Basic and applied research on multiple aminoglycoside antibiotic resistance of actinomycetes: an old-timer's recollection.

A list of our research achievements on multiple aminoglycoside antibiotic (AG) resistance in AG-producing actinomycetes is outlined. In 1979, the author discovered a novel AG (istamycin)-producing Streptomyces tenjimariensis SS-939 by screening actinomycetes with kanamycin (KM)-resistance and plasmid profiles. This discovery directed our biochemical and genetic approaches to multiple AG resistance (AGR) of AG producers. In this article, the following discoveries will be outlined: (1) AGR profiles correlating with the productivity of AGs in AG-producers, (2) Wide distribution of multiple AG resistance in AG-nonproducing actinomycetes, (3) Involvement of ribosomal resistance and AG-acetylating enzymes as underlying AGR factors, (4) Activation by single nucleotide substitution of a silent gene coding for aminoglycoside 3-N-acetyltransferase, AAC(3), in S. griseus, (5) Discovery of a novel antibiotic indolizomycin through protoplast fusion treatment between S. tenjimariensis and S. griseus strains with different AGR phenotypes, and (6) Double stage-acting activity of arbekacin (ABK; an anti-MRSA semisynthetic AG) discovered by acetylation of ABK with cloned AACs; that is both ABK and its acetylated derivatives showed remarkable antibiotic activities.

Epoch-making milestones in antibiotic exploratory researches in Japan.

Professor Satoshi Omura was awarded the 2015 Nobel Prize in Physiology or Medicine. He is the third to win the award for research on antibiotic, following Fleming' (UK, 1945, discovery of penicillin) and Waksman (USA, 1952, discovery of streptomycin), and the second person after Waksman to receive the award for research on actinomycetes. By focusing his research on macrolides stemming from leucomycin research rather than ß-lactams like penicillin or aminoglycosides like streptomycin, Prof. Omura realized many scientific achievements. These efforts finally led to the discovery of avermectin and its semi-synthetic derivative, ivermectin, considered a monumental contribution to the human race. In this manuscript, I will outline the chronicles of the epoch-making antibiotic exploratory researches preceding Prof. Omura.

Highly purified hypochlorous acid water facilitates glucose metabolism and memory formation in type 2 diabetic mice associated with altered-gut microbiota.

Hypochlorous acid (HOCl) is an endogenous oxidant and chlorinating agent in mammals that is effective against a broad range of microorganisms. However, the effects of exogenous HOCl on biological processes have not been reported. In this study, the effects of highly purified slightly acidic hypochlorous acid water (HP-HAW) were investigated. After the safety of oral administration of HP-HAW was confirmed, the effects of HP-HAW on glucose homeostasis were assessed in mice. HP-HAW treatment significantly improved blood glucose levels in hyperglycemic condition. Based on the 16S rRNA sequencing, HP-HAW treatment significantly increased the diversity and changed the composition of gut microbiota by decreasing the abundance of genus Romboutsia in mice fed normal chow. In obese mice, HP-HAW administration tended to improve glucose tolerance. HP-HAW also attenuated memory impairments and changes N-methyl-d-aspartate (NMDA) receptor mRNA expression in obese mice. HP-HAW treatment suppressed Il-6 mRNA expression in the hippocampus in type 2 diabetic mice. Overall, these results support HP-HAW as a potential therapeutic agent to improve or prevent glucose tolerance and memory decline via gut microbiota alteration.

Electrolyzed hypochlorous acid water exhibits potent disinfectant activity against various viruses through irreversible protein aggregation.

It is essential to employ efficient measures to prevent the transmission of pathogenic agents during a pandemic. One such method involves using hypochlorous acid (HClO) solution. The oxidative properties of HClO water (HAW) can contribute to its ability to eliminate viral particles. Here, we examined a highly purified slightly acidic hypochlorous acid water (Hp-SA-HAW) obtained from the reverse osmosis membrane treatment of an electrolytically-generated SA-HAW for its anti-viral activity and mode of action on viral proteins. Hp-SA-HAW exhibited broad-spectrum antiviral effects against various viruses, including adenovirus, hepatitis B virus, Japanese encephalitis virus (JEV), and rotavirus. Additionally, Hp-SA-HAW treatment dose-dependently resulted in irreversibly aggregated multimers of the JEV envelope and capsid proteins. However, Hp-SA-HAW treatment had no discernible effect on viral RNA, indicating that Hp-SA-HAW acts against amino acids rather than nucleic acids. Furthermore, Hp-SA-HAW substantially reduced the infectivity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), including the ancestral variant and other multiple variants. Hp-SA-HAW treatment induced the aggregation of the SARS-CoV-2 spike and nuclear proteins and disrupted the binding of the purified spike protein of SARS-CoV-2 to human ACE2. This study demonstrates that the broad-spectrum virucidal activity of highly purified HClO is attributed to viral protein aggregation of virion via protein oxidation.

The neutralizing effect of mouth rinsing with alkaline electrolyzed water on different regions of the oral cavity acidified by acidic beverages.

PURPOSE: Acids contained in foods and drinks are involved in the initiation and progression of dental erosion which causes tooth wear. The authors evaluated the neutralizing activity of alkaline electrolyzed water (AEW) in vitro and in vivo. METHODS: The neutralizing ability of AEW against eight beverages was investigated in vitro. Furthermore, in an in vivo study, the effect of mouth rinsing with AEW on the pH of the oral cavity acidified with acidic beverages was examined. Following mouth rinsing, the oral pH was measured using a pH meter with a micro pH-sensor at the cervical surface of the mandibular teeth and the surface of soft tissues. RESULTS: Regarding the neutralizing ability, the higher the pH of the AEW, the lower the volume required for neutralization of the tooth surface. Changes in the pH by mouth rinsing with AEW and purified tap water (PTW) were analyzed. The degree of increase of pH with AEW was greater than with PTW at almost all sites. CONCLUSION: These results suggest that AEW has the potential to prevent the initiation and progression of dental erosion. Mouth rinsing with AEW could be a simple and effective way to prevent the initiation and progression of dental erosion and dental caries.

Kanamycin and its derivative, arbekacin: significance and impact.

On the occasion of the 60th anniversary of the discovery (1957) of kanamycin (KM), a series of research achievements on KM and its semisynthetic derivative Arbekacin (ABK) are outlined. KM was first used clinically in 1958 and was appreciated for its remarkable curing effect on various bacterial infections, especially tuberculosis. ABK is a KM derivative rationally semisynthesized to overcome KM resistance due to enzymatic phosphorylation and acetylation. Since its approval in 1990 as an anti-MRSA drug, ABK has been and still is effectively used in chemotherapy because MRSA rarely develops high ABK-resistance. Research that illuminated the unique features of ABK enabling it to resist the development of resistance by MRSA are also described.

Bispolides, novel 20-membered ring macrodiolide antibiotics from microbispora.

Seven new related compounds named bispolides A1, A2, A3, B1, B2a, B2b and B3, have been found in a culture of Microbispora sp. A34030, isolated from a Malaysian soil sample. The planar structures were elucidated to be new 20-membered ring macrodiolide antibiotics on the basis of MS and NMR spectroscopic analyses. These antibiotics showed a good anti-MRSA activity in vitro.

Trends of arbekacin-resistant MRSA strains in Japanese hospitals (1979 to 2000).

A total of 472 clinical strains of methicillin-resistant Staphylococcus aureus (MRSA) isolated in Japan between 1979 and 2000 were investigated for resistance to 8 aminoglycosides, 4 aminoglycoside-modifying enzyme gene profiles, and AluI-restriction fragment length polymorphism of the coagulase gene determined by polymerase chain reaction assay. The majority of MRSA strains tested belonged to 4 groups based on coa-RFLP: L21, L22, L31, and M22. About 90% of recent isolates belonged to type L21, indicating the spread of a specific type of MRSA in Japan. Of the type L21 strains, 41.9% included the aac(6')/aph(2") gene, which was one of the risk factors of arbekacin (ABK) resistance, but only 5.5% were resistant to ABK. In contrast, all of the type M22 strains carried aac(6')/aph(2") and 70.1% showed ABK resistance. Among the other types, less than 20% of strains showed ABK resistance. These results suggested that ABK has maintained potent activity. If the predominance of type L21 continues, there will be no progression to ABK resistance in MRSA. However, it may be necessary to monitor the trends in type M22 continuously.

Synthesis and inhibitory activity of 8-substituted 2-deoxy-beta-KDO against CMP-KDO synthetase.

3-Deoxy-D-manno-octulosonate cytidyltransferase (CMP-KDO synthetase) is involved in the biosynthesis of lipopolysaccharide (LPS) which is an essential component of the outer membrane of gram-negative bacteria. New CMP-KDO synthetase inhibitors, 8-substituted derivatives of 2-deoxy-beta-KDO (2) have been prepared. Compounds 8, 11, 15 and 16 in which the 8-hydroxyl group of 2 is replaced by guanidine, di(carbamoylethyl)amino, p-methoxy- or p-nitro-benzyloxycarbonylamino, respectively affect moderately the CMP-KDO synthetase activity.

Development of a separate tank with an electrolysis-dependent bacteria controlling system for the long term storage of potable water.

For the long term storage of tap water, we developed a separate type of tank (5 m3) equipped with an electrolysis system to control bacterial growth. The electrolysis conditions using 20A direct current and a water flow rate of 10 L/min were capable of producing available chlorine (AC) at the rate of 5-8mg/min and raising the AC level of the stored tap water by about 0.2 mg/kg within 20-30 min The electrolyzed tap water with 0.2 mg/kg AC showed a capability per ml of killing 10(5)-10(6) cfu of bacteria such as Escherichia coli and Pseudomonas aeruginosa within 15 sec. A 6-month trial operation of the storage system with an automatic electrolysis control to keep AC level ranging 0.2-0.4 mg/kg demonstrated that the system worked well for the stored tap water in suppressing bacterial growth as well as in keeping good potable quality with reference to the 46 parameters specified for Japanese tap water. Actually, the electrolysis treatment was administered intermittently with an interval of about two weeks. Thus we believe the developed system has good potential to secure a potable water supply not only in the occasion of emergencies but also in countries having problems in the supply of safe drinking water.

Decontamination of aflatoxin-forming fungus and elimination of aflatoxin mutagenicity with electrolyzed NaCl anode solution.

Electrolysis of a 0.1% (17.1 mM) solution of NaCl using separate anode and cathode compartments gives rise to solutions containing active chemical species. The strongly acidic "anode solution" (EW+) has high levels of dissolved oxygen and available chlorine in a form of hypochlorous acid (HOCl) with a strong potential for sterilization, which we have investigated here. Exposing Aspergillus parasiticus at an initial density of 10(3)spores in 10 microL to a 50-fold volume (500 microL) of EW+ containing ca. 390 micromol HOCl for 15 min at room temperature resulted in a complete inhibition of fungal growth, whereas the cathode solution (EW-) had negligible inhibitory effects. Moreover, the mutagenicity of aflatoxin B(1) (AFB(1)) for Salmonella typhimurium TA-98 and TA-100 strains was strongly reduced after AFB(1) exposure to the EW+ but not with the EW-. In high-performance liquid chromatography analysis, the peak corresponding to AFB(1) disappeared after treatment with the EW+, indicating decomposition of the aflatoxin. In contrast, the routinely used disinfectant sodium hypochlorite, NaOCl, of the same available chlorine content as that of EW+ but in a different chemical form, hypochlorite (OCl-) ion, did not decompose AFB(1) at pH 11. However, NaOCl did decompose AFB(1) at pH 3, which indicated that the principle chemical formula to participate in the decomposition of AFB(1) is not the OCl- ion but HOCl. Furthermore, because the decomposition of AFB(1) was suppressed by pretreating the EW+ with the OH radical scavenger thiourea, the chemical species responsible for the AFB(1)-decomposing property of the EW+ should be at least due to the OH radical originated from HOCl. The OH in EW+ was proved by electron spin resonance analysis.

Characterization of a bifunctional aminoglycoside-modifying enzyme with novel substrate specificity and its gene from a clinical isolate of methicillin-resistant Staphylococcus aureus with high arbekacin resistance.

A clinical isolate (designated PRC104) of methicillin-resistant Staphylococcus aureus was discovered with a novel aminoglycoside resistance profile, including unusually high resistance (MIC 128 microg/ml) to arbekacin (an effective anti-MRSA drug in Japan). We characterized the activity and gene of its bifunctional aminoglycoside-modifying enzyme, AAC(6')/APH(2"), in comparison with those of a regular one that has been known as the critical resistance basis to both gentamicin and arbekacin in methicillin-resistant Staphylococcus aureus. The aac(6')/aph(2") gene of strain PRC104 contained a single base alteration at a novel site (G1126A) resulting in one amino acid substitution (S376N) in the phosphorylation catalytic motif. The phosphorylation activity of the PRC104 enzyme was enhanced for arbekacin and reduced for gentamicin. Both strain PRC104 and S. aureus RN4220 containing the cloned gene were identical in terms of the substrate specificity of the enzyme as well as the aminoglycoside resistance profile, although both mRNA and aminoglycoside resistance levels were markedly high in strain PRC104. Therefore, the cloned aac(6')/aph(2") gene may represent the molecular basis for the novel aminoglycoside modification capability as well as novel aminoglycoside resistance profile of S. aureus PRC104.

Semisynthetic aminoglycoside antibiotics: Development and enzymatic modifications.

The critical resistance mechanisms of aminoglycoside antibiotics in bacteria of clinical importance are the enzymatic N-acetylation, O-phosphorylation, and O-nucleotidylation that generally result in the inactivation of aminoglycosides. To overcome such resistance mechanisms, dibekacin (3',4'-dideoxykanamycin B) was developed as the first rationally designed semisynthetic aminoglycoside, based on the enzymatic 3'-O-phosphorylation of kanamycin. Subsequently, amikacin, netilmicin, and isepamicin were developed by introducing (S)-4-amino-2-hydroxybutyryl (AHB), ethyl, and (S)-3-amino-2-hydroxypropionyl side chains into the 1-amino group of kanamycin, sisomicin, and gentamicin B, respectively. These side chains are believed to block the access of a variety of aminoglycoside-modifying enzymes to their target sites. The latest semisynthetic aminoglycoside of clinical use in Japan is arbekacin (1-N-AHB-dibekacin), which has been extensively used since its approval as an anti-methicillin-resistant Staphylococcus aureus (MRSA) agent in 1990. Although it has several possible modification sites for aminoglycoside acetyltransferases (AACs), arbekacin-resistant MRSA strains that have emerged in the past 8 years have been those with a low or moderate level of resistance, due to a bifunctional enzyme, AAC(6')/APH(2"), at low incidence. To overcome AAC(6')/APH(2")-dependent arbekacin-resistant MRSA strains, 2"-amino-2"-deoxyarbekacin and its 5-epiamino derivative have been already synthesized. However, simulative modification studies using AACs from aminoglycoside-producing Streptomyces strains have revealed that AAC(3) and AAC(2') converted arbekacin to 3"-N-acetyl and 2'-N-acetyl derivatives, respectively, which retain high antibiotic activity. By contrast, the same acetylations of amikacin (3"-N-) and dibekacin (3-N-) resulted in their inactivation. Thus, these new findings confirmed the steric hindrance effect of the 1-N-acyl side chain and illuminated the novel aspect of arbekacin distinct from the other semisynthetic aminoglycosides, indicating that MRSA strains cannot be arbekacin-resistant even if they have acquired the aac(3) or aac(2') gene.

Usefulness of PCR-restriction fragment length polymorphism typing of the coagulase gene to discriminate arbekacin-resistant methicillin-resistant Staphylococcus aureus strains.

We compared the results of two typing methods for 678 strains of methicillin-resistant Staphylococcus aureus and methicillin-susceptible S. aureus. PCR-restriction fragment length polymorphism typing of the coagulase gene was a more reliable method than coagulase serotyping from the viewpoint of arbekacin resistance.

The complete genomic sequence of Nocardia farcinica IFM 10152.

We determined the genomic sequence of Nocardia farcinica IFM 10152, a clinical isolate, and revealed the molecular basis of its versatility. The genome consists of a single circular chromosome of 6,021,225 bp with an average G+C content of 70.8% and two plasmids of 184,027 (pNF1) and 87,093 (pNF2) bp with average G+C contents of 67.2% and 68.4%, respectively. The chromosome encoded 5,674 putative protein-coding sequences, including many candidate genes for virulence and multidrug resistance as well as secondary metabolism. Analyses of paralogous protein families suggest that gene duplications have resulted in a bacterium that can survive not only in soil environments but also in animal tissues, resulting in disease.