Interaction of Odoroside A, A Known Natural Cardiac Glycoside, with Na+/K+-ATPase.

The nature of odoroside A, a cardiac glycoside (CG) extracted from Nerium oleander, as well as its chemical structure is quite similar to a well-known CG, ouabain possessing a steroid skeleton, a five-membered unsaturated lactone ring, and a sugar moiety as a common structure. Like ouabain, odoroside A inhibits the activity of Na+/K+-ATPase (NKA) and shows significant anticancer activity, however its inhibitory mechanism remains unknown. CGs show various physiological activities, including cardiotonic and anticancer activities, through the inhibition of NKA by direct interaction. Additionally, X-ray crystallographic analysis revealed the inhibitory mechanism of ouabain and digoxin in relation to NKA. By using different molecular modeling techniques, docking simulation of odoroside A and NKA was conducted based on the results of these X-ray crystallographic analyses. Furthermore, a comparison of the results with the binding characteristics of three known CGs (ouabain, digoxin, and digitoxin) was also conducted. Odoroside A fitted into the CG binding pocket on the α-subunit of NKA revealed by X-ray crystallography. It had key interactions with Thr797 and Phe783. Also, three known CGs showed similar interactions with Thr797 and Phe783. Interaction modes of odoroside A were quite similar to those of ouabain, digoxin, and digitoxin. Docking simulations indicated that the sugar moiety enhanced the interaction between NKA and CGs, but did not show enhanced NKA inhibitory activity because the sugar moiety was placed outside the entrance of active site. Thus, these results suggest that the inhibitory mechanism of odoroside A to NKA is the same as the known CGs.

Antibiotic resistance and the presence of bla CfxA and bla CSP genes in ß-lactamase-producing clinical Capnocytophaga isolates from a university hospital in Japan.

Introduction . Capnocytophaga species are common inhabitants of the oral cavity and can be responsible for systemic diseases in immunocompromised patients with granulocytopenia. Furthermore, it has been reported that some clinical isolates of Capnocytophaga species produce extended-spectrum ß-lactamases (ESBLs).Gap statement. Information is lacking about the types of ß-lactamase genes possessed by Capnocytophaga spp. and the antimicrobial susceptibility of Capnocytophaga spp. possessing each ß-lactamase gene.Aim. The aim of this study was to investigate the presence of ß-lactamase genes in clinical strains of ß-lactamase-producing Capnocytophaga species isolated from clinical samples acquired at Shinshu University Hospital and examine the antimicrobial susceptibility of those strains.Methodology. The ß-lactamase-producing Capnocytophaga species (n=49) were obtained from clinical specimens. PCR assays were used to detect bla CfxA, bla CSP, bla TEM, bla CepA/CblA and transposon Tn4555 genes. Southern hybridization assays were used to detect bla CfxA and bla CSP. The minimum inhibitory concentration of some ß-lactams was determined using the E-test method.Results. PCR analysis indicated that the bla CfxA gene was present in 15 (30.6 %) and the bla CSP gene in 35 (69.3 %) of the 49 Capnocytophaga strains investigated, . Both bla CfxA and bla CSP genes were detected in a Capnocytophaga gingivalis strain. The PCR results were confirmed by Southern hybridization assays. Transposon Tn4555 was only detected in Capnocytophaga spp. harbouring the bla CfxA gene. All the ß-lactamase-producing Capnocytophaga isolates were susceptible to ceftazidime-clavulanic acid, cefoxitin and imipenem. In contrast, most of the isolates were resistant to amoxicillin.Conclusions. The clinical isolates of Capnocytophaga spp. showed a high prevalence of the bla CSP gene in Japan. The presence of the bla CSP gene was distributed in Capnocytophaga sputigena as well as other Capnocytophaga spp. These results seem to suggest the dissemination of bla CfxA and bla CSP ß-lactamase genes among Capnocytophaga species.

Isolation of thymidine-dependent and extended-spectrum-ß-lactamase-producing Escherichia coli small-colony variant from urine of a septuagenarian female patient with recurrent cystitis: A case report with genetic investigation.

Thymidine-dependent small-colony variant (TD-SCV) of Escherichia coli was isolated from urine of a septuagenarian female patient on hemodialysis suffering from recurrent cystitis. The patient had been treated with frequent administrations of trimethoprim sulfamethoxazole (SXT), every time her cystitis symptoms developed. In the TD-SCV isolate, the deletion was detected in the thyA gene associated with thymidylate synthase. Interestingly, the isolate was found to produce extended-spectrum ß-lactamase (ESBL), and the experiment on conjugational transfer of the resistance trait was successful. By means of genetic analysis, the isolate was found to carry blaCTX-M-1 group. To the best of our knowledge, this is the first report of urinary tract infection caused by the transmissible ESBL-producing TD-SCV of E. coli. MICs of the TD-SCV were obtained only on the Mueller Hinton agar media supplemented with appropriate concentrations of thymidine, which might lead to the difficulty for proper chemotherapy in daily medicine. Furthermore, transmission of the ESBL gene via plasmid should be of concern.

Isolation of a Capnophilic and Extended-Spectrum ß-Lactamase-Producing Proteus mirabilis Strain from the Urine of an Octogenarian Male Patient with Acute Pyelonephritis.

A capnophilic Gram-negative rod-shaped bacterium was recovered from the urine of an octogenarian male patient with acute pyelonephritis. The isolate was found to produce CTX-M-2-type extended-spectrum ß-lactamase. Interestingly, the isolate failed to grow on modified Drigalski (BTB) and MacConkey agar media, even under CO2-enriched atmosphere. Our analysis revealed that the pH-indicator dyes, bromothymol blue, and/or crystal violet that were incorporated into the agar media inhibited the growth of the isolate. Although routine identification methods using Vitek® 2 Compact systems were unsuccessful, the isolate was identified as Proteus mirabilis by 16S rRNA sequencing and MALDI-TOF MS analysis. The carbonic anhydrase (CA) region spanning approximately 2,000 bp upstream to 2,000 bp downstream, which is responsible for the CO2 requirement, was not amplified, which could be attributed to the large-scale deletion or mutation of the DNA sequences containing the CA gene region. In fact, revertants with the ability to grow without CO2 were not detected. However, a revertant that was capable of growing in both BTB and MacConkey agar was detected at frequencies less than 10-9. Therefore, the genes responsible for the highly sensitive reactions of the isolate to pH indicator dyes is not likely to be linked to the CA genes.

[Therapeutic Effects of Low-Dose Bevacizumab for the Treatment of Recurrent Brain Metastases].

OBJECTIVE: Molecularly targeted therapy has been adopted to treat a number of cancers. Bevacizumab, a recombinant humanized monoclonal antibody against vascular endothelial growth factor, is a representative agent used in molecularly targeted therapeutic regimens. However, the therapeutic effect of bevacizumab for the treatment of brain metastases remains unknown. We report the clinical effects of low dose bevacizumab(≤2.5mg/kg/week)to treat recurrent brain metastases. METHODS: We retrospectively analyzed patients with brain metastases who had been treated with bevacizumab between 2012 and 2016 at our institution. We identified clinical characteristics, including age, gender, primary tumor site, dose of bevacizumab, therapeutic and adverse effects, and magnetic resonance imaging results. The lesions were assessed with the RECIST criteria based on gadolinium-enhanced T1-weighted, T2-weighted, and FLAIR images. Statistical analysis was performed using t-test and Fisher's exact test. RESULTS: The cohort comprised 26 patients(8 men, 18 women)with a median age of 61 years(range 39-82 years). There were no significant clinical differences between the low dose and non-low dose groups. Patients in the low dose group did not report any adverse effects from bevacizumab. Three patients with brain metastases from colon cancer are illustrated to report the clinical course of low dose bevacizumab. CONCLUSION: Low dose bevacizumab may be a safe and effective therapeutic option to treat recurrent brain metastases from bevacizumab-sensitive cancers.

Characterization of first hemin-requiring Pseudomonas aeruginosa small-colony variants from the blood of an octogenarian male-patient with double pneumonitis.

A hemin-requiring Pseudomonas aeruginosa small-colony variant (SCV) was isolated from the blood of an octogenarian male-patient with double pneumonitis. The isolate was capable of growing on both sheep blood and chocolate agars but not on MacConkey agars without blood ingredient. Furthermore, the isolate revealed to grow only around the X-factor impregnated discs when examined using the X and V disc strips. However, not only RapID-NH system but also the VITEK2 system failed to identify the isolate. The isolate was finally identified as P. aeruginosa by the sequence of the 16S rRNA genes and the MALDI-TOF MS analysis. Interestingly, the isolate represented positive reaction for δ-aminolaevulinic acid (ALA)-test despite the requirement of hemin. Detailed analysis indicated that the isolate produced protoporphyrin IX from ALA. Therefore, the reason for the hemin dependence was deduced the dysfunction of hemH-encoded ferrochelatase behaving at the end of biosynthetic pathway of heme. However, the genetic analysis of hemH gene demonstrated no variations of both the DNA and the amino-acid sequences. To the best of our knowledge, this is the first clinical isolation of a hemin-dependent P. aeruginosa SCV from blood.

Four Sporadic Pediatric Cases of Yersinia enterocolitica O:8 Infection in a Rural Area of Japan.

In the spring of 2015, we experienced a cluster of 4 sporadic cases of yersiniosis in children in Nagano prefecture, a rural area of Japan. Two patients developed appendicitis-like episodes; one had acute gastroenteritis, and the other had bacteremia associated with liver abscess. The causative agent of these infections was Yersinia enterocolitica serogroup O:8. None of the patients had an underlying illness, and all have recovered completely. The patients were neither socially nor geographically related to each other. These 4 consecutive cases suggest that Y. enterocolitica O:8 has spread substantially in the middle part of Japan, and that this virulent strain might be more common than previously reported in our country.

Applicability assessment of ceramic microbeads coated with hydroxyapatite-binding silver/titanium dioxide ceramic composite earthplus™ to the eradication of Legionella in rainwater storage tanks for household use.

Water environments appear to be the habitats of Legionella species. Legionellosis is considered as a preventable illness because bacterial reservoirs can be controlled and removed. Roof-harvested rainwater has attracted significant attention not only as a groundwater recharge but also as a potential alternative source of nonpotable water. We successfully developed ceramic microbeads coated with hydroxyapatite-binding silver/titanium dioxide ceramic composite earthplus™ using the thermal spraying method. The ceramic microbeads were demonstrated to have bactericidal activities against not only Legionella but also coliform and heterotrophic bacteria. Immersing the ceramic microbeads in household rainwater storage tanks was demonstrated to yield the favorable eradication of Legionella organisms. Not only rapid-acting but also long-lasting bactericidal activities of the ceramic microbead were exhibited against Legionella pneumophila. However, time-dependent attenuation of the bactericidal activities against Legionella were also noted in the sustainability appraisal experiment. Therefore, the problems to be overcome surely remain in constantly managing the Legionella-pollution by means of immersing the ceramic microbeads. The results of our investigation apparently indicate that the earthplus™-coated ceramic microbeads would become the favorable tool for Legionella measures in household rainwater storage tanks, which may become the natural reservoir for Legionella species. Our investigation would justify further research and data collection to obtain more reliable procedures to microbiologically regulate the Legionella in rainwater storage tanks.

Notable alkaline tolerance of Kocuria marina isolate from blood of a pediatric patient with continuous intravenous epoprostenol therapy.

This study was the first to describe the hitherto deficiently evaluated alkaline tolerance of Kocuria marina isolate from a pediatric patient with continuous intravenous epoprostenol dosing therapy. Our isolate from blood of a 7-year-old Japanese boy was finally identified as K. marina by the morphological, cultural, and biochemical properties together with the comparative sequence analyses of the 16S rRNA genes. The K. marina isolate, the causative agent of catheter-related blood-stream infection, was not only revealed to be salt tolerant (NaCl 15%), but also demonstrated to be stably survived with no apparent decrease of cell counts for long periods (120 h) in an alkaline environment (pH 8, 9, 10, and 11) at 35 °C. Its remarkable tolerance to the stresses of high alkalinity compared with a clinical Staphylococcus aureus strain should provide consistent interpretation that the environment of high alkalinity (pH 10.2-10.8) measures should be insufficient to inactivate almost all the causative agents including K. marina strains in the solution of epoprostenol (pH 10.4) (Flolan(®), GlaxoSmithKline, Ltd., Tokyo, Japan.). To the best of our knowledge, the first description of the property of being tolerant to high alkalinity that the K. marina isolate exhibited was noteworthy and a useful piece of information. In conclusion, we believe that the present study should be a notification regarding the potential risk of catheter-related blood-stream infections due to K. marina, suggestive of an alkalophile, especially in patients receiving continuous intravenous epoprostenol dosing therapy.

A Pediatric Case of Bacteremia and Possible Cholecystitis Due to Moraxella osloensis.

We encountered a pediatric case of bacteremia and possible cholecystitis due to Moraxella osloensis that was treated successfully. We confirmed the diagnosis with the presence of a high serum titer of the antibody to the organism. Furthermore, 16S rRNA sequencing was performed to identify the bacteria.

Bath water contamination with Legionella and nontuberculous mycobacteria in 24-hour home baths, hot springs, and public bathhouses of Nagano Prefecture, Japan.

Bath water samples were collected from 116 hot springs, 197 public bathhouses, and 38 24-hour home baths in Nagano Prefecture, Japan, during the period of April 2009 to November 2011, for determining the presence and extent of contamination with Legionella and nontuberculous mycobacteria. Cultures positive for Legionella were observed in 123 of the 3,314 bath water samples examined. The distribution and abundance of Legionella and/or combined contamination with Legionella and nontuberculous mycobacteria were investigated to clarify the contamination levels. The abundance of Legionella was demonstrated to correlate considerably with the levels of combined contamination with Legionella and nontuberculous mycobacteria. Legionella spp. were obtained from 61% of the water samples from 24-hour home baths, but only from 3% of the samples from public bathhouses and hot springs. This is despite the fact that a few outbreaks of Legionnaires' disease in Nagano Prefecture as well as other regions of Japan have been traced to bath water contamination. The comparatively higher rate of contamination of the 24-hour home baths is a matter of concern. It is therefore advisable to routinely implement good maintenance of the water basins, particularly of the 24-hour home baths.

Helicobacter heilmannii sensu stricto-related gastric ulcers: a case report.

A spiral bacterium (SH9), morphologically different from Helicobacter pylori (H. pylori), was found in a 62-year-old woman's gastric mucosa. Gastroscopic examination revealed multiple gastric ulcers near the pyloric ring; mapping gastric biopsy showed mild mononuclear infiltration with large lymphoid follicles in the antrum, without corpus atrophy. Urea breath test and H. pylori culture were negative, but Giemsa staining of biopsies revealed tightly coiled bacteria that immunostained with anti-H. pylori antibody. Sequencing of SH9 16S rRNA and the partial urease A and B subunit genes showed that the former sequence had highest similarity (99%; 1302/1315 bp) to Helicobacter heilmannii (H. heilmannii) sensu stricto (H. heilmannii s.s.) BC1 obtained from a bobcat, while the latter sequence confirmed highest similarity (98.3%; 1467/1493 bp) to H. heilmannii s.s. HU2 obtained from a human. The patient was diagnosed with multiple gastric ulcers associated with H. heilmannii s.s. infection. After triple therapy (amoxicillin, clarithromycin, and lansoprazole) with regimen for eradicating H. pylori, gastroscopy showed ulcer improvement and no H. heilmannii s.s. upon biopsy.

Incidence of Legionella and heterotrophic bacteria in household rainwater tanks in Azumino, Nagano prefecture, Japan.

Many administrative agencies in Japan are encouraging installation of household rainwater-storage tanks for more effective use of natural rainwater. Water samples were collected periodically from 43 rainwater tanks from 40 households and tested for the presence of Legionella species and the extent of heterotrophic bacteria in Azumino city, Nagano prefecture, Japan. PCR assays indicated the presence of Legionella spp. in 12 (30%) of the 43 tank water samples. Attempts were made to identify correlations between PCR positive samples, topography, pH, chemical oxygen demand (COD), atmospheric temperature and the numbers of heterotrophic bacteria. Between June and October, 2012, the numbers of heterotrophic bacteria in rainwater tanks and the values of COD positively correlated with the presence of Legionella species. In most of the Legionella-positive cases, heterotrophic bacterial cell counts were >10(4) CFU/mL. Moreover, Legionella species were less frequently detected when the COD value was >5 mg KMnO(4)/L. Therefore, at least in Azumino, Japan between June and October 2012, both heterotrophic bacterial counts and COD values may be considered index parameters for the presence of Legionella cells in rainwater tanks. Much more accumulation of such data is needed to verify the accuracy of these findings.

A neonatal case of chronic granulomatous disease, initially presented with invasive pulmonary aspergillosis.

Chronic granulomatous disease (CGD) often presents with infectious illness, such as repeating bacterial and fungal infections, due to the inability to generate superoxide, which would destroy certain infectious pathogens, and is usually diagnosed in childhood. We describe a CGD case diagnosed in neonatal period, who initially presented with invasive aspergillosis. Neonatal invasive pulmonary aspergillosis is very rare and, to the best of our knowledge, this might be the youngest case in Japan.

First isolation of oleate-dependent Enterococcus faecalis small-colony variants from the umbilical exudate of a paediatric patient with omphalitis.

An oleate-dependent Enterococcus faecalis isolate representing small-colony variants (SCVs) was isolated from the umbilical exudate of a 31-month-old Japanese male patient in Nagano Children's Hospital, Azumino, Japan. The patient had been suffering from recurrent omphalitis since early infancy. The initial E. faecalis SCV isolate formed small colonies on sheep blood agar plates and tiny colonies on chocolate and modified Drigalski agar, although no visible growth was observed in HK-semi solid medium after 48 h incubation in ambient air. Moreover, the SCV isolate, the colonial morphology of which was reminiscent of Streptococcus species, could not be identified using the MicroScan WalkAway-40 and API 20 Strep systems, both of which yielded profile numbers that did not correspond to any bacterial species, probably as a result of insufficient growth of the isolate. The SCV isolate was subsequently identified as E. faecalis based on its morphological, cultural and biochemical properties, and this was confirmed by sequencing the 16S rRNA gene of the organism. Investigations revealed that the addition of oleate, an unsaturated fatty acid, enabled the isolate to grow on every medium with normal-sized colony morphology. Although it has long been known that long-chain fatty acids, especially unsaturated oleic acid, have a major inhibitory effect on the growth of a variety of microorganisms, including not only mycobacteria but also streptococci, this is, to the best of our knowledge, the first clinical isolation of an oleate-dependent E. faecalis SCV isolate. In addition, oleic acid might be considered to affect the cell membrane permeability of carbohydrates or antimicrobial agents such as ß-lactams.

First Isolation of carbon dioxide-dependent Proteus mirabilis from an uncomplicated cystitis patient with Sjögren's syndrome.

An uncomplicated cystitis caused by CO2-dependent Proteus mirabilis was observed in a 64-year-old Japanese female patient with Sjögren's syndrome in the Aomori Kyoritsu Hospital, Aomori, Japan. The initial P. mirabilis isolate came from a midstream urine specimen containing large numbers of Gram-negative, rod-shaped organisms that failed to grow on both Drigalski agar and sheep blood agar incubated in ambient air. The organism did grow when the urine was cultured overnight on blood agar under anaerobic conditions. Hence, we believed that the organism was an anaerobe. Further investigation revealed that the isolate grew on sheep blood agar along with swarming when the atmospheric CO2 concentrations were increased to 5%. Initially, we failed to characterize or identify the P. mirabilis isolate or determine its antimicrobial susceptibilities using the MicroScan WalkAway-40 System because the isolate did not grow in the system. However, the isolate was subsequently identified as P. mirabilis based on its morphological, cultural, and biochemical properties by using the commercially available kit systems, Quick ID-GN and ID-Test EB-20. This identification of the isolate was confirmed by sequencing the 16S rRNA gene of the organism. To our knowledge, this is the first clinical isolation of capnophilic P. mirabilis.

Isolation of an X-factor-dependent but porphyrin-positive Escherichia coli from urine of a patient with hemorrhagic cystitis.

An Escherichia coli isolate was recovered from a 92-year-old female patient with urinary tract infection. Gram-stained preparation of the urine sediment manifested some gram-negative rod-shaped cells, and the urine specimen culture yielded nonhemolytic colonies on sheep blood agar plate. However, no visible colonies appeared on modified Drigalski agar plate. The isolate was finally identified as an X-factor-dependent E. coli. The interesting finding was that the isolate revealed a positive reaction for porphyrin test despite the requirement of hemin. This finding suggested that some pyrrol-ring-containing porphyrin compounds or fluorescent porphyrins had been produced as chemical intermediates in the synthetic pathway from δ-amino-levulinic acid (ALA), although the isolate should be devoid of synthesizing hems from ALA. This was the first clinical isolation of such a strain, indicating that the E. coli isolate should possess incomplete synthetic pathways of hems from ALA.

Proportion of rectal carriage of extended-spectrum ß-lactamase-producing Enterobacteriaceae in the inpatients of a pediatric tertiary care hospital in Japan.

Extended-spectrum ß-lactamase (ESBL)-producing-Enterobacteriaceae strains were detected in 12% (6 out of 50) of fecal samples collected from the inpatients of a Japanese pediatric hospital. All the ESBLs belonged to the CTX-M-1 group. The proportion of carriage of ESBL producers was higher among patients who had received antibiotics within the past 3 months and among those who had cardiologic diseases.

Isolation and molecular characterization of catalase-negative Staphylococcus aureus from sputum of a patient with aspiration pneumonia.

Staphylococcus aureus produces various virulence factors. The catalase enzyme, in particular, is considered to be involved in oxidative stress resistance, and catalase activity is an important criterion for differentiating staphylococci from streptococci. In this report, we describe the catalase-negative S. aureus strain SH3064, which was isolated from the sputum of a patient with aspiration pneumonia. To evaluate the causes of the lack of catalase activity in S. aureus SH3064, we analyzed the sequence of katA gene encoding the catalase enzyme in this strain. We amplified the complete sequence of katA gene of S. aureus SH3064 by polymerase chain reaction using 2 sets of primers. The katA sequence showed 99.6% sequence identity (1512/1518 bp) with that of S. aureus ATCC 12600. We detected 2 mutations in the katA gene from S. aureus SH3064, an A217T substitution leading to a threonine 73-to-serine substitution and a single-base pair deletion (c.637delG) resulting in a frameshift mutation. The lack of catalase activity in this strain was attributed to the shift of the nucleotide reading frame.