Becton, Dickinson, and Company, Todd). Of the 288 specimens tested, GBS was recovered from 43 samples (14.9%) on direct agar media, with 82 (28.5%), positive on New Granada medium subculture, 67 (23.3%) on Lim broth subculture, and 61 (21.2%) on Todd, subculture. These results demonstrates that selective enrichment broth media provides more superior sensitivity than direct agar media for detection of GBS colonization in vaginal specimens, underscoring the usefulness of selective enrichment broth media in GBS screening for vaginal swabs in pregnant woman.

[Current biosafety in clinical laboratories in Japan: report of questionnaires' data obtained from clinical laboratory personnel in Japan].

To determine the status of biosafety in clinical laboratories in Japan, we conducted a survey using questionnaires on the biosafety of laboratory personnel in 2004. We obtained data from 431 hospitals (response: 59.5%). Respondents were 301 institutions (70%) having biological safety cabinets (BSCs). BSCs were held in 78% of microbiological laboratories, 7.9% of genetic laboratories, 2.7% of histopathological laboratories, and 1% or less at other laboratories. A clean bench in examination rooms for acid-fast bacilli was applied at 20 hospitals. We found 28 cases of possible laboratory-associated tuberculosis infection, 25 of which were associated with lack of BSC. Other risk factors were immature skills and insufficiently skilled eguipment operation. The frequency of rupture accidents during specimen centrifugation was 67% in dealing with blood and 9.7% in collecting acid-fast bacilli. Half or more accidents were related to inadequate sample tube materials. Technologists were shown to be working on blood collection in many hospitals (75%), and 1,534 events of self-inflicted needle puncture developed in the last 5 years. These results suggest that biosafety systems are woefully lacking or inadequate in clinical laboratories in Japan and must be established at the earliest possible opportunity.

Local regulation of skin blood flow during cooling involving presynaptic P2 purinoceptors in rats.

1. This study investigated a local effect of cooling on the plantar skin blood flow (PSBF) of tetrodotoxin-treated rats by laser-Doppler flowmetry. 2. When the air temperature around the left foot was locally cooled from 25 to 10 degrees C, the PSBF of the left foot decreased. 3. The response was inhibited by the alpha-adrenoceptor antagonist phentolamine, the alpha1-adrenoceptor antagonist bunazosin, the alpha2-adrenoceptor antagonist RS79948, and bretylium and guanethidine that inhibit noradrenaline release from sympathetic nerves. Adrenalectomy of the rats did not affect the cooling-induced response. 4. The P2 purinoceptor antagonists suramin and PPADS also significantly suppressed the cooling-induced reduction of PSBF. However, the inhibitory effect of PPADS on the cooling-induced response was abolished after the treatment with phentolamine. Intra-arterial injections of ATPgammaS, a stable P2 purinoceptor agonist, at 25 degrees C caused a transient decrease in PSBF in a dose-dependent manner, which was significantly inhibited by phentolamine and guanethidine. 5. These results suggest a novel mechanism for local cooling-induced reduction of skin blood flow in vivo; moderate cooling of the skin induces the release of ATP, which stimulates presynaptic P2 purinoceptors on sympathetic nerve terminals and facilitates the release of noradrenaline, thereby causing contractions of skin blood vessels via the activation of alpha1-and alpha2-adrenoceptors.

[Surveillance of antimicrobial resistance of Streptococcus pneumoniae isolates from the Kinki Region of Japan during 2003/2004. The Surveillance Program of Bacterial Resistance in the Kinki Region of Japan; SBRK].

Three hundred seventy five isolates of Streptococcus pneumoniae were collected from 14 medical institutions in the Kinki region of Japan between November 2003 and February 2004. We determined the minimum inhibitory concentration (MIC) of penicillin G (PCG) and 25 of other antimicrobial agents against these isolates according to the National Committee for Clinical Laboratory Standards (NCCLS). Overall, 71.5% of all isolates were resistant to PCG (intermediate and resistant categories were 51.7% and 19.8%, respectively). For the carbapenems and penem, the rank order of activity was PAPM (MIC90, 0.12 microg/ml) > IPM (0.25 microg/ml) > MEPM (0.5 microg/ml) = FRPM (0.5 microg/ml). For the cephems, the overall rank order of activity was CPR (MIC90, 0.5 microg/ml) = CDTR (0.5 microg/ml) > CTRX (1 microg/ml) = CTX (1 microg/ml) = CZOP (1 microg/ml) = CFPN (1 microg/ml). Rank order activity for six of fluoroquinolones was TFLX = MFLX (MIC90, 0.25 microg/ml) > GFLX (0.5 microg/ ml) = SPFX (0.5 microg/ml) > LVFX (1 microg/ml) > PZFX (4 microg/ml). The rate of resistance to fluoroquinolones per the NCCLS criteria were very low, ranging from 0.7% to 2.6%. Rate of resistance to other antimicrobiotics were CAM, 77.0%; CLDM, 41.7%; TEL, 0%; VCM, 0%; ST, 32.7%, and CP, 21.4%.

In vitro activity of beta-lactams and quinolones against AmpC beta-lactamase-producing Escherichia coli.

We studied the antimicrobial susceptibility of AmpC beta-lactamase-producing Escherichia coli isolates collected at ten medical institutions in the Kinki area of Japan during a 6-month period (November 2002 through April 2003). Of 2845 E. coli isolates tested, 29 (1.0%) showed a minimum inhibitory concentration (MIC) for cefazolin of more than 8 microg/ml and were three-dimensional extract test positive. In standard inoculum susceptibility tests against these 29 strains, the MIC90s for the four carbapenems tested ranged from 0.06 microg/ml to 0.5 microg/ml, and these compounds were more active than the other beta-lactams, with meropenem being the most active. The MIC90s for beta-lactams, except carbapenems, ranged from 4 microg/ml to 32 microg/ml, with cefepime being the most active. In high inoculum susceptibility tests against these strains, the MIC90s for the four carbapenems and cefepime were 8 microg/ml or less, and these compounds were more active than other beta-lactams. The MIC90s for beta-lactams, except carbapenems and cefepime, were 32 microg/ml or more. The MIC90s for the five quinolones tested ranged from 4 microg/ml to 16 microg/ml, and the order of increasing susceptibility was ciprofloxacin > levofloxacin, gatifloxacin and pazufloxacin > prulifloxacin.

Biphasic response of cutaneous blood flow induced by passive cutaneous anaphylaxis in rats.

In the immediate phase of passive cutaneous anaphylaxis, sensitized skin mast cells release various mediators when activated by antigen. The present study investigated the effects of the mediators on cutaneous blood flow at the antigen-antibody reaction site. Induction of passive cutaneous anaphylaxis produced a biphasic response consisting of an initial decrease, followed by a sustained increase, in the cutaneous blood flow. The initial phase was almost eliminated by the 5-hydroxytryptamine receptor antagonist methysergide, whereas the second phase was sensitive to the histamine H(2) receptor antagonist ranitidine. The histamine H(1) receptor antagonist chlorpheniramine, the denervation of sensory nerves with capsaicin, the cyclooxygenase inhibitor indomethacin, or the bradykinin B(2) receptor antagonist D-arginyl-L-arginyl-L-prolyl-trans-4-hydroxy-L-prolylglycyl-3-(2-thienyl)-L-alanyl-L-seryl-D-1,2,3,4-tetrahydro-3-isoquinolinecarbonyl-L-(2alpha,3beta,7abeta)-octahydro-1H-indole-2-carbonyl-L-arginine (HOE140) did not affect the blood-flow changes caused by the anaphylaxis. These results suggest that 5-hydroxytryptamine and histamine H(2) receptors mediate the initial decrease and the subsequent increase in cutaneous blood flow, respectively, induced by passive cutaneous anaphylaxis in rats.

Production of CTX-M-3 extended-spectrum beta-lactamase and IMP-1 metallo beta-lactamase by five Gram-negative bacilli: survey of clinical isolates from seven laboratories collected in 1998 and 2000, in the Kinki region of Japan.

The aim of this study was to research the distribution in the Kinki region of Japan of Enterobacteriaceae and Pseudomonas aeruginosa that produce extended-spectrum beta-lactamase (ESBL) and metallo beta-lactamase (MBL). One thousand isolates, 200 of each of four enterobacterial species (i.e. Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae and Serratia marcescens) and 200 of P. aeruginosa, were collected from seven different laboratories during two 2 month periods, one in 1998 and one in 2000. A double-disc synergy test (DDST) and 2-mercaptopropionic acid inhibition test (2-MPAT) were used to confirm beta-lactamase-producing isolates. The DDST was positive for one isolate of E. coli, five of K. pneumoniae, two of E. cloacae and 14 of S. marcescens. The 2-MPAT was positive for five isolates of S. marcescens and two of P. aeruginosa. We identified the beta-lactamase type of each isolate by molecular confirmatory tests (isoelectric focusing, PCR and DNA sequencing): CTX-M-3 ESBLs (three isolates of K. pneumoniae, two of E. cloacae and 13 of S. marcescens), CTX-M-2 ESBL (one isolate of K. pneumoniae), SHV-12 ESBLs (one isolate of E. coli and one of S. marcescens), CTX-M-3 and SHV-12 combination ESBL (one isolate of K. pneumoniae) and IMP-1 MBLs (five isolates of S. marcescens and two of P. aeruginosa). In conclusion, many species of Gram-negative bacilli that produce CTX-M-3 ESBLs and IMP-1 MBLs were disseminated widely in different hospitals of the Kinki region of Japan. Therefore, monitoring of laboratory bacterial ecology seems important to stop the spread of these strains through nosocomial outbreaks.

[Multicenter evaluation of a newly developed microdilution test, brothMIC NTM to determine minimum inhibitory concentrations of antimicrobial agents for nontuberculous mycobacteria].

We developed a new broth microdilution susceptibility test for nontuberculous mycobacteria to determine minimum inhibitory concentrations(MICs). The test method utilized air-dried microplates containing serially diluted antimicrobial agents and the modified Middlebrook 7H9 broth. The nine agents tested were rifampicin, isoniazid, ethambutol, streptomycin, kanamycin, levofloxacin, clarithromycin, ethionamide and amikacin. The test plates were reconstituted by inoculation of 0.1 ml of cell suspensions prepared in distilled water by a 1:100 dilution of the 0.5 McFarland suspension. After inoculation, the isolates resulted in incubation at 36 degrees C with clarithromycin at pH 7.4, and with the remaining agents at pH 6.6. The growth endpoints were visually read after 7-day and 10-day incubations for slowly growing nontuberculous mycobacteria, and after 3-day and 5-day incubations for rapidly growing mycobacteria, respectively. The reproducibility was evaluated with the five ATCC reference strains of nontuberculous mycobacteria. Of the 1,287 repeated tests of the four ATCC slowly growing strains(M. avium, M. intracellulare, M. kansasii and M. gordonae), 1,200(93.2%) of the MICs read after 7-day incubation fell within 3 log 2 dilutions. Also, a strain of rapidly growing mycobacteria, M. fortuitum, was repeatedly tested, and the reproducibility was estimated to be 93.3% after 3-day incubation. A total of 728 clinical isolates of nontuberculous mycobacteria comprising 14 species were tested against nine agents. The MICs against nontuberculous mycobacteria distributed in a wide range, and the activities of rifampicin, levofloxacin, clarithromycin were more potent. These results demonstrate this newly developed test method to be a practical, rapid, quantitative means to determine MICs for nontuberculous mycobacteria in clinical laboratories.

[Resistance to antimicrobiotics and mutation of PBPs in Streptococcus pneumoniae isolated from Kinki region of Japan].

We studied the susceptibility to penicillin G (PCG) and other antimicrobiotics in 235 clinical isolates of Streptococcus pneumoniae. Samples were collected between April 1 and June 30, 2000 from nine medical institutions of the Kinki Region of Japan. We classified the minimum inhibitory concentration (MIC) of PCG according to the National Committee for Clinical Laboratory Standards (NCCLS) criteria. The overall rate of all types of S. pneumoniae resistance was 53.2% (penicillin-susceptible S. pneumoniae (PSSP): 46.8%, penicillin-intermediate S. pneumoniae (PISP): 42.6%, penicillin-resistant S. pneumoniae (PRSP): 10.6%). In other antimicrobiotics, the resistance (R)/intermediate susceptibility (I) rates (R/I%) were as follows: ceftriaxone, 28.9%; cefotaxime, 7.7%; imipenem, 8.9%; meropenem, 9.8%; clarithromycin, 82.6%; clindamycin, 42.1%; levofloxacin, 0.4%; vancomycin, 0%. We used the polymerase chain reaction to study the mutations of the penicillin-binding proteins pbp1 a, pbp2b, and pbp2x in 140 strains of S. pneumoniae in the MIC for PCG was < 0.5 microgram/ml. Among the 109 strains of PSSP, 32 (29.4%) had no mutation and 77 (70.6%) showed mutation of more than one of the pbp mutations. Among the 31 strains of PISP, only 1 strain (3.2%) was not mutated. Since 70.6% of the strains classified as PSSP had pbp mutations, S. pneumoniae clearly can acquire resistance to anti-microbiotics. In the future, a comprehensive surveillance of S. pneumoniae is necessary.

[Antimicrobial susceptibility and beta-lactamase types among clinical isolates during January and February 2000 in the Kinki area of Japan].

We studied antimicrobial susceptibility and beta-lactamase types among clinical isolates in the Kinki area of Japan. Eight hundreds isolates of eight organisms were collected by seven medical institutions during January and February 2000. The rates of beta-lactamase producing by using the chromogenic nitrocephin test were 68.0% against Staphylococcus aureus isolates, 6.0% against Haemophilus influenzae isolates, 98.0% against Moraxella catarrhalis isolates. The rate of beta-lactamase negative ampicillin-resistant H. influenzae was 4.0% (4 out of 100). The result of beta-lactamase producing by using the acid-metric method were as follows the penicillinase and cephalosporinase: 27.0% and 37.0% against Escherichia coli isolates, 37.0% and 1.0% against Klebsiella pneumoniae isolates, 21.8% and 100% against Enterobacter cloacae isolates, 24.2% and 96.0% against Serratia marcescens isolates, 7.0% and 22.0% against Pseudomonas aeruginosa isolates. We identified beta-lactamase type of each isolate detected by polymerase chain reaction: SHV-derived extended-spectrum beta-lactamase (ESBLs) (1 isolate of E. coli and 1 isolate of K. pneumoniae), CTX-M-1-derived ESBLs (1 isolate of K. pneumoniae, 1 of E. cloacae and 4 of S. marcescens), and IMP-1-derived metallo beta-lactamases (2 isolates of S. marcescens).