[A Trial of Practical Training on Online Medication Instructions in Clinical Preparatory Education: Development of Human Resources to Provide New Forms of Medical Care after the COVID-19 Pandemic].

The coronavirus disease 2019 (COVID-19) pandemic has considerably affected several social services. The Ministry of Health, Labour, and Welfare has partially revised the Pharmaceuticals and Medical Devices Law and established legislations on permanent online medication instructions. Based on these social needs, the development of human resources to provide online medication instructions is vital. Therefore, we developed a training program for providing online medication instructions in preparatory clinical education. Pharmacy students who had conducted medical interviews with standardized patients participated in the training. Educational outcomes were evaluated using an objective multiple-choice test and free description before and after practical training. The median number of correct answers on objective tests on the legislation on online medication instructions increased significantly. Based on the free description analysis, students were able to comprehend the influence of communication environment on the quality of medication instructions. Based on the results of the direct evaluation using objective testing and indirect evaluation by analyzing the free descriptions, they also acquired the skills necessary for providing online medication instructions. Therefore, this training program can contribute to mastering the provision of online medication instructions.

Effects of the order of exposure to antimicrobials on the incidence of multidrug-resistant Pseudomonas aeruginosa.

Multidrug-resistant Pseudomonas aeruginosa (MDRP) is one of the most important pathogens in clinical practice. To clarify the mechanisms contributing to its emergence, we isolated MDRPs using the P. aeruginosa PAO1, the whole genome sequence of which has already been elucidated. Mutant strains resistant to carbapenems, aminoglycosides, and new quinolones, which are used to treat P. aeruginosa infections, were isolated; however, none met the criteria for MDRPs. Then, PAO1 strains were exposed to these antimicrobial agents in various orders and the appearance rate of MDRP varied depending on the order of exposure; MDRPs more frequently appeared when gentamicin was applied before ciprofloxacin, but were rarely isolated when ciprofloxacin was applied first. Exposure to ciprofloxacin followed by gentamicin increased the expression of MexCD-OprJ, an RND-type multidrug efflux pump, due to the NfxB mutation. In contrast, exposure to gentamicin followed by ciprofloxacin resulted in more mutations in DNA gyrase. These results suggest that the type of quinolone resistance mechanism is related to the frequency of MDRP and that the risk of MDRP incidence is highly dependent on the order of exposure to gentamicin and ciprofloxacin.

Complete Genome Sequence of Systemically Disseminated Sequence Type 8 Staphylococcal Cassette Chromosome mec Type IVl Community-Acquired Methicillin-Resistant Staphylococcus aureus.

Staphylococcus aureus JH4899, a community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) isolate collected from a patient with systematically disseminated infection, is classified as sequence type 8 and carries the staphylococcal cassette chromosome mec type IVl (SCCmecIVl). It produces TSST-1, SEC, a newly discovered enterotoxin (SE1), and epidermal cell differentiation inhibitor A (EDIN-A). Here, we present the complete genome sequence of the chromosome and a plasmid harboring the se1 and ednA genes.

Emergence of Daptomycin-Resistant Staphylococcus aureus during Treatment.

A 68-year-old man with persistent bacteremia accompanying a large iliopsoas abscess, vertebral osteomyelitis, discitis and central venous port infection caused by methicillin-resistant Staphylococcus aureus (MRSA) was admitted to our hospital. During the course of treatment, the emergence of a daptomycin (DAP)-resistant MRSA strain was confirmed; the minimum inhibitory concentration was 1 to 2 µg/mL for vancomycin and more than 1 µg/mL for DAP. Although the bacterial cell wall was not significantly thickened, an increased positive surface charge and single-nucleotide polymorphism within mprF have been confirmed in DAP-resistant strains. Still rare, but clinicians need to be cautious of the emergence of DAP-resistant MRSA during treatment.

Comprehensive analysis of systemically disseminated ST8/non-USA300 type community-acquired methicillin-resistant Staphylococcus aureus infection.

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) is genetically heterogeneous and various genotypes are spreading worldwide. We herein report a case of systematically disseminated Japan-intrinsic CA-MRSA infection that was successfully treated. A genetically identical single strain was isolated from a total of 11 different parts of the patient's body, and the pathogen was found to be multilocus sequence type 8, staphylococcal cassette chromosome mec IV, coagulase type III and negative for both Panton-Valentine leukocidin and arginine catabolic mobile element. The epidemiology and pathogenicity of the Japan-intrinsic CA-MRSA strain remain unknown, and further investigation of this life-threatening organism is warranted.

Is wet swab superior to dry swab as an intranasal screening test?

Methicillin-resistant Staphylococcus aureus is still a great concern, and recognition of the carrier is essential for appropriate infection control in intensive care units. The utility of wet swab compared to dry swab as an intranasal screening test has not been well assessed yet. A comparative study of the wet and dry swab in its ability to detect the organism was performed against critically ill patients, and it was found that there were no statistically significant differences between the two different methods. The wet swab did not show increased sensitivity compared to dry one.

Synergistic effect of kaempferol glycosides purified from Laurus nobilis and fluoroquinolones on methicillin-resistant Staphylococcus aureus.

In a previous study, we reported that two kaempferol glycosides isolated from Laurus nobilis L., kaempferol-3-O-alpha-L-(2'',4''-di-E-p-coumaroyl)-rhamnoside (C2) and kaempferol-3-O-alpha-L-(2''-E-p-coumaroyl-4''-Z-p-coumaroyl)-rhamnoside (C3), showed strong antibacterial activities against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci. Thereafter we found that these compounds greatly reduced the minimum inhibitory concentrations (MICs) of some fluoroquinolones in MRSA. In other words, C2 and C3 greatly potentiated anti-MRSA activity of fluoroquinolones. The effect of C2 and C3 with fluoroquinolones was found to be synergistic. The potentiation activity was observed with hydrophilic fluoroquinolones, such as norfloxacin and ciprofloxacin, but not with hydrophobic quinolones. We also found that norfloxacin reduced MICs of C2 and C3. The effect was synergistic. Possible mechanism of the synergistic effect was discussed.

Anti-methicillin resistant Staphylococcus aureus (MRSA) compounds isolated from Laurus nobilis.

We found that an extract from Laurus nobilis L. (Lauraceae) leaves showed antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). We purified two flavonoids as the effective compounds and identified them as kaempferol 3-O-alpha-L-(2'',4''-di-E-p-coumaroyl)-rhamnoside (C2) and kaempferol 3-O-alpha-L-(2''-Z-p-coumaroyl-4''-E-p-coumaroyl)-rhamnoside (C3). Both compounds showed strong antibacterial activity not only against MRSA but also against vancomycin-resistant enterococci (VRE). There was low or no antibacterial activity of C2 and C3 for Streptococcus pneumoniae, Pseudomonas aeruginosa and Serratia marcescens.

Enhancement of antibacterial effects of epigallocatechin gallate, using ascorbic acid.

Although plant polyphenols such as (-)-epigallocatechin gallate (EGCG) have antibacterial activity towards methicillin-resistant Staphylococcus aureus (MRSA), such polyphenols are unstable in solution. Because the instability of polyphenols is attributable to their oxidation, we examined the effects of antioxidants and inhibitors of polyphenol oxidation on the maintenance of polyphenol antibacterial activity. The antibacterial activity of EGCG was enhanced in the presence of ascorbic acid, and ascorbic acid was the most effective for retaining the concentration of stable EGCG. On the other hand, the antibacterial activity of EGCG was lowered in the presence of casein in spite of its suppressing effect on the EGCG decrease. The effect of EGCG on the antibiotic resistance of MRSA was also enhanced in the presence of ascorbic acid. The addition of an antioxidant may affect other pharmacological effects of polyphenols in analogous ways, although this does not mean the clinical usefulness of the addition directly.

Antimicrobial activity of oleanolic acid from Salvia officinalis and related compounds on vancomycin-resistant enterococci (VRE).

An extract from Salvia officinalis (Sage) leaves showed antimicrobial activity against vancomycin-resistant enterococci (VRE). We isolated the effective compound and identified it as oleanolic acid, a triterpenoid. We also tested antimicrobial activity of similar triterpenoids, ursolic acid, uvaol, betulinic acid and betulin. We found that ursolic acid also showed antimicrobial activity against VRE. The minimum inhibitory concentrations (MICs) of oleanolic acid and ursolic acid were 8 and 4 microg/ml, respectively. These two compounds also showed antimicrobial activity against Streptococcus pneumoniae and methicillin-resistant Staphylococcus aureus (MRSA). These compounds showed bactericidal activity against VRE at least for 48 h when added at concentrations that were two-times higher than their MICs.

Potentiation of antimicrobial activity of aminoglycosides by carnosol from Salvia officinalis.

We found that a crude extract from Salvia officinalis (sage) reduced the minimum inhibitory concentrations (MICs) of aminoglycosides in vancomycin-resistant enterococci (VRE). We isolated the effective compound from the extract and identified it as carnosol, one of diterpenoids. Carnosol showed a weak antimicrobial activity, and greatly reduced the MICs of various aminoglycosides (potentiated the antimicrobial activity of aminoglycosides) and some other types of antimicrobial agents in VRE. Carnosic acid, a related compound, showed the similar activity. The effect of carnosol and carnosic acid with gentamicin was synergistic.

Polyphenolic constituent structures of Zanthoxylum piperitum fruit and the antibacterial effects of its polymeric procyanidin on methicillin-resistant Staphylococcus aureus.

Zanthoxylum piperitum (Rutaceae) is used as a spice and a natural medicine in Japan. Our study found that ZP-CT-A, a polymeric proanthocyanidin purified from the fruit of this species, noticeably decreased the minimum inhibitory concentrations of beta-lactam antibiotics for methicillin-resistant Staphylococcus aureus (MRSA). The structure of ZP-CT-A was characterized on the basis of (13)C NMR and size exclusion chromatographic data and the results of thiolytic degradation. A mechanistic study of the effects of ZP-CT-A indicated that it suppressed the activity of beta-lactamase and largely decreased the stability of the bacterial cell membrane of MRSA, as shown by a reduction in the tolerance of MRSA to low osmotic pressure and high ionic strength solutions.

Functional gene cloning and characterization of MdeA, a multidrug efflux pump from Staphylococcus aureus.

A DNA fragment conferring drug resistance was cloned from the chromosomal DNA of Staphylococcus aureus N315 using a drug hypersensitive Escherichia coli KAM32 as the host. Although E. coli KAM32 cells were sensitive to many antimicrobial agents, transformed cells harboring a recombinant plasmid carrying the DNA region became resistant to several structurally unrelated antimicrobial agents, such as tetraphenylphosphonium chloride, Hoechst 33342 and norfloxacin. These results suggest that the cloned DNA fragment carries a gene(s) encoding a multidrug efflux pump. We partially determined the nucleotide sequence of the cloned DNA and found the mdeA gene within it. The E. coli cells transformed with the mdeA gene showed efflux activity of Hoechst 33342. On the other hand, S. aureus cells transformed with mdeA showed elevated resistance to doxorubicin, daunorubicin, tetraphenylphosphonium chloride, Hoechst 33342, ethidium bromide and rhodamine 6G. Elevated energy-dependent efflux of ethidium was observed with transformed S. aureus. We found that the mdeA gene was expressed under normal growth conditions in S. aureus N315.

Synergistic effect of [10]-gingerol and aminoglycosides against vancomycin-resistant enterococci (VRE).

An extract from ginger (root of Zingiber officinale) reduced the minimum inhibitory concentrations (MICs) of aminoglycosides in vancomycin-resistant enterococci (VRE). The effective compound was isolated and identified as [10]-gingerol. In the presence of [10]-gingerol at 1/10 concentration of its own MIC, the MIC of arbekacin was lowered by 1/32 to 1/16. [10]-Gingerol also reduced the MICs of other aminoglycosides, and of bacitracin and polymixin B, but not of other antimicrobial agents tested. Because [10]-gingerol reduced the MICs of several aminoglycosides both in strains possessing or lacking aminoglycoside-modification enzymes, it seems that the effect of [10]-gingerol is not related to these enzymes, which mainly confer bacterial resistance against aminoglycosides. It seemed that a detergent-like effect of [10]-gingerol potentiated the antimicrobial activity of the aminoglycosides. In fact, some detergents such as sodium dodecyl sulfate (SDS) and Triton X-100 reduced the MICs of aminoglycosides, bacitracin and polymixin B in VRE. Since the intrinsic resistance to aminoglycosides in enterococci is due to low level of entry of the drugs into the cells, increase in the membrane permeability caused by [10]-gingerol will enhance the influx of aminoglycosides into enterococcal cells.

Gene cloning and characterization of SdrM, a chromosomally-encoded multidrug efflux pump, from Staphylococcus aureus.

There are more than 30 genes for putative multidrug efflux pumps in the chromosome of Staphylococcus aureus. Only a few of these have been analyzed so far. Here we cloned a new gene, SA1972, using a PCR method, from the chromosome of S. aureus N315. We found that the product SA1972 could lead to elevated resistance against several antimicrobial agents such as norfloxacin, acriflavine and ethidium bromide. We designated the gene as sdrM. We observed elevated energy-dependent efflux of acriflavine in S. aureus cells introduced with the sdrM gene. We conclude that SdrM is a multidrug efflux pump belonging to the major facilitator (MF) superfamily.

Effects of tannins and related polyphenols on methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) often acquires multi-drug resistance and is involved in many cases of disease in hospitals. We investigated natural substances directly effective against MRSA or that influence antibiotic resistance. Aloe-emodin, an anthraquinone, and several licorice flavonoids showed potent antibacterial effects against MRSA. Like some hydrolysable tannins (corilagin and tellimagrandin I) and a tea polyphenol [(-)-epicatechin gallate], the licorice flavonoid licoricidin also restored the effects of oxacillin, a beta-lactam antibiotic against MRSA. Further study revealed that theasinensin A, a polyphenol formed from (-)-epigallocatechin gallate, proanthocyanidins obtained from fruits of Zizyphus jujuba var. inermis, and polymeric proanthocyanidins from fruit peels of Zanthoxylum piperitum also suppressed the antibiotic resistance of MRSA.

Remarkable synergies between baicalein and tetracycline, and baicalein and beta-lactams against methicillin-resistant Staphylococcus aureus.

During the screening of compounds that potentiate the effect of antimicrobial agents against methicillin-resistant Staphylococcus aureus(MRSA), we found that an extract of thyme (Thymus vulgaris L) leaves greatly reduced the minimum inhibitory concentration (MIC) of tetracycline against MRSA. We isolated the effective compound and identified it as baicalein (5, 6, 7-trihydroxyflavone). One of the clinically isolated MRSA strains possessed tetK, a gene encoding active efflux pump for tetracycline. We examined the effect of baicalein on the efflux of tetracycline, using Escherichia coli KAM32/pTZ1252 carrying the tetK. The E. coli KAM32/pTZ1252 showed 8 to 16 times higher MIC than E. coli KAM32. We observed strong inhibition of transport of tetracycline by baicalein with membrane vesicles prepared from E. coli KAM32/pTZ1252. Baicalein also showed synergy with tetracycline in a MRSA strain that doesn't possess tetK, or with beta-lactams. Thus, mechanisms of the synergies seem to be versatile.

Mechanisms of action of corilagin and tellimagrandin I that remarkably potentiate the activity of beta-lactams against methicillin-resistant Staphylococcus aureus.

Corilagin and tellimagrandin I are polyphenols isolated from the extract of Arctostaphylos uvaursi and Rosa canina L. (rose red), respectively. We have reported that corilagin and tellimagrandin I remarkably reduced the minimum inhibitory concentration (MIC) of beta-lactams in methicillin-resistant Staphylococcus aureus(MRSA). In this study, we investigated the effect of corilagin and tellimagrandin I on the penicillin binding protein 2 '(2a) (PBP2 '(PBP2a)) which mainly confers the resistance to beta-lactam antibiotics in MRSA. These compounds when added to the culture medium were found to decrease production of the PBP2 '(PBP2a) slightly. Using BOCILLIN FL, a fluorescent-labeled benzyl penicillin, we found that PBP2 '(PBP2a) in MRSA cells that were grown in medium containing corilagin or tellimagrandin I almost completely lost the ability to bind BOCILLIN FL. The binding activity of PBP2 and PBP3 were also reduced to some extent by these compounds. These results indicate that inactivation of PBPs, especially of PBP2 '(PBP2a), by corilagin or tellimagrandin I is the major reason for the remarkable reduction in the resistance level of beta-lactams in MRSA. Corilagin or tellimagrandin I suppressed the activity of beta-lactamase to some extent.

Induction of mexCD-oprJ operon for a multidrug efflux pump by disinfectants in wild-type Pseudomonas aeruginosa PAO1.

Induction of the MexCD-OprJ multidrug efflux pump was investigated in wild-type Pseudomonas aeruginosa PAO1. MexCD-OprJ was induced by clinically important disinfectants such as benzalkonium chloride and chlorhexidine gluconate, and by some cytotoxic agents such as tetraphenylphosphonium chloride, ethidium bromide and rhodamine 6G. MexCD-OprJ was not induced by norfloxacin, tetracycline, chloramphenicol, streptomycin, erythromycin or carbenicillin, although they are substrates for the pump. Cells of PAO1 showed increased resistance to norfloxacin when grown in the presence of the inducers of the mexCD-oprJ operon mentioned above. These results indicate that MexCD-OprJ plays an important role in intrinsic multidrug resistance in wild-type P. aeruginosa in hospitals where disinfectants are used frequently.