Arborcandins A, B, C, D, E and F, novel 1,3-beta-glucan synthase inhibitors: production and biological activity

Arborcandins A, B, C, D, E and F, which possess potent 1,3-beta-glucan synthase inhibitory activity, were isolated from the culture broth of a filamentous fungus, strain SANK 17397. Arborcandins are novel cyclic peptides, that are structurally different from known glucan synthase inhibitors such as echinocandins. The 1,3-beta-glucan synthases of Candida albicans and Aspergillus fumigatus were inhibited by arborcandins with IC50 ranging from 0.012 to 3 microg/ml. The apparent Ki value of arborcandin C for C. albicans and A. fumigatus were 0.12 microM and 0.016 microM, respectively. The inhibition against these two 1,3-beta-glucan synthases by arborcandin C was noncompetitive. These compounds exhibited potent fungicidal activity against Candida spp. with MIC ranging from 0.25 to 8 microg/ml. The growth of A. fumigatus was suppressed by arborcandins with concentrations ranging from 0.063 to 4 microg/ml.     

Stemphones, novel potentiators of imipenem activity against methicillin-resistant staphylococcus aureus, produced by Aspergillus sp. FKI-2136

A fungal strain FKI-2136 identified as genus Aspergillus was found to produce potentiators of imipenem activity against methicillin-resistant Staphylococcus aureus (MRSA). Two new compounds designated stemphones B and C were isolated along with a structurally related known compound cochlioquinone D from the fermentation broth of the producing strain by solvent extraction, silica gel column chromatography and preparative HPLC. These compounds have a common tetracyclic quinone skeleton. Stemphone C potentiated imipenem activity against the MRSA 512 fold by decreasing MIC value of imipenem from 16 microg/ml to 0.03 microg/ml.     

Comparison of daptomycin MIC results by DIN, NCCLS, SFM, and SRGA methods for 297 Gram-positive organisms

Daptomycin is a novel lipopeptide antibiotic with potent in vitro antibacterial activity against Gram-positive pathogens. For daptomycin minimal inhibitory concentration (MIC) testing, National Committee for Clinical Laboratory Standards (NCCLS) recommends the use of broth containing physiological levels of calcium (50 microg/ml). The daptomycin susceptibility of 297 organisms was determined by NCCLS (Mueller-Hinton (MH) broth), Deutsches Institut für Normung (DIN; isotonic broth), Société Fran?aise de Microbiologie (SFM; three batches MH agar), and Swedish Reference Group for Antibiotics (SRGA; PDM agar). All media were supplemented to 50 microg/ml Ca(2+). There was good correlation between DIN and SFM methods (for staphylococci) with NCCLS results. Enterococci MICs using SFM methods were one to three dilutions lower and pneumococci results were one dilution higher than NCCLS. SRGA results were higher than NCCLS by one to four dilutions. Use of isotonic agar is an accepted alternative to isosensitest agar for the DIN method.     

Detection of ticarcillin-clavulonic acid susceptibility with microdilution method in Citrobacter, Hafnia, Proteus and some gram negative bacteria

The broth dilution method has been regarded as a good alternative test for detection of susceptibilities to antimicrobial agents. In this study, the antimicrobial activity of ticarcillin-clavulonic acid (TIM) was investigated by the minimal inhibitory concentration (MIC) method on strains of Aeromonas, Citrobacter, Hafnia, Morganella, Proteus, Pseudomonas and gram negative bacteria isolated from raw milk. The isolate collection included 91 gram negative strains. Fifty-one (56.04%) isolates were found sensitive (MIC < or = 8 microg/ml), 12 (13.19%) isolates were found intermediately sensitive (MIC 16-32 microg/ml), and 28 (30.77%) isolates were found resistant (MIC > or = 64 microg/ml) to TIM.     

Mulundocandin, an echinocandin-like lipopeptide antifungal agent: biological activities in vitro.

Mulundocandin (MCN) is an antifungal lipopeptide which belongs to the echinocandin class of antimycotic agents. MCN exhibited good in vitro activity against Candida albicans and C. glabrata isolates with MIC ranges of 0.5-4.0 microg/ml and 2.0-4.0 microg/ml, respectively. MCN also exhibited some activity against C. tropicalis isolates (MIC range 1.0-8.0 microg/ml). However, MCN was poorly active against other non-albicans isolates and was inactive against Cryptococcus neoformans, Aspergillus species and Trichophyton. MCN appeared to exert its antifungal activity through preferential inhibition of germ tube formation (MIC-HY 0.015-0.03 microg/ml) and was typically less active on the yeast form (MIC 0.5-4.0 microg/ml). In kill-curve experiments 99.9% reductions in cell viability were observed following 8 hours exposure to MCN at 4 x MIC and 8 x MIC and after 5 hours exposure to 16 x MIC.     

Relationship between protein binding and antimicrobial activities of antibiotics against Streptococcus pneumoniae and Haemophilus influenzae

Fifty isolates of Streptococcus pneumoniae and 42 isolates of Haemophilus influenzae were isolated from the blood of children admitted to pediatric wards of hospitals in subprefucture between January 1998 and December 2005. The susceptibilities were measured by a microbroth dilution method using a standard broth and a broth containing 4.5% albumin. Against S. pneumoniae, penicillin G, ampicillin, cefotaxime, ceftriaxone, panipenem, meropenem, vancomycin, cefditoren, cefcapene, cefteram, faropenem and tebipenem were used and against H. influenzae, ampicillin, piperacillin, cefotaxime, ceftriaxone, panipenem, meropenem, clavulanic acid/ amoxicillin, cefditoren, cefcapene, cefteram, faropenem and tebipenem were used. Against S. pneumoniae, tebipenem was the highest antimicrobial activity in oral antibiotics (MIC90; < or = 0.06 microg/ml) and panipenem showed the highest activity for intravenous antibiotics (MIC90; < or = 0.12 microg/ml). Against H. influenzae, cefditoren was the highest activity for oral antibiotics (MIC90; < or = 0.06 microg/ml) and meropenem showed the highest activity for intravenous antibiotics (MIC90; < or = 50.06 microg/ml). The MIC90s measured by albumin containing broth were higher than those measured by standard broth. Protein binding rates of ceftriaxone, cefditoren, and faropenem were greater than 90%, and the MIC90 of these antibiotics measured by albumin addition methods were over 4-fold higher than those measured by standard methods.     

Susceptibilities of bacteria isolated from patients with lower respiratory infectious diseases to antibiotics (2002)

From October 2002 to September 2003, we collected the specimen from 476 patients with lower respiratory tract infections in 16 institutions in Japan, and investigated the susceptibilities of isolated bacteria to various antibacterial agents and patients' characteristics. Of 584 strains that were isolated from specimen (mainly from sputum) and assumed to be bacteria causing in inflammation, 578 strains were examined. The breakdown of the isolated bacteria were: Staphylococcus aureus 77, Streptococcus pneumoniae 103, Haemophilus influenzae 95, Pseudomonas aeruginosa (non-mucoid) 61, P. aeruginosa (mucoid) 23, Klebsiella pneumoniae 36, Moraxella subgenus Branhamella catarrhalis 29, etc. Of 77 S. aureus strains, those with 2 microg/ml or less of MIC of oxacillin (MPIPC) [methicillin-susceptible S. aureus: MSSA] was 34 strains (44.2%) and those with 4 microg/ml or more of MIC of oxacillin (methicillin-resistant S. aureus: MRSA) was 43 strains (55.8%). Against MSSA, imipenem (IPM) and minocycline (MINO) had the most potent antibacterial activity and inhibited the growth of all the strains at 0.25 microg/ml. Although clindamycin (CLDM) and aminoglycosides also had the potent activity, the resistant strains against those agents were detected. Cefotiam (CTM) inhibited the growth of all the strains at 1 microg/ml without the low sensitive strains. Against MRSA, vancomycin (VCM) showed the most potent activity and inhibited the growth of all the strains at 2 microg/ml. Arbekacin (ABK) also showed the relatively potent activity and inhibited the growth of all the strains at 4 microg/ml. Carbapenems showed the most potent activities against S. pneumoniae and inhibited the growth of all the strains at 0.25-0.5 microg/ml. Cefozopran (CZOP) also had a preferable activity (MIC90: 1 microg/ml) and inhibited the growth of all the strains at 2 microg/ml. In contrast, the resistant strains for cefaclor (CCL), erythromycin (EM), CLDM, and tetracycline (TC) were detected in 50.5%, 76.7%, 50.5%, and 80.6% of all the strains, respectively. Against H. influenzae, LVFX showed the most potent activity and inhibited the growth of 92 of all the strains (96.8%) at 0.063 microg/ml. Tobramycin (TOB) showed the most potent activity against P. aeruginosa (both mucoid and non-mucoid) and inhibited the growth of all the strains at 2 microg/ml. The antibacterial activity of CZOP was good and its MIC90 against mucoid and non-mucoid strains was 8 and 16 microg/ml, respectively. CZOP and cefpirome (CPR) were the most potent against K. pneumoniae with 0.125 microg/ml of MIC90. Also, all the agents generally showed potent activities against M. (B.) catarrhalis and the MIC90 of all drugs were 4 microg/ml or less. The approximately half the number (47.5%) of the patients with respiratory infection were aged 70 years or older. As for the incidence by the diseases, bacterial pneumonia and chronic bronchitis were the highest, being noted in 35.7 and 33.8% of all the patients, respectively. The bacteria frequently isolated from the patients with bacterial pneumonia were S. pneumoniae (22.6%). In contrast, S. aureus (16.6%) and P. aeruginosa (13.7%) were relatively frequently isolated from the patients with chronic bronchitis. Before the drug administration, the bacteria frequently isolated from all the patients were H. influenzae (24.5%) and S. pneumoniae (24.2%). In comparison of the isolated bacteria by pretreatment agents, P. aeruginosa was relatively frequently isolated from the patients pretreated with cephems or macrolides and H. influenzae was relatively frequently isolated from the patients pretreated with penicillins.     

Antifungal activities of azole agents against the Malassezia species

In this paper, we identified 95 Malassezia isolates by morphological and biochemical criteria and assessed the in vitro activity of fluconazole, itraconazole, ketoconazole and voriconazole by broth microdilution against these species using slightly modified Leeming-Notman medium. The Malassezia isolates were identified as M. furfur (74), M. sympodialis (11), M. obtusa (8) and M. globosa (2). The modified Leeming-Notman medium used for susceptibility testing allowed good growth of Malassezia spp. Visual reading of the minimal inhibitory concentration (MIC) was readily achieved until Day 5 of incubation at 32 degrees C. Although high MIC values of 16 microg/mL for fluconazole were observed in 9.5% of Malassezia isolates, in general these microorganisms were susceptible to all drugs studied. Interestingly, one M. globosa isolate showed high MIC values for voriconazole, itraconazole and fluconazole. For the 95 strains, the MIC ranges were <0.03-4 microg/mL for ketoconazole, <0.03 to >16 microg/mL for voriconazole, <0.125 to >64 microg/mL for fluconazole and <0.03-16 microg/mL for itraconazole. In summary, the good reproducibility and visual readings obtained using modified Leeming-Notman medium suggest that this medium should be proposed for antifungal testing of drugs against Malassezia spp.     

Yaequinolones, new insecticidal antibiotics produced by Penicillium sp. FKI-2140. I. Taxonomy, fermentation, isolation and biological activity

New nine insecticidal antibiotics designated yaequinolones were isolated from the culture broth of the fungal strain Penicillium sp. FKI-2140 by solvent extraction, centrifugal partition chromatography and HPLC. Yaequinolones showed growth inhibitory activity against brine shrimp (Artemia salina). Among them, yaequinolone F has the most potent activity with MIC value of 0.19 microg/ml.     

A novel colorimetric broth microdilution method to determine the minimum inhibitory concentration (MIC) of antibiotics and essential oils against Helicobacter pylori

Helicobacter pylori infections have been associated with the pathogenesis of a number of stomach and gastroduodenal diseases. In order to find alternative drugs for their treatment the search is increasingly focused on new antimicrobial products. However, no standardized methods are available to test the anti-Helicobacter pylori activity in particular of natural substances. Therefore we developed a broth microdilution assay to investigate the susceptibility of this fastidious slow growing bacterium against 15 essential oils widely used to treat disorders of the gastrointestinal tract. The MIC values were determined colorimetrically using p-iodonitrophenyltetrazolium violet (INT) as an indicator for bacterial cell viability. The test sytem was evaluated with three common antibiotics: amoxicillin, ampicillin and levofloxacin. The antibiotic MICs were controlled by Etest. The Helicobacter reference strain was remarkably susceptible to both the antibiotics (amoxicillin MIC: 0.02 microg/ml, ampicillin MIC: 0.064 microg/ml, levofloxacin MIC: 0.39 microg/ml) and the essential oils. Most of their MICs ranged from 0.015 to 0.064% (v/v) and about 140.0 to 280.0 microg/ml, respectively. Interestingly, chamomile oil, orange flower oil and ginger oil inhibited the bacterial growth in extraordinarily low concentrations of 0.0075% (v/v) and about 65 microg/ml, respectively. The bactericidal concentrations were generally one to two dilution steps higher. In conclusion, we could develop an innovative assay for the MIC determination of essential oils and antibiotics against Helicobacter pylori, which is simple to handle, accurate, reproducible and not as time- and material-consuming as traditional agar dilution techniques.     

Two new members of streptothricin class antibiotics from Streptomyces qinlingensis sp. nov

Four streptothricin-group antibiotics (1~4) were isolated from the fermentation broth of Streptomyces qinlingensis sp. nov. Along with the known antibiotics streptothricins F (1) and D (3), two new members of this class (2, 4) were identified as 12-carbamoyl derivatives of 1 and 3, respectively, mainly by analysis of the IR, HR-MS and NMR spectral data. The antibacterial activities of 1~4 against Escherichia coli (MICs 3.1, 25.0, 3.1 and 12.5 microg/ml), Bacillus subtilis (MICs 6.3, 25.0, 3.1 and 50 microg/ml), Staphylococcus aureus (MICs 12.5, >100.0, 6.3, >100.0 microg/ml), Bacillus cereus (MICs 25.0, 50.0, 25.0 and 50.0 microg/ml) and Pseudomonas aeruginosa (MICs 50.0, >100.0, 50.0, >100.0 microg/ml) were assayed by micro-broth dilution. The results based on MIC data indicated that 2 and 4 exhibited significantly less potent antibacterial activities when compared to that of 1 and 3.     

CJ-15,801, a novel antibiotic from a fungus, Seimatosporium sp

A novel antibiotic, CJ-15,801 (I), was isolated from the fermentation broth of a fungus, Seimatosporium sp. CL28611. The structure was determined to be a pantothenic acid analog having an alpha,beta-unsaturated carboxylic acid moiety by spectroscopic analyses. The compound inhibits the growth of multi-drug resistant (MDR) Staphylococcus aureus strains with MIC ranging from 6.25 to 50 microg/ml.     

Novel cytocidal antibiotics, glucopiericidinols A1 and A2. Taxonomy, fermentation, isolation, structure elucidation and biological characteristics

Two novel antibiotics, glucopiericidinols A1 (1) and A2 (2) were isolated from the cultured broth of Streptomyces sp. OM-5689. The structures of these two compounds were deduced employing spectroscopic analyses. These antibiotics showed potent cytocidal activities against HeLa S3 cells in vitro (MIC 1: 0.39 microgram/ml, 2: 0.10 microgram/ml) when the cells were exposed to the antibiotics for 3 days. Although 1 and 2 showed no activity at 1,000 micrograms/ml against various Gram-positive and Gram-negative bacteria, yeast or fungi, they did have inhibitory activity against Piricularia oryzae (MIC of 1: 125 micrograms/ml, of 2: 31 micrograms/ml).     

Clethramycin, a new inhibitor of pollen tube growth with antifungal activity from Streptomyces hygroscopicus TP-A0623. I. Screening, taxonomy, fermentation, isolation and biological properties

The cytoskeletal proteins, actin and myosin, play a central role in pollen tube growth. The pollen tube growth is inhibited by cytochalasin, which interferes with actin polymerization. In the screening of pollen tube growth inhibitors, clethramycin was found from the fermentation broth of an actinomycete strain TP-A0623. The producing strain was isolated from a root of Clethra barbinervis collected in Toyama, Japan and identified as Streptomyces hygroscopicus based on the taxonomic study. Clethramycin showed in vitro antifungal activity against yeast such as Candida albicans and C. glabrata with the MIC of 0.5 approximately 8 microg/ml, but weak activity against Gram-positive and negative bacteria (MIC > or = 64 microg/ml). Cytotoxicity of clethramycin was moderate and the IC50 was 57 microg/ml against HeLa cells and 120 microg/ml against WI-38 cells.     

In vitro antimicrobial activity of telavancin against methicillin-resistant Staphylococcus aureus clinical isolates from Japan (2006)

In vitro antimicrobial activity of telavancin, a rapidly bactericidal lipoglycopeptide, was evaluated against 1500 strains of MRSA recently isolated in Japan. Telavancin had potent activity, with MIC values that ranged from 0.12 microg/ml to 0.5 microg/ml and a MIC90 value of 0.5 microg/ml. The MIC90s of vancomycin and linezolid were 1.0microg/ml and 2 microg/ml, respectively. No vancomycin intermediate resistant or vancomycin-resistant MRSAs were detected in this surveillance study.     

YM-181741, a novel benz[a]anthraquinone antibiotic with anti-Helicobacter pylori activity from Streptomyces sp

A novel benz[a]anthraquinone, YM-181741, was isolated from the culture broth of actinomycete strain Q57219. The strain was identified as Streptomyces sp. by morphological and chemotaxonomic characterization. YM-181741 was purified from the culture supernatant by serial column chromatography. The structure of YM-181741 was determined by spectroscopic analysis including one- and two-dimensional NMR experiments. YM-181741 showed selective anti-Helicobacterpylori activity with a MIC value of 0.2 microg/ml.     

Discrepancies between mecA PCR and conventional tests used for detection of methicillin resistant Staphylococcus aureus

Conventional and molecular techniques are being used in the detection of methicillin resistance in Staphylococcus aureus but they do not always show concordant results. In this study, a mecA PCR-based amplification was compared with the 1 microg oxacillin disk diffusion test and the Epsilometer test (E-test) for detection of MICs. Among 31 isolates initially characterized as MRSA by the disk diffusion test, mecA was detected in only 13 (42%) isolates. The E-test showed a wide range of oxacillin MICs (0.5 - > 256 microg/ml) among these 31 MRSA isolates: seven isolates had an MIC of > 256 microg/ml, one had 64 microg/ml, two had 4 microg/ml, two had 3 microg/ml, one had 2.5 microg/ml, nine had 2 microg/ml, three had 1.5 microg/ml, five had 1 microg/ml and one had 0.5 microg/ml. Comparing the mecA PCR results with the E-test oxacillin MIC findings revealed that mecA was detected in seven of eight isolates (87.5%) with an MIC of > or = 64 microg/ml, in three of 14 isolates (21.4%) with an MIC of 2-4 microg/ml and in three of nine isolates (33.3%) with an MIC of < 2 microg/ml. Beta-lactamase production was positive in 28/31 isolates (90.3%). Because of this variation between tests and since several resistance mechanisms are known to mediate methicillin resistance in S. aureus, the reliable detection of MRSA cannot be solely based on detection of mecA gene in S. aureus. At this stage and until new guidelines are introduced by an official body, such as NCCLS, a combination of conventional methods alone or together with a molecular method should be used every time S. aureus is tested for detection of methicillin resistance.     

Susceptibilities of bacteria isolated from patients with lower respiratory infectious diseases to antibiotics (2003)

From October 2003 to September 2004, we collected the specimen from 399 patients with lower respiratory tract infections in 12 institutions in Japan, and investigated the susceptibilities of isolated bacteria to various antibacterial agents and patients' characteristics. Of 474 strains that were isolated from specimen (mainly from sputum) and assumed to be bacteria causing in inflammation, 469 strains were examined. The breakdown of the isolated bacteria were: Staphylococcus aureus 76, Streptococcus pneumoniae 81, Haemophilus influenzae 84, Pseudomonas aeruginosa (non-mucoid) 56, P. aeruginosa (mucoid) 11, Klebsiella pneumoniae 36, Moraxella subgenus Branhamella catarrhalis 24, etc. Of 76 S. aureus strains, those with 2 microg/ml or less of MIC of oxacillin (methicillin-susceptible S. aureus: MSSA) and those with 4 microg/ml or more of MIC of oxacillin (methicillin-resistant S. aureus: MRSA) were both 38 strains (50.0%). Against MSSA, imipenem had the most potent antibacterial activity and inhibited the growth of all the strains at 0.063 microg/mL. Against MRSA, vancomycin showed the most potent activity and inhibited the growth of all the strains at 2 microg/mL. Arbekacin also showed the potent activity and inhibited the growth of all the strains at 4 microg/mL. Carbapenems showed the most potent activities against S. pneumoniae and inhibited the growth of all the strains at 0.125-0.5 microg/mL. Cefozopran (CZOP) also had a preferable activity (MIC90:2 microg/ mL) and inhibited the growth of all the strains at 4 microg/mL. In contrast, there were high-resistant strains (MIC: 128 microg/mL or more) for cefaclor (11.1%), erythromycin (43.2%), and clindamycin (40.7%). Against H. influenzae, levofloxacin showed the most potent activity and inhibited the growth of 83 of all the strains (98.8%) at 0.063 microg/mL. Tobramycin showed the most potent activity against P. aeruginosa (both mucoid and non-mucoid) and its MIC90 was 2 microg/mL. The activity of CZOP also was preferable and its MIC90 was 4 microg/mL for the mucoid-type and 8 microg/mL for the non-mucoid type. CZOP was the most potent activities against K. pneumoniae and inhibited the growth of all the strains at 0.125 microg/mL. Also, all the agents generally showed potent activities against M. (B.) catarrhalis and the MIC90 of them were 4 microg/mL or less. The approximately half the number (54.1%) of the patients with respiratory infection were aged 70 years or older. Bacterial pneumonia and chronic bronchitis accounted for 46.1% and 30.6% of all the respiratory infection, respectively. The bacteria frequently isolated from the patients with bacterial pneumonia were S. aureus (18.6%) and H. influenzae (18.1%). In contrast, S. aureus (16.9%) and S. pneumoniae (14.9%) were frequently isolated from the patients with chronic bronchitis. Before the drug administration, the bacteria frequently isolated from the patients were S. pneumoniae (20.6%) and H. influenzae (21.5%). The bacteria relatively frequently isolated from the patients treated with cephems or macrolides were P. aeruginosa, and S. aureus was relatively frequently isolated from the patients treated with quinolones.     

Susceptibility of ESBL-producing Escherichia coli and Klebsiella pneumoniae to various antibacterial agents

With the increasing use of broad-spectrum antibacterial agents, the increase in various drug-resistant bacterial strains has become a concern in recent years. Especially, the development of drug-resistance by Enterobacteriaceae which significantly affects therapy and prognosis in sepsis and lower gastrointestinal post-operative infection. The extended spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae strains isolated in the Surveillance Program of Bacterial Resistance in Kinki region of Japan (SBRK) were supplied between November 2000 and March 2003. The susceptibilities of them to 16 kinds of antimicrobial agents were investigated. The number of them was 48 strains consisting of 36 Escherichia coli strains (75%) and 12 Klebsiella pneumoniae strains (25%). Our focus was on carbapenem and the new quinolone antibacterial agents. Among the 16 major antibacterial agents examined, carbapenem had low MIC50/90 values. Meropenem had a MIC50/90 of 0.03/0.06microg/ml, followed by biapenem (0.12/0.5), imipenem (0.25/0.5) and panipenem (0.25/0.5). Among cephem, ceftazidime had the lowest MIC50 at 4 microg/ml. All four of the cephem agents had a MIC90 of greater than 128microg/ml. Among beta-lactamase inhibitors, tazobactam/piperacillin had the lowest MIC50 at 4 microg/ml, and sulbactam/cefoperazone had a MIC50 of 32 microg/ml. Among the new quinolones, prulifloxacin had the lowest MIC50 at 1 microg/ml, and the other drugs had a MIC50 of 2 microg/ml. The resistance rate of ciprofloxacin was 61.1% in E. coli and 16.6% in K. pneumoniae. Comparison of drug-sensitivity to cephem by ESBL-gene type revealed that cefpirome, cefepime and cefozopran had higher MIC50/90 values against the CTX-M group with a MIC50 of greater than 128microg/ml. Ceftazidime and aztreonam had higher MIC50/90 values against the TEM/SHV group than those against the CTX-M group. In the CTX-M group, the MIC50 was 4 and 16microg/ml, respectively.