Surveillance of antimicrobial awareness among patients visiting community pharmacies.

OBJECTIVE: Although antimicrobial resistance (AMR) measures have been progressing, cases of patients requesting their doctors to prescribe antimicrobial agents and patients mistakenly believing that these agents are effective against viruses occasionally occur. In the AMR action plan (2023-2027) in Japan, one of the primary goals are public awareness and education. However, public understanding of AMR and antimicrobial agents has been reported to be at an unsatisfactory level. Here, we conducted a surveillance of antimicrobial awareness among patients visiting community pharmacies. MATERIAL AND METHODS: A questionnaire survey was conducted among patients visiting nine pharmacies in Hachioji, Tokyo, Japan. A total of 1887 active questionnaires were collected. The relationship between answers was analyzed using logistic regression analysis. RESULTS: Of the patients, 72% were unaware of AMR, and 68% believed that antimicrobials are effective against viruses. In addition, 28% of the patients answered that they did not take antimicrobial agents as prescribed by their physicians. Seventeen percent of the patients had never received appropriate instruction of antimicrobial use from pharmacists. Analysis of the relationship between answers showed that patients with correct knowledge were 1.65 times more likely to take antimicrobial agents as prescribed by their physicians (P < 0.01). Furthermore, the factors that led to the inappropriate behaviors of patients were associated with preliminary antimicrobial prescriptions from physicians (odds ratio, 3.18; 95% CI, 2.12-4.76) (P < 0.01). CONCLUSION: This study strongly suggests that physician and pharmacist interventions regarding the appropriate use of antimicrobial agents are important to improve awareness of antimicrobial agents.

Surveillance of Antimicrobial Prescriptions in Community Pharmacies Located in Tokyo, Japan.

An antimicrobial resistance (AMR) Action Plan was launched in 2016 to prevent the spread of antimicrobial-resistant bacteria in Japan. Additional support for the appropriate use of pediatric antimicrobial agents was initiated in 2018 to promote the appropriate use of antimicrobial agents in the community. To evaluate the effectiveness of the AMR Action Plan in the community, we investigated antimicrobial prescriptions in community pharmacies. Data on prescriptions for antimicrobial agents dispensed in 42 community pharmacies located in the Tama district, Tokyo, Japan, were collected between April 2013 and December 2019. In this study, we employed the DPY, which was calculated as defined daily doses (DDDs)/1000 prescriptions/year. The DPY is the number of antimicrobial agents used (potency) per 1000 antimicrobial prescriptions dispensed in pharmacies per year. The number of prescriptions for third-generation cephalosporins, fluoroquinolones, and macrolides decreased after the initiation of the AMR Action Plan; the DPYs of these antimicrobial agents decreased significantly by 31.4%, increased by 15.8%, and decreased by 23.6%, respectively (p < 0.05). The number of antimicrobial prescriptions for pediatric patients has been decreasing since 2018. Declines in the DPYs of third-generation cephalosporins, fluoroquinolones, and macrolides were higher in pediatric pharmacies than in other pharmacies. Our data suggest that the AMR Action Plan and additional support for the appropriate use of antimicrobial agents in children influenced the number of antimicrobial prescriptions in community pharmacies in Japan.

MALDI-TOF MS Approaches for the Identification of the Susceptibility of Extended-Spectrum ß-Lactamases in Escherichia coli.

The increase in multidrug-resistant microorganisms that produce extended-spectrum ß-lactamases (ESBLs) and carbapenemases is a serious problem worldwide. Recently, matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) has been used for the rapid detection of antibiotic-resistant bacteria. The objective of this study was to establish a method to detect ESBL-producing Escherichia coli by monitoring the hydrolyzation of cefotaxime (CTX) using MALDI-TOF MS. According to the ratio of the peak intensity of CTX and hydrolyzed-CTX-related compounds, the ESBL-producing strains could be clearly distinguished after 15 min of incubation. Moreover, the minimum inhibitory concentration (MIC) values for E. coli were 8 µg/mL and lower than 4 µg/mL, which could be distinguished after 30 min and 60 min of incubation, respectively. The enzymatic activity was determined using the difference in the signal intensity of the hydrolyzed CTX at 370 Da for the ESBL-producing strains incubated with or without clavulanate. The ESBL-producing strains with low enzymatic activity or blaCTX-M genes could be detected by monitoring the hydrolyzed CTX. These results show that this method can rapidly detect high-sensitivity ESBL-producing E. coli.

Rapid detection of bacteria that produce extended-spectrum ß-lactamase by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

OBJECTIVES: Proper use of antibacterial agents is necessary to prevent the spread of drug-resistant bacteria. To support clinicians, laboratories need to rapidly determine bacterial drug susceptibility/resistance. We have established a method to distinguish extended-spectrum ß-lactamase (ESBL)-producing clinical isolates by capturing structural changes in ß-lactam antibiotics using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). METHODS: Clinical isolates of Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis, classified into ESBL-producing strains and sensitive strains based on the presence or absence of a CTX-M-type gene, were used. Test bacteria were cultured aerobically in solid-phase wells of Eiken DPD1 dry plates at 35°C for 15 min or 30 min with the antibiotics cefotaxime (CTX), cefpodoxime (CPDX) or piperacillin (PIPC). Culture supernatants were then used for analysis with a MALDI Biotyper. RESULTS: Signals derived from non-hydrolyzed products of antibiotics were observed in all strains. In the case of ESBL-producing strains, signals derived from the hydrolysis products of antibiotics were also observed. Since the ratio of signal intensity derived from hydrolysis products divided by the total signal intensity detected was ≥11% for CTX and ≥6% for CPDX and PIPC, all strains were determined to be ESBL-producing bacteria. CONCLUSION: The short incubation time of 15 min suggests that this method can identify ESBL-producing strains much more rapidly than conventional methods.

pH Behavior of Polymer Complexes between Poly(carboxylic acids) and Poly(acrylamide derivatives) Using a Fluorescence Label Technique.

In order to clarify the local environment during interpolymer complex formation between poly(carboxylic acids) and poly(acrylamide derivatives) with different N-substitutions, a fluorescence label technique was used. 3-(2-propenyl)-9-(4-N,N-dimethylaminophenyl) phenanthrene (VDP) was used as an intramolecular fluorescence probe. All polymers were synthesized by free radical polymerization. Interpolymer complexation was monitored by charge transfer emission from the VDP unit. Both of the poly(carboxylic acids) formed interpolymer complexes with poly(N,N-dimethylacrylamide) (polyDMAM). The micro-environments around the VDP unit in the acidic pH region for the poly(methacrylic acid) (polyMAAc) and polyDMAM mixed systems were more hydrophobic than those of the poly(acrylic acid) (polyAAc) and polyDMAM mixed systems, as the α-methyl group of the MAAc unit contributed to hydrophobicity around the polymer chain during hydrogen bond formation. This suggests that, when the poly(carboxylic acids) and poly(acrylamide derivatives) were mixed, with a subsequent decrease in the solution pH, a hydrogen bond was partially formed, following which the hydrophobicity of the micro-environment around the polymer chains was changed, resulting in the formation of interpolymer complexes. Moreover, the electron-donating ability of the carbonyl group in the poly(acrylamide derivatives) had an effect on complexation with poly(carboxylic acids).

Influence of Gas Temperature in Atmospheric Non-Equilibrium Plasma on Bactericidal Effect.

In this study, the relationship between plasma gas temperature and the bactericidal effects on five of bacteria (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecalis and Bacillus cereus (spore)) in liquid was investigated using a temperature-controllable plasma source. We determined that the bactericidal ability improved as the plasma gas temperature increased. Specifically, the bactericidal ability on E. coli of 80-℃ plasma was enhanced by as much as 6.3 times compared to that of 10-℃ plasma. The relationship between plasma gas temperature and the amount of hydroxyl radical, singlet oxygen, hydrogen peroxide, and ozone introduced into the solution was investigated. Our results also showed that each reactive species production increased by 2.1, 9.0, 1.6, and 17 times, respectively, with 80-℃ compared to 10-℃ plasma. The relationship between the bactericidal ability and amount of reactive species indicated that singlet oxygen and ozone introduced to the solution mostly influenced the bactericidal ability as the plasma gas temperature increased. We conclude that the plasma gas temperature is the crucial parameter for plasma sterilization.

Indigo Naturalis Ameliorates Oxazolone-Induced Dermatitis but Aggravates Colitis by Changing the Composition of Gut Microflora.

BACKGROUND: Indigo naturalis (IND) is an herbal medicine that has been used as an anti-inflammatory agent to treat diseases including dermatitis and inflammatory bowel disease in China. However, the mechanism by which IND exerts its immunomodulatory effect is not well understood. METHODS: A murine model of dermatitis and inflammatory bowel disease, both induced by oxazolone (OXA), was treated with IND. The severity of dermatitis was evaluated based on ear thickness measurements and histological scoring. The severity of colitis was evaluated by measuring body weight, histological scoring, and endoscopic scoring. The expression of inflammatory cytokines in ear and colon tissue was evaluated using real-time PCR. 16S rRNA DNA sequencing of feces from OXA-induced colitis mice was performed before and after IND treatment. The effects of IND on OXA-induced colitis were also evaluated after depleting the gut flora with antibiotics to test whether alteration of the gut flora by IND influenced the course of intestinal inflammation in this model. RESULTS: IND treatment ameliorated OXA dermatitis with a reduction in IL-4 and eosinophil recruitment. However, OXA colitis was significantly aggravated in spite of a reduction in intestinal IL-13, a pivotal cytokine in the induction of the colitis. It was found that IND dramatically altered the gut flora and IND no longer exacerbated colitis when colitis was induced after gut flora depletion. CONCLUSIONS: Our data suggest that IND could modify the inflammatory immune response in multiple ways, either directly (i.e., modification of the allergic immune cell activity) or indirectly (i.e., alteration of commensal compositions).

Imaging of the Staphylococcus aureus Inactivation Process Induced by a Multigas Plasma Jet.

To identify mechanisms underlying the bacterial inactivation process by atmospheric nonthermal plasma using a unique plasma jet that can generate various gas plasmas, Staphylococcus aureus were irradiated with carbon dioxide plasma, which produces a large amount of singlet oxygens, and nitrogen plasma, which produces a large amount of OH radicals. And damaged areas of plasma-treated bacteria were observed by field emission scanning electron microscopy, transmission electron microscopy, and atomic force microscopy. As a result, bacteria were damaged by both gas plasmas, but the site of damage differed according to gas species. Therefore, it suggests that singlet oxygen generated by carbon dioxide plasma or other reactive species caused by singlet oxygen contributes to the damage of internal structures of bacteria through the cell wall and membrane, and OH radicals generated by nitrogen plasma or other reactive species derived from OH radicals contribute to damage of the cell wall and membrane.

Microbial Inactivation in the Liquid Phase Induced by Multigas Plasma Jet.

Various gas atmospheric nonthermal plasmas were generated using a multigas plasma jet to treat microbial suspensions. Results indicated that carbon dioxide and nitrogen plasma had high sterilization effects. Carbon dioxide plasma, which generated the greatest amount of singlet oxygen than other gas plasmas, killed general bacteria and some fungi. On the other hand, nitrogen plasma, which generated the largest amount of OH radical, killed ≥ 6 log of 11 species of microorganisms, including general bacteria, fungi, acid-fast bacteria, spores, and viruses in 1-15 min. To identify reactive species responsible for bacterial inactivation, antioxidants were added to bacterial suspensions, which revealed that singlet oxygen and OH radicals had greatest inactivation effects.

Identification of carotenoids from the extremely halophilic archaeon Haloarcula japonica.

The carotenoids produced by extremely halophilic archaeon Haloarcula japonica were extracted and identified by their chemical, chromatographic, and spectroscopic characteristics (UV-Vis and mass spectrometry). The composition (mol%) was 68.1% bacterioruberin, 22.5% monoanhydrobacterioruberin, 9.3% bisanhydrobacterioruberin, <0.1% isopentenyldehydrorhodopin, and trace amounts of lycopene and phytoene. The in vitro scavenging capacity of a carotenoid, bacterioruberin, extracted from Haloarcula japonica cells against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals was evaluated. The antioxidant capacity of bacterioruberin was much higher than that of ß -carotene.

A metabolomics-based approach for predicting stages of chronic kidney disease.

Chronic kidney disease (CKD) is a major epidemiologic problem and a risk factor for cardiovascular events and cerebrovascular accidents. Because CKD shows irreversible progression, early diagnosis is desirable. Renal function can be evaluated by measuring creatinine-based estimated glomerular filtration rate (eGFR). This method, however, has low sensitivity during early phases of CKD. Cystatin C (CysC) may be a more sensitive predictor. Using a metabolomic method, we previously identified metabolites in CKD and hemodialysis patients. To develop a new index of renal hypofunction, plasma samples were collected from volunteers with and without CKD and metabolite concentrations were assayed by quantitative liquid chromatography/mass spectrometry. These results were used to construct a multivariate regression equation for an inverse of CysC-based eGFR, with eGFR and CKD stage calculated from concentrations of blood metabolites. This equation was able to predict CKD stages with 81.3% accuracy (range, 73.9-87.0% during 20 repeats). This procedure may become a novel method of identifying patients with early-stage CKD.

Exploration of novel predictive markers in rat plasma of the early stages of chronic renal failure.

To identify blood markers for early stages of chronic kidney disease (CKD), blood samples were collected from rats with adenine-induced CKD over 28 days. Plasma samples were subjected to metabolomic profiling by liquid chromatography-mass spectrometry, followed by multivariate analyses. In addition to already-identified uremic toxins, we found that plasma concentrations of N6-succinyl adenosine, lysophosphatidylethanolamine 20:4, and glycocholic acid were altered, and that these changes during early CKD were more sensitive markers than creatinine concentration, a universal indicator of renal dysfunction. Moreover, the increase in plasma indoxyl sulfate concentration occurred earlier than increases in phenyl sulfate and p-cresol sulfate. These novel metabolites may serve as biomarkers in identifying early stage CKD.

The reactivity of α-oxoaldehyde with reactive oxygen species in diabetes complications.

The reactions of three α-oxoaldehydes (methylglyoxal, glyoxal, and pyruvic acid) with hydroxyl radicals generated by sonolysis of water were investigated using an electron spin resonance (electron paramagnetic resonance) spin-trapping method, and their reaction kinetics were investigated. It is apparent from our experimental results that methylglyoxal exhibits the highest reactivity of the three α-oxoaldehydes. These α-oxoaldehydes can react with hydroxyl radicals faster than other well-known antioxidants can. The reactivity of hydroxyl radicals is higher than that of hydrogen peroxides.

Reversible off-on fluorescence probe for hypoxia and imaging of hypoxia-normoxia cycles in live cells.

We report a fully reversible off-on fluorescence probe for hypoxia. The design employs QSY-21 as a Förster resonance energy transfer (FRET) acceptor and cyanine dye Cy5 as a FRET donor, based on our finding that QSY-21 undergoes one-electron bioreduction to the radical under hypoxia, with an absorbance decrease at 660 nm. At that point, FRET can no longer occur, and the dye becomes strongly fluorescent. Upon recovery of normoxia, the radical is immediately reoxidized to QSY-21, with loss of fluorescence due to restoration of FRET. We show that this probe, RHyCy5, can monitor repeated hypoxia-normoxia cycles in live cells.

Synthesis of oxovanadium complexes and their apoptosis-inducing activity in leukemia cells.

Vanadium complexes with different ligands were synthesized and evaluated for antiproliferative activity on U937 cells. The alkyl chain length of the ligands affected the antiproliferative activity, and two complexes-3b and 4-exhibited strong activities with IC(50) values of 6.02 and 3.90 µM respectively. Annexin V staining and DNA ladder formation indicated that these complexes induced apoptosis in U937 cells.

Oxovanadium complexes with quinoline and pyridinone ligands: syntheses of the complexes and effect of alkyl chains on their apoptosis-inducing activity in leukemia cells.

Vanadium complexes with quinoline ligands (1b-g) and pyridinone ligands (2b-d) were synthesized, and the effect of the length and shape of alkyl chains on the antiproliferative activity toward U937 cells was studied. For the synthesis of the vanadium complexes, quinoline and pyridinone ligands were prepared and then treated with VOSO(4) or VO(acac)(2). The vanadyl(IV) complexes were characterized by IR, ESR, and UV-vis spectroscopy and elemental analyses. The antiproliferative activity of 1a-g toward U937 cells showed little dependence on the length and shape of the alkyl chain. In contrast, a good correlation was found between the IC(50) values and partition coefficients (logP) values of 2a-c. Among them, 2c showed the highest inhibitory activity, and its IC(50) value was smaller than that of cisplatin. The apoptosis-inducing ability of 2b and 2c was supported by annexin V-propidium iodide staining experiments and agarose gel electrophoresis analysis. Inhibitors of caspase-3, -8, and -9 did not affect the antiproliferative activity of 2c, indicating that the apoptosis induced by 2c was via a caspase-independent pathway.

Synthesis and macrophage activation of lentinan-mimic branched amino polysaccharides: curdlans having N-Acetyl-d-glucosamine branches.

N-Acetyl-d-glucosamine branches were incorporated at the C-6 position of curdlan, a linear ß-1,3-d-glucan, and the resulting nonnatural branched polysaccharides were evaluated in terms of the immunomodulation activities in comparison with lentinan, a ß-1,3-d-glucan having d-glucose branches at C-6. To incorporate the amino sugar branches, we conducted a series of regioselective protection-deprotections of curdlan involving triphenylmethylation at C-6, phenylcarbamoylation at C-2 and C-4, and detriphenylmethylation. Subsequent glycosylation with a d-glucosamine-derived oxazoline, followed by deprotection gave rise to the branched curdlans with various substitution degrees. The products exhibited remarkable solubility in both organic solvents and water. Their immunomodulation activities were determined using mouse macrophagelike cells, and the secretions of both the tumor necrosis factor and nitric oxide proved to be significantly higher than those with lentinan. These results conclude that the amino sugar/curdlan hybrid materials are promising as a new type of polysaccharide immunoadjuvants useful for cancer chemotherapy.

[Fluorophothometric determination of aldehyde by utilizing condensation reaction with resorcinol].

A simple and sensitive fluorophotometric method for the determination of aldehyde was established by utilizing condensation reaction with resorcinol. In the determination of vanillin that is one of aldehydes, the calibration curve exhibited linearity over the vanillin concentration range of 3.0-7600 ng ml(-1) at an emission wavelength of 507 nm with an excitation of 410 nm and with the relative standard deviations (n=5) of 2.5%, 2.0% for 7.6 ng ml(-1), 760 ng ml(-1) of vanillin, respectively. This method was successfully applied in the assay of vanillin in cold medicine.