Details of a Researcher - YOKOTA Shinichi

Papers - YOKOTA Shinichi

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A unique tetrasaccharide-containing anchor glycolipid of lipoteichoic acid is commonly found in Lactobacillus gasseri and Lactobacillus paragasseri.

Tsukasa Shiraishi, Ryosuke Kutomi, Yamaha Sato, Akihito Endo, Satoru Fukiya, Satoshi Takahashi, Atsushi Yokota, Shin-Ichi Yokota

Bioscience, biotechnology, and biochemistry 2025.06 [International journal]

Lipoteichoic acid (LTA) is a cell surface molecule with physiologically important roles in Gram-positive bacteria. Previously, we identified a novel tetrasaccharide-containing anchor glycolipid in the Lactobacillus gasseri JCM 1131T LTA. However, it is unclear whether this tetrasaccharide-containing anchor glycolipid structure is characteristic of JCM 1131T alone or is commonly found in other L. gasseri-related isolates. Lactobacillus paragasseri has been proposed as a new species, separated from the L. gasseri. In the present study, we characterized chemical structures of LTA from two strains of L. gasseri and four strains of L. paragasseri. All six strains shared a polymer comprising repeating units of glycerophosphate with partial D-alanine substitutions, as well as L. gasseri JCM 1131T. Anchor glycolipid structure suggested that all strains shared a tetrasaccharide-containing glyceroglycolipid with two and three acyl groups. Focusing on LTA from L. gasseri and L. paragasseri species, a tetrasaccharide-containing anchor glycolipid is a specific feature.

DOI PubMed

Efficacy of disinfectants and endotoxin-retentive filters for the removal of bacterial DNA from dialysates

Minoru Nakamura, Ami Murata, Toru Yokoyama, Daisuke Furuya, Tomokazu Indo, Shin-ichi Yokota

Renal Replacement Therapy ( Springer Science and Business Media LLC ) 11 ( 1 ) 2025.05

Abstract Background Bacterial DNA (bDNA) fragments in dialysate lines can trigger inflammatory responses in patients on dialysis. However, no studies have reported the removal and inactivation of bDNA in dialysate lines using cleaning and disinfection, and management procedures to control bDNA contamination have yet to be established. Methods The efficiency of an endotoxin-retentive filter (ETRF) for the removal of bDNA was examined using an experimental dialysate line incorporating an ETRF and the solubilized materials derived from hot-water-disinfected Pseudomonas aeruginosa cells. To examine the inactivation of bDNA by disinfection, P. aeruginosa cell suspensions were disinfected with hot water, peracetic acid, or sodium hypochlorite, and the amount of bDNA remaining after the disinfection treatment was determined. Single-stranded and double-stranded bDNA were measured using Qubit® fluorometry. The molecular size of bDNA was analyzed by polyacrylamide gel electrophoresis. Results In the spike-and-recovery test of solubilized materials derived from hot-water-disinfected bacterial cells, bDNA leakage was observed when the circuit pressure of the inlet ETRF was elevated. bDNA was inactivated more during disinfection with sodium hypochlorite than with peracetic acid and hot water. Conclusions In addition to the ETRF, disinfection with sodium hypochlorite is an effective method for the management of bDNA in dialysates.

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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 2025.03 [International journal]

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

Chemical structure of lipoteichoic acid in the probiotic strain Latilactobacillus curvatus CP2998.

Tsukasa Shiraishi, Ryo Katsuki, Hiroyuki Kumeta, Shinji Sakata, Shin-Ichi Yokota

FEMS microbiology letters 372 2025.01 [International journal]

Latilactobacillus curvatus, found in various fermented foods, is a promising probiotic with unique health benefits. Lipoteichoic acid (LTA) is a characteristic amphiphilic surface polymer of Gram-positive bacteria and exhibits immunomodulatory activities. Despite the structural diversity of LTA among different bacterial species and strains, no information is available on the chemical structure of LTA in L. curvatus. In this study, we aimed to determine the structure of LTA isolated from L. curvatus CP2998. One- and two-dimensional nuclear magnetic resonance spectra of intact LTA revealed that LTA had a glycerolphosphate polymer as a hydrophilic main chain with partial substitutions of α-linked glucose and d-alanine at the hydroxy group at position 2 of the glycerol residue. The anchor glycolipid fraction was obtained by hydrofluoric acid treatment. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry spectrum of the anchor glycolipid revealed that it contained diglucosyldiacylglycerol and diglucosylmonoacylglycerol. Our results suggest that L. curvatus CP2998 possesses a typical type I LTA structure; however, the lactic acid bacteria-specific anchor glycolipid structures, such as tri- or tetra-saccharides and three fatty acid residues, were not identified.

DOI PubMed

Immunostimulatory activity of lipoteichoic acid with three fatty acid residues derived from Limosilactobacillus antri JCM 15950 T

Shino Yamasaki-Yashiki, Tsukasa Shiraishi, Mai Gyobu, Haruna Sasaki, Jun Kunisawa, Shin-ichi Yokota, Yoshio Katakura

Applied and Environmental Microbiology ( American Society for Microbiology ) 2024.09

ABSTRACT Some strains of lactic acid bacteria can regulate the host’s intestinal immune system. Bacterial cells and membrane vesicles (MVs) of Limosilactobacillus antri JCM 15950 ^T promote immunoglobulin A (IgA) production in murine Peyer’s patch cells via toll-like receptor (TLR) 2. This study aimed to investigate the role of lipoteichoic acid (LTA), a ligand of TLR2, in the immunostimulatory activity of these bacterial cells and their MVs. LTA extracted from bacterial cells was purified through hydrophobic interaction chromatography and then divided into fractions LTA1 and LTA2 through anion-exchange chromatography. LTA1 induced greater interleukin (IL)-6 production from macrophage-like RAW264 cells than LTA2, and the induced IL-6 production was suppressed by TLR2 neutralization using an anti-TLR2 antibody. The LTAs in both fractions contained two hexose residues in the glycolipid anchor; however, LTA1 was particularly rich in triacyl LTA. The free hydroxy groups in the glycerol phosphate (GroP) repeating units were substituted by d -alanine ( d -Ala) and α-glucose in LTA1, but only by α-glucose in LTA2. The dealanylation of LTA1 slightly suppressed IL-6 production in RAW264 cells, whereas deacylation almost completely suppressed IL-6 production. Furthermore, IL-6 production induced by dealanylated LTA1 was markedly higher than that induced by dealanylated LTA2. These results indicated that the critical moieties for the immunostimulatory activity of L. antri -derived LTA were the three fatty acid residues rather than the substitution with d -Ala in GroP. LTA was also detected in MVs, suggesting that the triacyl LTA, but not the diacyl LTA, translocated to the MVs and conferred immunostimulatory activity. IMPORTANCE Some lactic acid bacteria activate the host intestinal immune system via toll-like receptor (TLR) 2. Lipoteichoic acid (LTA) is a TLR2 ligand; however, the moieties of LTA that determine its immunostimulatory activity remain unclear because of the wide diversity of LTA partial structures. We found that Limosilactobacillus antri JCM 15950 ^T has three types of LTAs (triacyl, diacyl, and monoacyl LTAs). Specifically, structural analysis of the LTAs revealed that triacyl LTA plays a crucial role in immunostimulation and that the fatty acid residues are essential for the activity. The three acyl residues are characteristic of LTAs from many lactic acid bacteria, and our findings can explain the immunostimulatory mechanisms widely exhibited by lactic acid bacteria. Furthermore, the immunostimulatory activity of membrane vesicles released by L. antri JCM 15950 ^T is due to the transferred LTA, demonstrating a novel mechanism of membrane vesicle-mediated immunostimulation.

DOI

Preparation and Structural Analysis of Lipoteichoic Acid on Cell Membranes Derived from Lactic Acid Bacteria

Tsukasa Shiraishi, Shin-ichi Yokota

Methods in Molecular Biology ( Springer US ) 39 - 60 2024.08

DOI

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 14 ( 1 ) 15181 - 15181 2024.07 [International journal]

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 PubMed

Development of a natural product optimization strategy for inhibitors against MraY, a promising antibacterial target.

Kazuki Yamamoto, Toyotaka Sato, Aili Hao, Kenta Asao, Rintaro Kaguchi, Shintaro Kusaka, Radhakrishnam Raju Ruddarraju, Daichi Kazamori, Kiki Seo, Satoshi Takahashi, Motohiro Horiuchi, Shin-Ichi Yokota, Seok-Yong Lee, Satoshi Ichikawa

Nature communications 15 ( 1 ) 5085 - 5085 2024.06 [International journal]

MraY (phospho-N-acetylmuramoyl-pentapeptide-transferase) inhibitory natural products are attractive molecules as candidates for a new class of antibacterial agents to combat antimicrobial-resistant bacteria. Structural optimization of these natural products is required to improve their drug-like properties for therapeutic use. However, chemical modifications of these natural products are painstaking tasks due to complex synthetic processes, which is a bottleneck in advancing natural products to the clinic. Here, we develop a strategy for a comprehensive in situ evaluation of the build-up library, which enables us to streamline the preparation of the analogue library and directly assess its biological activities. We apply this approach to a series of MraY inhibitory natural products. Through construction and evaluation of the 686-compound library, we identify promising analogues that exhibit potent and broad-spectrum antibacterial activity against highly drug-resistant strains in vitro as well as in vivo in an acute thigh infection model. Structures of the MraY-analogue complexes reveal distinct interaction patterns, suggesting that these analogues represent MraY inhibitors with unique binding modes. We further demonstrate the generality of our strategy by applying it to tubulin-binding natural products to modulate their tubulin polymerization activities.

DOI PubMed

Traces of pandemic fluoroquinolone-resistant Escherichia coli clone ST131 transmitted from human society to aquatic environments and wildlife in Japan

Toyotaka Sato, Kojiro Uemura, Mitsuru Yasuda, Aiko Maeda, Toshifumi Minamoto, Kazuki Harada, Michiyo Sugiyama, Shiori Ikushima, Shin-ichi Yokota, Motohiro Horiuchi, Satoshi Takahashi, Testuo Asai

One Health ( Elsevier BV ) 18 100715 2024.06 [Refereed]

DOI

Lipoteichoic acid composed of poly-glycerolphosphate containing l-lysine and involved in immunoglobulin A-inducing activity in Apilactobacillus genus.

Tsukasa Shiraishi, Chiaki Matsuzaki, Tai-Ying Chiou, Hiroyuki Kumeta, Manami Kawada, Kenji Yamamoto, Tomoya Takahashi, Shin-Ichi Yokota

International journal of biological macromolecules 271 ( Pt 1 ) 132540 - 132540 2024.06 [International journal]

Lipoteichoic acid (LTA) in the gram-positive bacterial cell wall acts as an immunomodulatory factor in host cells. The chemical structures vary among bacterial species and strains, and may be related to biological activities. In our previous work, much higher immunoglobulin A (IgA)-inducing activity was observed in cells of the Apilactobacillus genus (Apilactobacillus kosoi 10HT, Apilactobacillus apinorum JCM 30765T, and Apilactobacillus kunkeei JCM 16173T) than other lactic acid bacteria, and their LTA was responsible for the activity. In the present study, we elucidated the chemical structures of LTA from these Apilactobacillus strains to explore the structure-function relationship of the IgA-inducing activity. The 1H-nuclear magnetic resonance spectra suggested that their LTA structures were similar. All have a poly-glycerolphosphate main chain, which comprised 12 to 20 average number of the repeating units, with partial substitutions of glucose(α1-, glucosyl(α1-2)glucose(α1- (α-linked-kojibiose), and l-lysine at the C-2 hydroxy group of the glycerol residue. l-Lysine is a substituent never seen before in LTA, and is a probable characteristic of the Apilactobacillus genus. Removal of l-lysine residue from LTA by mild alkaline treatment decreased IgA induction in murine Peyer's patch experiments. The novel l-lysine residue in Apilactobacillus LTA plays a crucial role in the remarkably high IgA-inducing activity.

DOI PubMed

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

Noriteru Yamada, Go Kamoshida, Tsukasa Shiraishi, Daiki Yamaguchi, Momoko Matsuoka, Reika Yamauchi, Nana Kanda, Roku Kamioka, Norihiko Takemoto, Yuji Morita, Masahiro Fujimuro, Shin-Ichi Yokota, Kinnosuke Yahiro

Journal of bacteriology 206 ( 5 ) e0043523 2024.05 [International journal]

UNLABELLED: 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.

DOI PubMed J-GLOBAL

Effects of metal corrosion in the pump of a dialysis machine on the sterility of the terminal dialysate by spike-and-recovery testing with bacteria

Minoru Nakamura, Masaya Okayama, Seya Hagiwara, Tomoyasu Nawa, Shin-ichi Yokota

Renal Replacement Therapy ( Springer Science and Business Media LLC ) 10 ( 1 ) 2024.02

Abstract Background Dialysis units have been concerned that the corroded metal parts of pumps used in hemodialysis might not allow sterility of the pump to be ensured due to bacterial contamination, even after cleaning and disinfection are performed after dialysis treatment. The purpose of this study was to clarify the effectiveness of the cleaning/disinfection process in eliminating bacterial contamination of the dialysate in pumps with and without metal corrosion. Methods A suspension of Pseudomonas aeruginosa [10 colony-forming unit (CFU)/mL] was introduced into pumps without or with corrosion of the metal parts, and the flow in the dialysis circuit was stopped for 6, 12, or 18 h. Then, after cleaning and disinfection of the circuit with a sodium-hypochlorite-containing reagent, the amounts of live bacteria in the terminal dialysate and on the surface of the metal parts of the pump were counted. Results Irrespective of the presence or absence of metal corrosion, bacteria were detected, even after cleaning and disinfection, on the surfaces of the pump parts that had been left in contact with the bacterial suspension for more than 12 h. However, on the surfaces of the pump parts showing metal corrosion, the bacterial numbers increased dramatically after 18 h of flow stoppage time following introduction of bacteria, and bacteria were even detected in the terminal dialysate despite cleaning/disinfection of the pump. Conclusions Corrosion of the metal parts used in pumps used for dialysis increases the risk of bacterial contamination of not only the pump parts and flow path of the dialysis machine but also the terminal dialysate, even if cleaning/disinfection is performed. For sterility assurance of the dialysis circuit and dialysate during routine use, it is necessary to eliminate corrosion of the metal parts of dialysis pumps during scheduled maintenance.

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 2024.01 [International journal]

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.) 2024.01 [International journal]

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

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 2023.09 [International journal]

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

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 2023.07 [International journal]

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

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 2023.06 [International journal]

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

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

Yutaro Nagano, Koji Kuronuma, Yasuo Kitamura, Kanami Nagano, Hayato Yabe, Sayaka Kudo, Toyotaka Sato, Shinya Nirasawa, Mami Nakae, Motohiro Horiuchi, Shin-Ichi Yokota, Yoshihiro Fujiya, Atsushi Saito, Satoshi Takahashi, Hirofumi Chiba

Infection control and hospital epidemiology 1 - 7 2023.04 [International journal]

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.

DOI PubMed

Corrigendum to 'Clonal/subclonal changes and accumulation of CTX-M-type β-lactamase genes in fluoroquinolone-resistant Escherichia coli ST131 and ST1193 strains isolated during the past 12 years, Japan' [Journal of Global Antimicrobial Resistance 27 (2021) 150-155].

Yukari Fukushima, Toyotaka Sato, Naoyuki Tsukamoto, Chie Nakajima, Yasuhiko Suzuki, Satoshi Takahashi, Shin-Ichi Yokota

Journal of global antimicrobial resistance 32 195 - 195 2023.03 [International journal]

DOI PubMed

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

Rintaro Kaguchi, Akira Katsuyama, Toyotaka Sato, Satoshi Takahashi, Motohiro Horiuchi, Shin-ichi Yokota, Satoshi Ichikawa

Journal of the American Chemical Society ( American Chemical Society (ACS) ) 2023.01 [Refereed]

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Papers - YOKOTA Shinichi