PmrAB, the two-component system of Acinetobacter baumannii, controls the phosphoethanolamine modification of lipooligosaccharide in response to metal ions.

Acinetobacter baumannii is highly resistant to antimicrobial agents, and XDR strains have become widespread. A. baumannii has developed resistance to colistin, which is considered the last resort against XDR Gram-negative bacteria, mainly caused by lipooligosaccharide (LOS) phosphoethanolamine (pEtN) and/or galactosamine (GalN) modifications induced by mutations that activate the two-component system (TCS) pmrAB. Although PmrAB of A. baumannii has been recognized as a drug resistance factor, its function as TCS, including its regulatory genes and response factors, has not been fully elucidated. In this study, to clarify the function of PmrAB as TCS, we elucidated the regulatory genes (regulon) of PmrAB via transcriptome analysis using pmrAB-activated mutant strains. We discovered that PmrAB responds to low pH, Fe2+, Zn2+, and Al3+. A. baumannii selectively recognizes Fe2+ rather than Fe3+, and a novel region ExxxE, in addition to the ExxE motif sequence, is involved in the environmental response. Furthermore, PmrAB participates in the phosphoethanolamine modification of LOS on the bacterial surface in response to metal ions such as Al3+, contributing to the attenuation of Al3+ toxicity and development of resistance to colistin and polymyxin B in A. baumannii. This study demonstrates that PmrAB in A. baumannii not only regulates genes that play an important role in drug resistance but is also involved in responses to environmental stimuli such as metal ions and pH, and this stimulation induces LOS modification. This study reveals the importance of PmrAB in the environmental adaptation and antibacterial resistance emergence mechanisms of A. baumannii. IMPORTANCE: Antimicrobial resistance (AMR) is a pressing global issue in human health. Acinetobacter baumannii is notably high on the World Health Organization's list of bacteria for which new antimicrobial agents are urgently needed. Colistin is one of the last-resort drugs used against extensively drug-resistant (XDR) Gram-negative bacteria. However, A. baumannii has become increasingly resistant to colistin, primarily by modifying its lipooligosaccharide (LOS) via activating mutations in the two-component system (TCS) PmrAB. This study comprehensively elucidates the detailed mechanism of drug resistance of PmrAB in A. baumannii as well as its biological functions. Understanding the molecular biology of these molecules, which serve as drug resistance factors and are involved in environmental recognition mechanisms in bacteria, is crucial for developing fundamental solutions to the AMR problem.

Affinity of ß-Lactam Antibiotics for Neisseria gonorrhoeae Penicillin-Binding Protein 2 Having Wild, Cefixime-Reduced-Susceptible, and Cephalosporin (Ceftriaxone)-Resistant penA Alleles.

Multidrug-resistant Neisseria gonorrhoeae is a serious concern worldwide. Resistance to ß-lactam antibiotics occurs through mutations in penicillin-binding proteins (PBPs), acquisition of ß-lactamases, and alteration of antibiotic penetration. Mosaic structures of penA, which encodes PBP2, play a major role in resistance to ß-lactams, especially cephalosporins. Ceftriaxone (CRO) is recognized as the only satisfiable antibiotic for the treatment of gonococcal infections; however, CRO-resistant isolates have emerged in the community. Here, we examined the affinity of ß-lactam antibiotics for recombinant PBP2 in a competition assay using fluorescence-labeled penicillin. We found no or little difference in the affinities of penicillins and meropenem (MEM) for PBP2 from cefixime (CFM)-reduced-susceptible strain and cephalosporin-resistant strain. However, the affinity of cephalosporins, including CRO, for PBP2 from the cephalosporin-resistant strain was markedly lower than that for PBP2 from the CFM-reduced-susceptible-resistant strain. Notably, piperacillin (PIP) showed almost the same affinity for PBP2 from penicillin-susceptible, CFM-reduced-susceptible, and cephalosporin (including CRO)-resistant strains. Thus, PIP/tazobactam and MEM are candidate antibiotics for the treatment of CRO-resistant/multidrug-resistant N. gonorrhoeae.

The clarithromycin-binding proteins NIPSNAP1 and 2 regulate cytokine production through mitochondrial quality control.

The mechanism underlying the anti-inflammatory effect of macrolide antibiotics, such as clarithromycin (CAM), remains to be clarified. The CAM-binding proteins 4-nitrophenylphosphatase domain and non-neuronal synaptosomal associated protein 25 (SNAP25)-like protein homolog (NIPSNAP) 1 and 2 are involved in the immune response and mitochondrial homeostasis. However, the axis between CAM-NIPSNAP-mitochondria and Toll-like receptor (TLR) and their molecular mechanisms remain unknown. In this study, we sought to elucidate the relationship between mitochondrial homeostasis mediated by NIPSNAP1 and 2 and the immunomodulatory effect of CAM. NIPSNAP1 or 2 knockdown (KD) by RNA interference impaired TLR4-mediated interleukin-8 (IL-8) production. Similar impairment was observed upon treatment with mitochondrial function inhibitors. However, IL-8 secretion was not impaired in NIPSNAP1 and 2 individual knockout (KO) and double KO (DKO) cells. Moreover, the oxygen consumption rate (OCR) in mitochondria measured using a flex analyzer was significantly reduced in NIPSNAP1 or 2 KD cells, but not in DKO cells. CAM also dose-dependently reduced the OCR. These results indicate that CAM suppresses the IL-8 production via the mitochondrial quality control regulated by temporary functional inhibition of NIPSNAP1 and 2. Our findings provide new insight into the mechanisms underlying cytokine production, including the TLR-mitochondria axis, and the immunomodulatory effects of macrolides.

Human pathogenic bacteria on high-touch dry surfaces can be controlled by warming to human-skin temperature under moderate humidity.

Healthcare-associated infections have become a major health issue worldwide. One route of transmission of pathogenic bacteria is through contact with "high-touch" dry surfaces, such as handrails. Regular cleaning of surfaces with disinfectant chemicals is insufficient against pathogenic bacteria and alternative control methods are therefore required. We previously showed that warming to human-skin temperature affected the survival of pathogenic bacteria on dry surfaces, but humidity was not considered in that study. Here, we investigated environmental factors that affect the number of live bacteria on dry surfaces in hospitals by principal component analysis of previously-collected data (n = 576, for CFU counts), and experimentally verified the effect of warming to human-skin temperature on the survival of pathogenic bacteria on dry surfaces under humidity control. The results revealed that PCA divided hospital dry surfaces into four groups (Group 1~4) and hospital dry surfaces at low temperature and low humidity (Group 3) had much higher bacterial counts as compared to the others (Group 1 and 4) (p<0.05). Experimentally, warming to human-skin temperature (37°C with 90% humidity) for 18~72h significantly suppressed the survival of pathogenic bacteria on dry surfaces, such as plastic surfaces [p<0.05 vs. 15°C (Escherichia coli DH5α, Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, and blaNDM-5 E. coli)] or handrails [p<0.05 vs. 15~25°C (E. coli DH5α, S. aureus, P. aeruginosa, A. baumannii)], under moderate 55% humidity. Furthermore, intermittent heating to human-skin temperature reduced the survival of spore-forming bacteria (Bacillus subtilis) (p<0.01 vs. continuous heating to human-skin temperature). NhaA, an Na+/H+ antiporter, was found to regulate the survival of bacteria on dry surfaces, and the inhibitor 2-aminoperimidine enhanced the effect of warming at human-skin temperature on the survival of pathogenic bacteria (E. coli DH5α, S. aureus, A. baumannii) on dry surfaces. Thus, warming to human-skin temperature under moderate humidity is a useful method for impairing live pathogenic bacteria on high-touch surfaces, thereby helping to prevent the spread of healthcare-associated infections.

Serotype replacement and an increase in non-encapsulated isolates among community-acquired infections of Streptococcus pneumoniae during post-vaccine era in Japan.

Objectives: It is feared that the serotype replacement of Streptococcus pneumoniae occurred by the introduction of pneumococcal vaccines as periodical inoculation leads to reduced efficacy of the approved vaccines and altered antimicrobial susceptibility. Methods: We determined serotypes of 351 S. pneumoniae isolates collected at a commercial clinical laboratory in Hokkaido prefecture, Japan, from December 2018 to February 2019 by using the polymerase chain reaction procedure of the US Centers for Disease Control and Prevention. Antimicrobial susceptibility and resistance gene profiles were also examined. Results: Vaccine coverage rates were 7.9% for 13-valent conjugate vaccine, and 32.5% for 23-valent polysaccharide vaccine, respectively. Non-typable strains were 19.7%. cpsA-positive isolates (group I), and null capsule clade (NCC)1, NCC2 and NCC3 (group II) comprised 31.3%, 28.4%, 32.8%, and 7.5% of the 69 non-typable strains, respectively. No penicillin-resistant/intermediate isolates were found; however, serotypes 35B and 15A/F showed low susceptibility to ß-lactams. Only five strains (1.4%) were levofloxacin-resistant, and all were from the older persons, and three strains were serotype 35B. Conclusion: The progression of serotype replacement in non-invasive pneumococcal infections has occurred during the post-vaccine era in Japan, and non-encapsulated isolates, such as NCC, have increased. Antimicrobial susceptibility is not worsened.

High prevalence of colistin heteroresistance in specific species and lineages of Enterobacter cloacae complex derived from human clinical specimens.

BACKGROUND: Colistin (CST) is a last-line drug for multidrug-resistant Gram-negative bacterial infections. CST-heteroresistant Enterobacter cloacae complex (ECC) has been isolated. However, integrated analysis of epidemiology and resistance mechanisms based on the complete ECC species identification has not been performed. METHODS: Clinical isolates identified as "E. cloacae complex" by MALDI-TOF MS Biotyper Compass in a university hospital in Japan were analyzed. Minimum inhibitory concentrations of CST were determined by the broth microdilution method. The population analysis profiling (PAP) was performed for detecting the heteroresistant phenotype. The heat shock protein 60 (hsp60) cluster was determined from its partial nucleotide sequence. From the data of whole-genome sequencing, average nucleotide identity (ANI) for determining ECC species, multilocus sequence type, core genome single-nucleotide-polymorphism-based phylogenetic analysis were performed. phoPQ-, eptA-, and arnT-deleted mutants were established to evaluate the mechanism underlying colistin heteroresistance. The arnT mRNA expression levels were determined by reverse transcription quantitative PCR. RESULTS: Thirty-eight CST-resistant isolates, all of which exhibited the heteroresistant phenotype by PAP, were found from 138 ECC clinical isolates (27.5%). The prevalence of CST-resistant isolates did not significantly differ among the origin of specimens (29.0%, 27.8%, and 20.2% for respiratory, urine, and blood specimens, respectively). hsp60 clusters, core genome phylogeny, and ANI revealed that the CST-heteroresistant isolates were found in all or most of Enterobacter roggenkampii (hsp60 cluster IV), Enterobacter kobei (cluster II), Enterobacter chuandaensis (clusters III and IX), and Enterobacter cloacae subspecies (clusters XI and XII). No heteroresistant isolates were found in Enterobacter hormaechei subspecies (clusters VIII, VI, and III) and Enterobacter ludwigii (cluster V). CST-induced mRNA upregulation of arnT, which encodes 4-amino-4-deoxy-L-arabinose transferase, was observed in the CST-heteroresistant isolates, and it is mediated by phoPQ pathway. Isolates possessing mcr-9 and mcr-10 (3.6% and 5.6% of total ECC isolates, respectively) exhibited similar CST susceptibility and PAP compared with mcr-negative isolates. CONCLUSIONS: Significant prevalence (approximately 28%) of CST heteroresistance is observed in ECC clinical isolates, and they are accumulated in specific species and lineages. Heteroresistance is occurred by upregulation of arnT mRNA induced by CST. Acquisition of mcr genes contributes less to CST resistance in ECC.

Pseudo-outbreak of Mycobacterium lentiflavum at a general hospital in Japan.

BACKGROUND: Mycobacterium lentiflavum is a slow-growing nontuberculous mycobacterium that is widely distributed in soil and water systems, but it is sometimes pathogenic to humans. Although cases of M. lentiflavum infections are rare, 22 isolates of M. lentiflavum were identified at a single hospital in Japan. We suspected a nosocomial outbreak; thus, we conducted transmission pattern and genotype analyses. METHODS: Cases of M. lentiflavum isolated at Kushiro City General Hospital in Japan between May 2020 and April 2021 were analyzed. The patient samples and environmental culture specimens underwent whole-genome sequencing (WGS). Additionally, we retrospectively collected clinical data from patient medical records. RESULTS: Altogether, 22 isolates of M. lentiflavum were identified from sputum and bronchoalveolar lavage samples. Clinically, the instances with M. lentiflavum isolates were considered contaminants. In the WGS analysis, 19 specimens, including 18 patient samples and 1 environmental culture from the hospital's faucet, showed genetic similarity. The frequency of M. lentiflavum isolation decreased after we prohibited the use of taps where M. lentiflavum was isolated. CONCLUSIONS: WGS analysis identified that the cause of M. lentiflavum pseudo-outbreak was the water used for patient examinations, including bronchoscopy.

Rapid, simple, and cost-effective plaque assay for murine norovirus using microcrystalline cellulose.

Murine norovirus (MNV) is used widely as a practical alternative to human norovirus (HuNoV). Plaque-forming assays for MNV are important for developing therapeutic agents against HuNoV infections. Although agarose-overlay MNV assays have been reported, recent improvements in cellulose derivatives suggest that they could be optimized further, particularly with respect to improving the overlay material. To determine which overlay material is optimal for the MNV plaque assay, we compared four typical cellulose derivatives [microcrystalline cellulose (MCC), hydroxyethyl cellulose (HEC), hydroxypropyl methylcellulose (HPMC), and carboxymethyl cellulose (CMC)] with conventional agarose. We found that 3.5% (w/v) MCC-containing medium provided clear round-shaped plaques on RAW 264.7 cells 1 day after inoculation; the visibility of plaques was comparable with that of the original agarose-overlay assay. Removing residual MCC powder from the MCC-overlay assay before fixing was important for obtaining distinct plaques that are clearly countable. Finally, after calculating the plaque diameter as a percentage of well diameter, we found that 12- and 24-well plates were better than other plates for accurate plaque counting. The MCC-based MNV plaque assay is cost-effective and rapid, and produces plaques that are easy to count. Accurate virus quantification using this optimized plaque assay will enable reliable estimation of norovirus titers.

Discovery of Biologically Optimized Polymyxin Derivatives Facilitated by Peptide Scanning and In Situ Screening Chemistry.

Peptides can be converted to highly active compounds by introducing appropriate substituents on the suitable amino acid residue. Although modifiable residues in peptides can be systematically identified by peptide scanning methodologies, there is no practical method for optimization at the "scanned" position. With the purpose of using derivatives not only for scanning but also as a starting point for further chemical functionalization, we herein report the "scanning and direct derivatization" strategy through chemoselective acylation of embedded threonine residues by a serine/threonine ligation (STL) with the help of in situ screening chemistry. We have applied this strategy to the optimization of the polymyxin antibiotics, which were selected as a model system to highlight the power of the rapid derivatization of active scanning derivatives. Using this approach, we explored the structure-activity relationships of the polymyxins and successfully prepared derivatives with activity against polymyxin-resistant bacteria and those with Pseudomonas aeruginosa selective antibacterial activity. This strategy opens up efficient structural exploration and further optimization of peptide sequences.

Synthesis of macrocyclic nucleoside antibacterials and their interactions with MraY.

The development of new antibacterial drugs with different mechanisms of action is urgently needed to address antimicrobial resistance. MraY is an essential membrane enzyme required for bacterial cell wall synthesis. Sphaerimicins are naturally occurring macrocyclic nucleoside inhibitors of MraY and are considered a promising target in antibacterial discovery. However, developing sphaerimicins as antibacterials has been challenging due to their complex macrocyclic structures. In this study, we construct their characteristic macrocyclic skeleton via two key reactions. Having then determined the structure of a sphaerimicin analogue bound to MraY, we use a structure-guided approach to design simplified sphaerimicin analogues. These analogues retain potency against MraY and exhibit potent antibacterial activity against Gram-positive bacteria, including clinically isolated drug resistant strains of S. aureus and E. faecium. Our study combines synthetic chemistry, structural biology, and microbiology to provide a platform for the development of MraY inhibitors as antibacterials against drug-resistant bacteria.

Wall teichoic acid-dependent phagocytosis of intact cell walls of Lactiplantibacillus plantarum elicits IL-12 secretion from macrophages.

Selected lactic acid bacteria can stimulate macrophages and dendritic cells to secrete IL-12, which plays a key role in activating innate and cellular immunity. In this study, we investigated the roles of cell wall teichoic acids (WTAs) displayed on whole intact cell walls (ICWs) of Lactiplantibacillus plantarum in activation of mouse macrophages. ICWs were prepared from whole bacterial cells of several lactobacilli without physical disruption, and thus retaining the overall shapes of the bacteria. WTA-displaying ICWs of several L. plantarum strains, but not WTA-lacking ICWs of strains of other lactobacilli, elicited IL-12 secretion from mouse bone marrow-derived macrophages (BMMs) and mouse macrophage-like J774.1 cells. The ability of the ICWs of L. plantarum to induce IL-12 secretion was abolished by selective chemical elimination of WTAs from ICWs, but was preserved by selective removal of cell wall glycopolymers other than WTAs. BMMs prepared from TLR2- or TLR4-deficient mouse could secret IL-12 upon stimulation with ICWs of L. plantarum and a MyD88 dimerization inhibitor did not affect ICW-mediated IL-12 secretion. WTA-displaying ICWs, but not WTA-lacking ICWs, were ingested in the cells within 30 min. Treatment with inhibitors of actin polymerization abolished IL-12 secretion in response to ICW stimulation and diminished ingestion of ICWs. When overall shapes of ICWs of L. plantarum were physically disrupted, the disrupted ICWs (DCWs) failed to induce IL-12 secretion. However, DCWs and soluble WTAs inhibited ICW-mediated IL-12 secretion from macrophages. Taken together, these results show that WTA-displaying ICWs of L. plantarum can elicit IL-12 production from macrophages via actin-dependent phagocytosis but TLR2 signaling axis independent pathway. WTAs displayed on ICWs are key molecules in the elicitation of IL-12 secretion, and the sizes and shapes of the ICWs have an impact on actin remodeling and subsequent IL-12 production.

Colistin Susceptibility in Companion Animal-Derived Escherichia coli, Klebsiella spp., and Enterobacter spp. in Japan: Frequent Isolation of Colistin-Resistant Enterobacter cloacae Complex.

Transmission of colistin-resistant Enterobacterales from companion animals to humans poses a clinical risk as colistin is a last-line antimicrobial agent for treatment of multidrug-resistant Gram-negative bacteria including Enterobacterales. In this study, we investigated the colistin susceptibility of 285 Enterobacterales (including 140 Escherichia coli, 86 Klebsiella spp., and 59 Enterobacter spp.) isolated from companion animals in Japan. We further characterized colistin-resistant isolates by multilocus sequence typing (MLST), phylogenetic analysis of hsp60 sequences, and population analysis profiling, to evaluate the potential clinical risk of companion animal-derived colistin-resistant Enterobacterales to humans in line with the One Health approach. All E. coli isolates were susceptible to colistin, and only one Klebsiella spp. isolate (1.2%, 1/86 isolates) was colistin resistant. Enterobacter spp. isolates were frequently colistin resistant (20.3%, 12/59 isolates). In colistin-resistant Enterobacter spp., all except one isolate exhibited colistin heteroresistance by population analysis profiling. These colistin-heteroresistant isolates belonged to clusters I, II, IV, VIII, and XII based on hsp60 phylogeny. MLST analysis revealed that 12 colistin-resistant Enterobacter spp. belonged to the Enterobacter cloacae complex; five Enterobacter kobei (four ST591 and one ST1577), three Enterobacter asburiae (one ST562 and two ST1578), two Enterobacter roggenkampii (ST606 and ST1576), and Enterobacter hormaechei (ST1579) and E. cloacae (ST765) (each one strain). Forty-two percent of the colistin-resistant E. cloacae complex isolates (predominantly ST562 and ST591) belonged to lineages with human clinical isolates. Four E. kobei ST591 isolates were resistant to third-generation cephalosporines, aminoglycosides, and fluroquinolones but remained susceptible to carbapenems. In conclusion, our study is the first to our knowledge to report the frequent isolation of the colistin-resistant E. cloacae complex from companion animals. Furthermore, a subset of isolates belonged to human-associated lineages with resistance to multiple classes of antibiotics. These data warrant monitoring carriage of the colistin-resistant E. cloacae complex in companion animals as part of a domestic infection control procedure in line with the One Health approach.

Investigating the role of heat shock protein 47 in fibrosis in Crohn's disease.

Crohn's disease (CD) and ulcerative colitis (UC) are chronic inflammatory disorders of the gastrointestinal tract that share similar genetic risk factors. However, while fibrotic stricture of the intestine is a major characteristic of CD; it is rarely observed in UC. Deposition of collagen in the extracellular matrix contributes to the formation of fibrotic strictures in CD, but the underlying mechanisms are unknown. In the present study, we found that heat shock protein 47 (HSP47), a stress-response protein that acts as a molecular chaperone during the processing and secretion of collagen, expressed in the intestinal tissue from patients with CD. Serum HSP47 levels and anti-HSP47 antibody titers were significantly higher in patients with CD than in those with UC. Furthermore, anti-HSP47 antibody levels correlated significantly with fibrosis in CD. In addition, HSP47 inhibition significantly suppressed collagen production in fibroblasts in vitro. These findings suggest that HSP47 is a biomarker for differentiating fibrotic from non-fibrotic forms of CD. Additionally, we propose that HSP47 could be a potential target for treating fibrosis in patients with CD.

Role of Lipoteichoic Acid from the Genus Apilactobacillus in Inducing a Strong IgA Response.

Lactic acid bacterium-containing fermentates provide beneficial health effects by regulating the immune response. A naturally fermented vegetable beverage, a traditional Japanese food, reportedly provides health benefits; however, the beneficial function of its bacteria has not been clarified. Apilactobacillus kosoi is the predominant lactic acid bacterium in the beverage. Using murine Peyer's patch cells, we compared the immunoglobulin A (IgA)-inducing activity of A. kosoi 10HT to those of 29 other species of lactic acid bacteria and found that species belonging to the genus Apilactobacillus (A. kosoi 10HT, A. apinorum JCM30765T, and A. kunkeei JCM16173T) possessed significantly higher activity than the others. Thereafter, lipoteichoic acids (LTAs), important immunostimulatory molecules of Gram-positive bacteria, were purified from the three Apilactobacillus species, and their IgA-inducing activity was compared to those of LTAs from Lactiplantibacillus plantarum JCM1149T and a probiotic strain, Lacticaseibacillus rhamnosus GG. The results revealed that LTAs from Apilactobacillus species had significantly higher activity than others. We also compared the LTA structure of A. kosoi 10HT with that of L. plantarum JCM1149T and L. rhamnosus GG. Although d-alanine or both d-alanine and carbohydrate residues were substituents of free hydroxyl groups in the polyglycerol phosphate structure in LTAs from strains JCM1149T and GG, d-alanine residues were not found in LTA from strain 10HT by 1H nuclear magnetic resonance (NMR) analysis. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) analysis of the glycolipid structure of LTA revealed that LTA from strain 10HT contained dihexosyl glycerol, whereas trihexosyl glycerol was detected in LTAs from other strains. These structural differences may be related to differences in IgA-inducing activity. IMPORTANCE The components of lactic acid bacteria that exert immunostimulatory effects are of increasing interest for therapeutic and prophylactic options, such as alternatives to antibiotics, cognitive enhancements, and vaccine adjuvants. LTAs act as immunostimulatory molecules in the host innate immune system by interacting with pattern recognition receptors. However, as LTA structures differ among species, detailed knowledge of the structure-function relationship for immunostimulatory effects is required. Comparisons of the IgA-inducing activity of LTAs have demonstrated that LTAs from the genus Apilactobacillus possess distinctive activities to stimulate mucosal immunity. The first analysis of the LTA structure from the genus Apilactobacillus suggests that it differs from structures of LTAs of related species of lactic acid bacteria. This knowledge is expected to aid in the development of functional foods containing lactic acid bacteria and pharmaceutical applications of immunostimulatory molecules from lactic acid bacteria.

Solid-Phase Total Synthesis of Plusbacin A3.

The total synthesis of the depsipeptide natural product plusbacin A3 (1) utilizing solid-phase peptide synthesis (SPPS) was disclosed. A 3-hydroxy-proline derivative compatible with Fmoc SPPS was prepared by a diastereoselective Joullié-Ugi three-component reaction (JU-3CR)/hydrolysis sequence. After peptide elongation on the solid support, cleavage of the peptide from the resin, followed by macrolactamization and global deprotection, gave plusbacin A3 (1).

A hydroxypropyl methylcellulose plaque assay for human respiratory syncytial virus.

Quantifying proliferative virus particles is one of the most important experimental procedures in virology. Compared with classical overlay materials, newly developed cellulose derivatives enable a plaque-forming assay to produce countable clear plaques easily. HEp-2 cells are widely used in plaque assays for human respiratory syncytial virus (RSV). It is crucial to use an overlay material to keep HEp-2 cell proliferation and prevent RSV particles from spreading over the fluid. Among four cellulose derivatives, carboxymethyl cellulose sodium salt (CMC), hydroxypropyl methylcellulose (HPMC), microcrystalline cellulose (MCC), and hydroxyethyl cellulose (HEC), we found that HPMC was the optimal overlay material because HPMC maintained HEp-2 cell proliferation and RSV infectivity. Although MCC was unsuitable for RSV, it assisted the plaque-forming by human metapneumovirus in TMPRSS2-expressing cells. Therefore, depending on the cells and viruses, it is necessary to use different overlay materials at varying concentrations.

Self-reported Smell and Taste Disorders in Patients With COVID-19: A Japanese Single-center Study.

BACKGROUND/AIM: Smell and taste disorders are among the most common symptoms of COVID-19. However, the relationship between smell and taste disorders and systemic symptoms is not fully understood in Japan. PATIENTS AND METHODS: Questionnaires were mailed to 105 of 111 COVID-19 patients who were hospitalized at our hospital between March and July 2020 in Japan. RESULTS: A total of 74 patients (response rate: 70.5%) completed the survey. Of these, six patients (8.1%) presented with smell disorders only, 16 (21.6%) presented with taste disorders only, and 17 (23.0%) presented with both smell and taste disorders. The mean Visual Analog Scale for smell and taste was 0.5 and 20, respectively, at the time of the most severe symptoms. CONCLUSION: Among COVID-19 patients in Japan, smell and taste disorders are often followed by fever and may not be the first symptoms. Sense of smell is particularly impaired. These symptoms often improve, although they sometimes persist for a long time as sequelae.

Establishment of a reference panel of Helicobacter pylori strains for antimicrobial susceptibility testing.

BACKGROUND: Eradication treatment for Helicobacter pylori gastritis is covered by national health insurance since 2013 in Japan. However, eradication failure due to the increase of antimicrobial resistance has become a serious problem. The present study aims to establish a reference panel of Japanese H. pylori strains for antimicrobial susceptibility testing. METHOD: A total of 28 strains were collected from 4 medical facilities in Japan. Antimicrobial susceptibility tests (ASTs) to clarithromycin (CLR), amoxicillin (AMX), and metronidazole (MNZ), were used to select standard reference strains. Complete genome sequences were also determined. RESULTS: Three H. pylori strains (JSHR3, JSHR6 and JSHR31) were selected as standard reference strains by the Japanese Society for Helicobacter Research (JSHR). The minimum inhibitory concentrations (MICs) of the antibiotics against these 3 strains by agar dilution method with Brucella-based horse-serum-containing agar medium were as follows: JSHR3 (CLR 16 µg/ml, AMX 0.032 µg/ml and MNZ 4 µg/ml), JSHR6 (CLR 0.016 µg/ml, AMX 0.032 µg/ml and MNZ 4 µg/ml), and JSHR31 (CLR 16 µg/ml, AMX 1 µg/ml and MNZ 64 µg/ml). CONCLUSIONS: A reference panel of H. pylori JSHR strains was established. The panel consisted of JSHR6, which was antibiotic-susceptible, JSHR3, which was CLR-resistant, and JSHR31, which was multi-resistant. This reference panel will be essential for standardized ASTs before the optimal drugs are selected for eradication treatment.