Biology subjects

Yoshii, Y.

Publications and source records attributed to Yoshii, Y..

5 recordsLinked to original sources

bioRxiv2026

A Novel Conjugation System in AMR-associated pELF-type Linear Plasmids of Enterococcus faecium.

The pELF-type linear plasmid is a unique transconjugative plasmid that disseminates various antimicrobial resistance (AMR) genes (e.g., vancomycin resistance in vancomycin-resistant Enterococcus faecium, [VRE]), leading to hospital outbreaks worldwide. Despite evidence of its role in AMR gene expansion, the molecular mechanisms underlying conjugative transfer of pELF-type plasmids have been poorly understood due to their unique linear structure and the lack of convenient genetic engineering tools adapted to E. faecium strain. Herein, we focused on a putative transconjugation-associated gene cluster, named the trapELF cluster, encoded on pELF2, a vanA-type vancomycin resistance gene-harboring plasmid. RNA-seq analysis of the pELF2 plasmid identified a putative operon containing an ftsK-like ATPase-encoding gene and an additional 10 genes as candidates for conjugative transfer of pELF-type linear plasmids. We constructed isogenic deletion mutants for these 10 genes and revealed that two genes, designated traC and traD, are essential for the conjugative transfer of pELF2 to a recipient strain. Inducible overexpression of the tra genes in trans from an ectopic shuttle vector confirmed the essentiality of traC and traD for conjugative transfer. Promoter assay using a luciferase reporter plasmid revealed that the upstream region of the putative tra operon possesses functional promoter activity characterized by constitutive strong transcriptional expression. Furthermore, we observed membrane localization for a fluorescent protein fusion of TraC, but not for that of TraD. Based on these findings, we propose a working model for the unique molecular machinery for the transconjugation of pELF-type linear plasmids. Author SummaryEnterococci are among the opportunistic pathogens of greatest global concern due to their multidrug resistance, and it is well established that various plasmids mediate horizontal transfer of resistance genes in this species, converting susceptible strains into resistant ones. While most enterococcal plasmids have a circular structure, a linear pELF-type plasmid was recently discovered. Since then, pELF-type plasmids have been reported worldwide as important vehicles for antimicrobial resistance genes, but their mechanism of transfer has remained unknown. In this study, we have, for the first time, successfully identified the genes required for the conjugative transfer of pELF-type plasmids. We confirmed that these genes are absent from known conjugative plasmids, yet are highly conserved among pELF-type plasmid sequences deposited in public databases. Taken together, these findings suggest that pELF-type plasmids possess a unique transfer mechanism that reflects their distinctive molecular architecture.

microbiology↗

bioRxiv2025

Exploring mutational possibilities of KPC variants to reach high level resistance to cefiderocol

IntroductionKlebsiella pneumoniae carbapenemase (KPC) is one of the most widespread carbapenemases, with over 242 variants identified. Some, like KPC-33 (D179Y), resist both ceftazidime-avibactam (CZA) and cefiderocol but show only a moderate cefiderocol MIC increase. KPCs success stems from its genetic adaptability, enabling mutation accumulation and necessitating proactive resistance monitoring. This study investigates the mutational possibilities for three KPC variants to develop high-level cefiderocol resistance. MethodUsing random mutagenesis and enrichment selection over a 10-day cefiderocol exposure at increasing concentration, we investigated the mutational potential of KPC-2, KPC-3, and KPC-33 to develop high-level cefiderocol resistance. Resistance mechanisms were then analyzed through phenotypic testing and sequencing. ResultsRandom mutagenesis generated 10, 10, and 105 mutants for blaKPC-2, blaKPC- 3, and blaKPC-33, respectively. After ten days of increasing cefiderocol exposure, MICs rose significantly, with KPC-2 mutants reaching >32 mg/L. Phenotypic analysis revealed a common resistance profile across all tested mutants, with resistance to ceftazidime, ceftazidime-avibactam, cefixime, and piperacillin, but restored susceptibility to carbapenems and most other {beta}-lactams. Sequencing identified key mutations (D179Y- D209V in KPC-2, L169P in KPC-3), while chromosomal changes, particularly cirA and ybiX disruptions, played a crucial role in resistance evolution. DiscussionOur results show that the enriched mutations in KPC genes are not sufficient to confer high cefiderocol MICs, suggesting that, in the studied variants, no simple mutational pathway allows the KPC enzyme to efficiently hydrolyze cefiderocol. These findings underscore the interplay between enzymatic and iron transport mutations in cefiderocol resistance, highlighting the importance of surveillance to anticipate emerging resistance in KPC-producing pathogens.

microbiology↗

bioRxiv2022

Escherichia coli membrane microdomain SPFH protein HflC interacts with YajC and contributes to aminoglycoside and oxidative stress tolerance

Many eukaryotic membrane-dependent functions are often spatially and temporally regulated by membrane microdomains (FMMs) also known as lipid rafts. These domains are enriched in polyisoprenoid lipids and scaffolding proteins belonging to the Stomatin, Prohibitin, Flotillin, and HflK/C (SPFH) protein superfamily that was also identified in Gram-positive bacteria. By contrast, little is still known about FMMs in Gram-negative bacteria. In Escherichia coli K12, 4 SPFH proteins, YqiK, QmcA, HflK, and HflC, were shown to localize in discrete polar or lateral inner-membrane locations, raising the possibility that E. coli SPFH proteins could contribute to the assembly of inner-membrane FMMs and the regulation of cellular processes. Here we studied the determinant of the localization of QmcA and HflC and showed that FMM-associated cardiolipin lipid biosynthesis is required for their native localization pattern. Using Biolog phenotypic arrays, we showed that a mutant lacking all SPFH genes displayed increased sensitivity to aminoglycosides and oxidative stress that is due to the absence of HflKC. Our study therefore provides further insights into the contribution of SPFH proteins to stress tolerance in E. coli. IMPORTANCEEukaryotic cells often segregate physiological processes in cholesterol-rich functional membrane micro-domains. These domains are also called lipid rafts and contain proteins of the Stomatin, Prohibitin, Flotillin, and HflK/C (SPFH) superfamily, which are also present in prokaryotes but were mostly studied in Gram-positive bacteria. Here, we showed that the cell localization of the SPFH proteins QmcA and HflKC in the Gram-negative bacteria E. coli is altered in absence of cardiolipin lipid synthesis. This suggests that cardiolipins contribute to E. coli membrane microdomain assembly. Using a broad phenotypic analysis, we also showed that HflKC contribute to E. coli tolerance to aminoglycosides and oxidative stress. Our study, therefore, provides new insights into the cellular processes associated with SPFH proteins in E. coli.

microbiology↗

bioRxiv2022

Intermittent antibiotic treatment of bacterial biofilms favors the rapid evolution of resistance

The rise of antibiotic resistance in bacterial pathogens is a major health concern and the determinants of this emergence are actively studied. By contrast, although biofilms are an important cause of infections due to their high tolerance to a broad range of antimicrobials, much less is known on the development of antibiotic resistance within the biofilm environment, an issue potentially aggravating the current antibiotic crisis. Here, we compared the occurrence of resistance mutations in pathogenic Escherichia coli planktonic and biofilm populations exposed to clinically relevant cycles of lethal treatments with the aminoglycoside antibiotic amikacin. This experimental evolution approach revealed that mutations in sbmA and fusA are rapidly selected in biofilm but not in planktonic populations. The apparition of these bona fide resistance --and not tolerance-- mutations is favored by the biofilm preexisting tolerance and high mutation rate. Moreover, we showed that while fusA mutations displayed a high fitness cost in planktonic conditions, these mutations were maintained in biofilms, a phenomenon further possibly amplified by the selection of fimH mutations favoring biofilm formation itself. Our study therefore provides new insights into the dynamic evolution of antibiotic resistance in biofilms, which could lead to clinically practical antibiotic regimen limiting biofilm-associated infections, while mitigating the emergence of worrisome antibiotic resistance mutations.

microbiology↗

bioRxiv2017

Multidisciplinary field research in Kabwe, Zambia, towards better understanding of lead contamination of the city - A short report from a field survey

Heavy metal contamination is a serious issue in many post-mining regions around the world. Kabwe town, Zambia, is known as one of the most polluted cities in the world, where high lead (Pb) levels have been reported in soils, plants, animals and human blood. Multidisciplinary approaches are critically needed to understand the current situation and to remediate the polluted area. In the current research, a large-scale preliminary field survey was performed to understand the current situation in Kabwe and to plan future mitigation approaches. Three aspects were mainly observed; 1) plant communities during the dry season in Kabwe city, 2) spectral images of the land surfaces in various locations in Kabwe and 3) Pb concentrations in soils and water. Overall, >15 different plant species were observed and many of them maintained their green colour even during the dry season. Some tree species, for example, Caesalpiniaceae and Fabaceae families may be utilised as phytostabilization approaches although their impacts on the soil Pb mobility should be further studied. For the spectral images, we used a handmade portable spectrometer, and our obtained spectral images showed typical curves observed from soils. These data may be used to understand the distribution of different soil types in this area, using aboveground images such as satellite images. For Pb concentrations in soils, extremely high total Pb levels (>1,000 ppm) was observed only within 2 km from the mining site. There was a weak but a positive correlation between the total and soluble Pb thus further study should also focus on the mobility of Pb from soils to plant ecosystems.

ecology↗