研究者詳細 - 小笠原　徳子

論文 - 小笠原　徳子

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TNF induces production of type 2 cytokines in human group 2 innate lymphoid cells.

Noriko Ogasawara, Julie A Poposki, Aiko I Klingler, Bruce K Tan, Kathryn E Hulse, Whitney W Stevens, Anju T Peters, Leslie C Grammer, Kevin C Welch, Stephanie S Smith, David B Conley, Ken-Ichi Takano, Tetsuo Himi, Robert C Kern, Robert P Schleimer, Atsushi Kato

The Journal of allergy and clinical immunology 145 ( 1 ) 437 - 440 2020年01月 [査読有り] [国際誌]

DOI PubMed

Role of RANK-L as a potential inducer of ILC2-mediated type 2 inflammation in chronic rhinosinusitis with nasal polyps.

Noriko Ogasawara, Julie A Poposki, Aiko I Klingler, Bruce K Tan, Kathryn E Hulse, Whitney W Stevens, Anju T Peters, Leslie C Grammer, Kevin C Welch, Stephanie S Smith, David B Conley, Joseph R Raviv, Pejman Soroosh, Ken-Ichi Takano, Tetsuo Himi, Robert C Kern, Robert P Schleimer, Atsushi Kato

Mucosal immunology 13 ( 1 ) 86 - 95 2020年01月 [査読有り] [国際誌]

Chronic rhinosinusitis with nasal polyps (CRSwNP) is characterized by type 2 inflammation with accumulation of activated group 2 innate lymphoid cells (ILC2s) and elevation of thymic stromal lymphopoietin (TSLP). A member of the TNF superfamily (TNFSF), TNFSF15, is known to induce the production of type 2 cytokines in ILC2s. Although ILC2s have been implicated in CRSwNP, the presence and role of TNFSFs in ILC2-mediated type 2 inflammation in CRSwNP has not been elucidated. Here, we investigate the involvement of TNFSFs in ILC2-mediated type 2 inflammation in CRSwNP. We found that receptor activator of NF-κB (RANK) ligand (RANK-L (TNFSF11)) was significantly elevated in nasal polyps (NPs), and that the receptor of RANK-L, RANK, was expressed on ILC2s in human peripheral blood and NPs. An agonistic antibody against RANK induced production of type 2 cytokines in human ILC2s, and TSLP significantly enhanced this reaction. The membrane-bound RANK-L was detected mainly on CD45 + immune cells, including TH2 cells in NPs. The co-culture of NP-derived ILC2s and TH2 cells significantly enhanced production of type 2 cytokines, and anti-RANK-L monoclonal antibody suppressed this enhancement. In conclusion, RANK-L, together with TSLP, may play an inductive role in the ILC2-mediated type 2 inflammation in CRSwNP.

DOI PubMed

Epithelial activators of type 2 inflammation: Elevation of thymic stromal lymphopoietin, but not IL-25 or IL-33, in chronic rhinosinusitis with nasal polyps in Chicago, Illinois.

Noriko Ogasawara, Aiko I Klingler, Bruce K Tan, Julie A Poposki, Kathryn E Hulse, Whitney W Stevens, Anju T Peters, Leslie C Grammer, Kevin C Welch, Stephanie S Smith, David B Conley, Robert C Kern, Robert P Schleimer, Atsushi Kato

Allergy 73 ( 11 ) 2251 - 2254 2018年11月 [査読有り] [国際誌]

DOI PubMed

Up-regulation of serum periostin and squamous cell carcinoma antigen levels in infants with acute bronchitis due to respiratory syncytial virus.

Hiroaki Nakamura, Kenichi Akashi, Masako Watanabe, Shoichiro Ohta, Junya Ono, Yoshinori Azuma, Noriko Ogasawara, Keisuke Yamamoto, Norikazu Shimizu, Hiroyuki Tsutsumi, Kenji Izuhara, Toshio Katsunuma

Allergology international : official journal of the Japanese Society of Allergology 67 ( 2 ) 259 - 265 2018年04月 [査読有り] [国際誌]

BACKGROUND: Periostin and squamous cell carcinoma antigen (SCCA) are involved in the pathogenesis of asthma. Acute bronchitis due to respiratory syncytial virus (RSV) infection during infancy exhibits an asthma-like pathogenesis, suggesting that it may be associated with the subsequent development of asthma. However, the mechanism by which RSV infection leads to development of asthma has not yet been fully elucidated. METHODS: Infants younger than 36 months were enrolled and classified into three groups. Group I included patients hospitalized with RSV-induced bronchitis. These patients were further stratified into two sub-groups according to whether the criteria for the modified Asthma Predictive Index (mAPI) had been met: Group I consisted of mAPI (+) and mAPI (-) patients; Group II included patients with food allergy as a positive control group; and Group III included children with no allergy as a negative control group. Serum periostin and SCCA levels were measured in the groups. This study was registered as a clinical trial (UMIN000012339). RESULTS: We enrolled 14 subjects in Group I mAPI (+), 22 in Group I mAPI (-), 18 in Group II, and 18 in Group III. In Group I, the serum periostin and SCCA levels were significantly higher during the acute phase compared with the recovery phase. However, no significant differences were found between Group I mAPI (+) and mAPI (-). CONCLUSIONS: The serum periostin and SCCA levels increased during acute RSV bronchitis. Both periostin and SCCA may play a role in the pathogenesis of acute bronchitis due to RSV.

DOI PubMed

IL-10, TGF-β, and glucocorticoid prevent the production of type 2 cytokines in human group 2 innate lymphoid cells.

Ogasawara N, Poposki JA, Klingler AI, Tan BK, Weibman AR, Hulse KE, Stevens WW, Peters AT, Grammer LC, Schleimer RP, Welch KC, Smith SS, Conley DB, Raviv JR, Soroosh P, Akbari O, Himi T, Kern RC, Kato A

The Journal of allergy and clinical immunology ( Mosby Inc. ) 141 ( 3 ) 1147 - 1151 2018年03月 [査読有り] [国際誌]

DOI PubMed

Evaluation of consistency in quantification of gene copy number by real-time reverse transcription quantitative polymerase chain reaction and virus titer by plaque-forming assay for human respiratory syncytial virus.

Keisuke Yamamoto, Noriko Ogasawara, Soh Yamamoto, Kenichi Takano, Tsukasa Shiraishi, Toyotaka Sato, Hiroyuki Tsutsumi, Tetsuo Himi, Shin-Ichi Yokota

Microbiology and immunology 62 ( 2 ) 90 - 98 2018年02月 [査読有り] [国際誌]

The plaque-forming assay is the standard technique for determining viral titer, and a critical measurement for investigating viral replication. However, this assay is highly dependent on experimental technique and conditions. In the case of human respiratory syncytial virus (RSV) in particular, it can be difficult to objectively confirm the accuracy of plaque-forming assay because the plaques made by RSV are often small and unclear. In recent studies, RT-qPCR methods have emerged as a supportive procedure for assessment of viral titer, yielding highly sensitive and reproducible results. In this report, we compare the viral replication, as determined by plaque-forming assay, and the copy numbers of RSV genes NS1, NS2, N, and F, as determined by RT-qPCR. Two real-time PCR systems, SYBR Green and TaqMan probe, gave highly similar results for measurement of copy numbers of RSV N genes of virus subgroups A. We determined the RSV gene copy numbers in the culture cell supernatant and cell lysate measured at various multiplicities of infection. We found that copy number of the RSV N gene in the culture supernatant and cell lysate was highly correlated with plaque-forming units. In conclusion, RT-qPCR measurement of RSV gene copy number was highly dependent on viral titer, and the detailed comparison between each gene copy number and virus titer should be useful and supportive in confirming RSV plaque-forming assay and virus dynamics. The technique may also be used to estimate the amount of RSV present in clinical specimens.

DOI PubMed

NIP-SNAP-1 and -2 mitochondrial proteins are maintained by heat shock protein 60.

Soh Yamamoto, Tomoya Okamoto, Noriko Ogasawara, Shin Hashimoto, Tsukasa Shiraishi, Toyotaka Sato, Keisuke Yamamoto, Hiroyuki Tsutsumi, Kenichi Takano, Testuo Himi, Hideaki Itoh, Shin-Ichi Yokota

Biochemical and biophysical research communications ( ACADEMIC PRESS INC ELSEVIER SCIENCE ) 483 ( 3 ) 917 - 922 2017年02月 [査読有り] [国際誌]

NIP-SNAP-1 and -2 are ubiquitous proteins thought to be associated with maintenance of mitochondrial function, neuronal transmission, and autophagy. However, their physiological functions remain largely unknown. To elucidate their functional importance, we screened for proteins that interact with NIP-SNAP-1 and -2, resulting in identification of HSP60 and P62/SQSTM1 as binding proteins. NIP-SNAP-1 and -2 localized in the mitochondrial inner membrane space, whereas HSP60 localized in the matrix. Native gel electrophoresis and filter trap assays revealed that human HSP60 prevented aggregation of newly synthesized NIP-SNAP-2 in an in vitro translation system. Moreover, expression levels of NIP-SNAP-1 and -2 in cells were decreased by knockdown of HSP60, but not HSP10. These findings indicate that HSP60 promotes folding and maintains the stability of NIP-SNAP-1 and -2.

DOI PubMed

Mitochondrial proteins NIP-SNAP-1 and -2 are a target for the immunomodulatory activity of clarithromycin, which involves NF-κB-mediated cytokine production.

Yamamoto S, Ogasawara N, Yamamoto K, Uemura C, Takaya Y, Shiraishi T, Sato T, Hashimoto S, Tsutsumi H, Takano K, Himi T, Yokota SI

Biochemical and biophysical research communications ( ACADEMIC PRESS INC ELSEVIER SCIENCE ) 483 ( 3 ) 911 - 916 2017年02月 [査読有り] [国際誌]

Macrolide antibiotics have immunomodulatory activities, including suppression of cytokine production, cell adhesion molecule expression, and mucin production. These immunomodulatory activities improve the symptoms of respiratory diseases associated with chronic inflammation. However, the underlying molecular mechanism(s) is not well understood yet. To address this, we prepared clarithromycin (CAM)-conjugated Sepharose and examined bound cellular proteins by proteome analysis. We identified mitochondrial proteins 4-nitrophenylphosphatase domain and non-neuronal synaptosomal associated protein 25-like protein homolog (NIP-SNAP)-1 and -2 and very long-chain acyl-CoA dehydrogenase (VLCAD) as CAM-binding proteins. Production of proinflammatory cytokines (IL-8 and IL-6) induced by lipopolysaccharides (LPSs) and Pam3-CSK4 in human epithelial cell lines BEAS-2B and T24 were suppressed by knockdown of NIP-SNAP-1 or -2, and partly by knockdown of VLCAD. Also, knockdown of NIP-SNAP-1 or -2 in various cell lines suppressed LPS-induced expression of IL-8 and IL-6 mRNA and NF-κB activity. Thus, CAM suppresses NF-κB-mediated proinflammatory cytokine production by interacting with mitochondrial proteins, NIP-SNAP-1 and -2.

DOI PubMed

Clarithromycin prevents human respiratory syncytial virus-induced airway epithelial responses by modulating activation of interferon regulatory factor-3

Keisuke Yamamoto, Soh Yamamoto, Noriko Ogasawara, Kenichi Takano, Tsukasa Shiraishi, Toyotaka Sato, Ryo Miyata, Takuya Kakuki, Ryuta Kamekura, Takashi Kojima, Hiroyuki Tsutsumi, Tetsuo Himi, Shin-ichi Yokota

PHARMACOLOGICAL RESEARCH ( ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD ) 111 804 - 814 2016年09月 [査読有り]

Macrolide antibiotics exert immunomodulatory activity by reducing pro-inflammatory cytokine production by airway epithelial cells, fibroblasts, vascular endothelial cells, and immune cells. However, the underlying mechanism of action remains unclear. Here, we examined the effect of clarithromycin (CAM) on pro-inflammatory cytokine production, including interferons (IFNs), by primary human nasal epithelial cells and lung epithelial cell lines (A549 and BEAS-2B cells) after stimulation by Toll-like receptor (TLR) and RIG-I-like receptor (RLR) agonists and after infection by human respiratory syncytial virus (RSV). CAM treatment led to a significant reduction in poly I:C-and RSV-mediated IL-8, CCL5, IFN-beta and -lambda production. Furthermore, IFN-beta promoter activity (activated by poly I:C and RSV infection) was significantly reduced after treatment with CAM. CAM also inhibited IRF-3 dimerization and subsequent translocation to the nucleus. We conclude that CAM acts a crucial modulator of the innate immune response, particularly IFN production, by modulating IRF-3 dimerization and subsequent translocation to the nucleus of airway epithelial cells. This newly identified immunomodulatory action of CAM will facilitate the discovery of new macrolides with an anti-inflammatory role. (C) 2016 Elsevier Ltd. All rights reserved.

DOI PubMed

Rapid and Integrated Bacterial Evolution Analysis unveils gene mutations and clinical risk of Klebsiella pneumoniae.

Kojiro Uemura, Toyotaka Sato, Soh Yamamoto, Noriko Ogasawara, Jirachaya Toyting, Kotaro Aoki, Akira Takasawa, Masayuki Koyama, Atsushi Saito, Takayuki Wada, Kaho Okada, Yurie Yoshida, Koji Kuronuma, Chie Nakajima, Yasuhiko Suzuki, Motohiro Horiuchi, Kenichi Takano, Satoshi Takahashi, Hirofumi Chiba, Shin-Ichi Yokota

Nature communications 16 ( 1 ) 2917 - 2917 2025年03月 [国際誌]

Bacteria continually evolve. Previous studies have evaluated bacterial evolution in retrospect, but this approach is based on only speculation. Cohort studies are reliable but require a long duration. Additionally, identifying which genetic mutations that have emerged during bacterial evolution possess functions of interest to researchers is an exceptionally challenging task. Here, we establish a Rapid and Integrated Bacterial Evolution Analysis (RIBEA) based on serial passaging experiments using hypermutable strains, whole-genome and transposon-directed sequencing, and in vivo evaluations to monitor bacterial evolution in a cohort for one month. RIBEA reveals bacterial factors contributing to serum and antimicrobial resistance by identifying gene mutations that occurred during evolution in the major respiratory pathogen Klebsiella pneumoniae. RIBEA also enables the evaluation of the risk for the progression and the development of invasive ability from the lung to blood and antimicrobial resistance. Our results demonstrate that RIBEA enables the observation of bacterial evolution and the prediction and identification of clinically relevant high-risk bacterial strains, clarifying the associated pathogenicity and the development of antimicrobial resistance at genetic mutation level.

DOI PubMed

Bacillaceae serine proteases and Streptomyces epsilon-poly-l-lysine synergistically inactivate Caliciviridae by inhibiting RNA genome release

Soh Yamamoto, Noriko Ogasawara, Yuka Sudo-Yokoyama, Sachiko Sato, Nozomu Takata, Nana Yokota, Tomomi Nakano, Kyoko Hayashi, Akira Takasawa, Mayumi Endo, Masako Hinatsu, Keitaro Yoshida, Toyotaka Sato, Satoshi Takahashi, Kenichi Takano, Takashi Kojima, Jun Hiraki, Shin-ich Yokota

Scientific Reports ( Springer Science and Business Media LLC ) 14 ( 1 ) 2024年07月

Abstract Human norovirus (HuNoV) is an enteric infectious pathogen belonging to the Caliciviridae family that causes occasional epidemics. Circulating alcohol-tolerant viral particles that are readily transmitted via food-borne routes significantly contribute to the global burden of HuNoV-induced gastroenteritis. Moreover, contact with enzymes secreted by other microorganisms in the environment can impact the infectivity of viruses. Hence, understanding the circulation dynamics of Caliciviridae is critical to mitigating epidemics. Accordingly, in this study, we screened whether environmentally abundant secretase components, particularly proteases, affect Caliciviridae infectivity. Results showed that combining Bacillaceae serine proteases with epsilon-poly-l-lysine (EPL) produced by Streptomyces—a natural antimicrobial—elicited anti-Caliciviridae properties, including against the epidemic HuNoV GII.4_Sydney_2012 strain. In vitro and in vivo biochemical and virological analyses revealed that EPL has two unique synergistic viral inactivation functions. First, it maintains an optimal pH to promote viral surface conformational changes to the protease-sensitive structure. Subsequently, it inhibits viral RNA genome release via partial protease digestion at the P2 and S domains in the VP1 capsid. This study provides new insights regarding the high-dimensional environmental interactions between bacteria and Caliciviridae, while promoting the development of protease-based anti-viral disinfectants.

DOI

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

Soh Yamamoto, Noriko Ogasawara, Yukari Mitsuhashi, Kenichi Takano, Shin-Ichi Yokota

Scientific reports 14 ( 1 ) 2354 - 2354 2024年01月 [国際誌]

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.

DOI PubMed

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

Yoshiki Hiyama, Soh Yamamoto, Toyotaka Sato, Noriko Ogasawara, Naoya Masumori, Satoshi Takahashi, Shin-Ichi Yokota

Microbial drug resistance (Larchmont, N.Y.) 2024年01月 [国際誌]

Multidrug-resistant Neisseria gonorrhoeae is a serious concern worldwide. Resistance to β-lactam antibiotics occurs through mutations in penicillin-binding proteins (PBPs), acquisition of β-lactamases, and alteration of antibiotic penetration. Mosaic structures of penA, which encodes PBP2, play a major role in resistance to β-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 β-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.

DOI PubMed

当院における人工内耳装用者(児)への遠隔地マッピングの取り組み

海崎文, 新谷朋子, 才川悦子, 小笠原徳子, 木村綾美, 佐藤楓, 高野賢一

Audiology Japan ( (一社)日本聴覚医学会 ) 66 ( 5 ) 370 - 370 2023年09月

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

Shin-Ichi Yokota, Naoyuki Tsukamoto, Toyotaka Sato, Yasuo Ohkoshi, Soh Yamamoto, Noriko Ogasawara

IJID regions 8 105 - 110 2023年09月 [国際誌]

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 β-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.

DOI PubMed

片耳伝音難聴の軟骨伝導補聴器試聴から装用の現状

木村綾美, 新谷朋子, 才川悦子, 小笠原徳子, 海崎文, 佐藤楓, 高野賢一

Audiology Japan ( (一社)日本聴覚医学会 ) 66 ( 5 ) 426 - 426 2023年09月

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

Shota Fukuzawa, Toyotaka Sato, Kotaro Aoki, Soh Yamamoto, Noriko Ogasawara, Chie Nakajima, Yasuhiko Suzuki, Motohiro Horiuchi, Satoshi Takahashi, Shin-Ichi Yokota

Annals of clinical microbiology and antimicrobials 22 ( 1 ) 60 - 60 2023年07月 [国際誌]

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.

DOI PubMed

Colistin-resistant bacteria poses few risks under physiological conditions

Soh Yamamoto, Masaru Usui, Noriko Ogasawara, Wataru Hayashi, Masato Suzuki, Noriyuki Nagano, Chie Nakajima, Yasuhiko Suzuki, Motohiro Horiuchi, Satoshi Takahashi, Shin-ichi Yokota, Yutaka Tamura, Toyotaka Sato

Research Square ( Research Square Platform LLC ) 2023年06月

Abstract Globally, 5.0 million people die annually from infections associated with antimicrobial-resistant bacteria, most commonly Escherichia coli¹. As colistin is a last-resort antibiotic for multidrug-resistant bacterial infections, the global spread of plasmid-mediated colistin resistance genes (mcr) gene is considered a major public health risk^2-4. However, the actual health risks of colistin resistance in hazardous bacteria have never been evaluated under physiological conditions. Here, we show that the fitness/virulence and colistin resistance of the pandemic multidrug-resistant E. coli clone ST131⁵ very depending on the acquired colistin resistance determinants and differ between physiological and in vitro conditions. The fitness/virulence of ST131 was unaffected by chromosomal-gene (pmrB) mutations or the acquisition of mcr-5-harbouring plasmids in mouse models. However, the acquisition of mcr-1- or mcr-3-harbouring plasmids attenuated fitness/virulence and promoted colistin susceptibility in human serum. We identified two virulence attenuation factors (vafA and vafB) on the pIncI2_mcr-1 plasmid that hijacked the ST131 transcriptome and inhibited nucleotide synthesis, attenuating colistin resistance. Our results demonstrate that colistin resistance poses much less of a threat than believed^6,7. We suggest that “nonresistance genes,” rather than resistance genes, are important antimicrobial resistance determinants for human health because they determine fitness/virulence and ultimately antimicrobial susceptibility under physiological conditions.

DOI

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

Soh Yamamoto, Yuka Sudo-Yokoyama, Noriko Ogasawara, Shin-Ichi Yokota

Journal of virological methods 316 114715 - 114715 2023年06月 [国際誌]

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.

DOI PubMed

抗菌性物質・薬剤耐性/生体防御大腸菌ST131におけるmcr-1保有プラスミドの獲得による病原性減弱機構(Antimicrobial agents and resistance/Host defense Virulent attenuation mechanism by acquisition of mcr-1-harboring plasmid into Escherichia coli ST131)

佐藤豊孝, 山本聡, 小笠原徳子, 臼井優, 長野則之, 土井洋平, 堀内基広, 高橋聡, 横田伸一, 田村豊

日本細菌学雑誌 ( 日本細菌学会 ) 78 ( 1 ) 48 - 48 2023年02月

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