Biology subjects

Gomi, R.

Publications and source records attributed to Gomi, R..

8 recordsLinked to original sources

bioRxiv2025

Miniature inverted-repeat transposable elements mobilize diverse antibiotic resistance genes in Enterobacteriaceae

Miniature inverted-repeat transposable elements (MITEs) are nonautonomous mobile genetic elements (MGEs) that can be mobilized by transposases provided by the relevant autonomous MGEs. MITEs originating from Tn3-family transposons were previously termed Tn3-derived inverted-repeat miniature elements (TIMEs). Composite transposon-like structures bounded by two copies of TIME, called TIME-COMPs, were shown to mobilize the intervening sequences. However, their association with antibiotic resistance genes (ARGs) has not yet been systematically studied. This study thus aimed to identify new TIME-COMP-like structures containing ARGs in the genomic sequences of the clinically important bacterial family Enterobacteriaceae in public databases. TIME-COMP-like structures were first searched for in the plasmid database PLSDB, focusing on small plasmids, using a self-against-self blastn approach to identify repeated elements. Then, newly and previously identified MITEs (including TIMEs) were searched for in the NCBI core nucleotide database to identify TIME-COMP-like structures located on other replicons. Bioinformatic analysis identified multiple previously unreported TIME-COMPs containing ARGs, which are bounded by directly or inversely oriented TIMEs, namely, IS101, MITESen1, and a novel 244-bp TIME termed TIME244. TIME244 contains a putative resolution site related to that of Tn21. These TIMEs were predominantly detected in plasmids and very rarely in chromosomes. The ARGs embedded in newly identified TIME-COMPs were blaKPC-2, floR, qnrS1, and tet(A). Notably, the blaKPC-2 carbapenemase gene was found in TIME-COMPs bounded by TIME244 and a TIME-COMP bounded by IS101. These findings highlight a potential role for TIMEs in the spread of diverse ARGs. IMPACT STATEMENTBacterial miniature inverted-repeat transposable elements (MITEs) are a group of short (50 bp-500 bp) nonautonomous transposable elements that are thought to have originated from insertion sequences or transposons. Although MITEs can theoretically mobilize antibiotic resistance genes (ARGs) in the presence of transposases, only a few studies have reported their association with ARGs, probably due to difficulties in identifying MITEs in genomic sequences. This study provides evidence, based on bioinformatic analysis of public Enterobacteriaceae genomes, that a subset of MITEs, called Tn3-derived inverted-repeat miniature elements (TIMEs), mobilizes ARGs by forming composite transposon-like structures. A novel 244-bp TIME, designated TIME244, was present in more than 100 Enterobacteriaceae plasmids in the current RefSeq database, suggesting its further transmission in bacterial populations through horizontal gene transfer. This study reveals that TIMEs were often overlooked when analyzing the genetic contexts of ARGs in previous studies. These findings highlight the importance of TIMEs in bacterial gene acquisition and underscore the need for new tools that can detect TIMEs in bacterial genomes for ARG surveillance. DATA SUMMARYAccession numbers of sequence data analyzed in this study are provided within the article or in supplementary data files.

microbiology↗

bioRxiv2025

Simultaneous detection of protozoa, bacteria, and viruses from environmental water through membrane-adsorption followed by direct nucleic acid extraction

Waterborne outbreaks caused by protozoan, bacterial, and viral pathogens continue to pose serious public health threats. However, comprehensive monitoring of all three major pathogen types remains uncommon, primarily due to the technical difficulty of simultaneously concentrating and characterizing these diverse microorganisms from a single water sample. Here, we evaluated a membrane adsorption followed by bead-beating-based direct nucleic acid extraction for its sensitivity in detecting waterborne pathogens. We further applied the optimized protocol to river water samples. Our results demonstrated that the use of magnesium chloride in combination with a mixed cellulose ester membrane filter (pore size: 0.8 m) achieved the highest concentration efficiency for Cryptosporidium parvum, Legionella pneumophila, and murine norovirus (MNV), outperforming other methods utilizing the membrane with alternative pore sizes or membrane with another material (i.e., nylon). Furthermore, targeting rRNA of C. parvum instead of rDNA, or incorporating a preamplification step for the L. pneumophila mip gene and MNV, significantly improved the sensitivity. Notably, the 50% limit of detection for C. parvum using our method was comparable to that of traditional immunofluorescent antibody testing. Application of this method to river water further revealed a higher abundance of Bacteroides rRNA markers compared to their corresponding rDNA, underscoring the potential of rRNA-based targets for more sensitive bacterial detection. Importantly, when the target is genomic DNA or RNA, preamplification enables the use of a larger nucleic acids input volume, thereby enhancing sensitivity without introducing measurement bias. Overall, the combination of membrane adsorption and bead-beating-based direct nucleic acid extraction offers a sensitive, all-in-one approach for simultaneous detection of protozoa, bacteria, and viruses in environmental water. This method serves as an effective screening tool for the presence of the three major types of waterborne microorganisms. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=71 SRC="FIGDIR/small/669200v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@d0e9f4org.highwire.dtl.DTLVardef@1afc9fcorg.highwire.dtl.DTLVardef@1f5c355org.highwire.dtl.DTLVardef@1eb14eb_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2024

Quinolone resistance genes qnr, aac(6')-Ib-cr, oqxAB, and qepA in environmental Escherichia coli: insights into their genetic contexts from comparative genomics

Previous studies have reported the occurrence of transferable quinolone resistance determinants in environmental Escherichia coli. However, little is known about their vectors and genetic contexts. To gain insights into these genetic characteristics, we analyzed the complete genomes of 53 environmental E. coli isolates containing one or more transferable quinolone resistance determinants, including 20 sequenced in this study and 33 sourced from RefSeq. The studied genomes carried the following transferable quinolone resistance determinants alone or in combination: aac(6')-Ib-cr, oqxAB, qepA1, qnrA1, qnrB4, qnrB7, qnrB19, qnrD1, qnrS1, and qnrS2, with qnrS1 being predominant. These resistance genes were detected on plasmids of diverse replicon types; however, aac(6')-Ib-cr, qnrS1, and qnrS2 were also detected on the chromosome. The genetic contexts surrounding these genes included not only those found in clinical isolates but also novel contexts, such as qnrD1 embedded within a composite transposon-like structure bounded by Tn3-derived inverted-repeat miniature elements (TIMEs). This study provides deep insights into mobile genetic elements associated with transferable quinolone resistance determinants, highlighting the importance of genomic surveillance of antimicrobial resistant bacteria in the environment.

microbiology↗

bioRxiv2023

Genomic surveillance of antimicrobial-resistant Escherichia coli in fecal sludge and sewage in Uganda

The global increase of antimicrobial resistance (AMR) is a major public health concern. An effective AMR surveillance tool is needed to track the emergence and spread of AMR. Wastewater surveillance has been proposed as a resource-efficient tool for monitoring AMR carriage in the community. Here, we performed genomic surveillance of antimicrobial-resistant Escherichia coli obtained from fecal sludge and sewage in Uganda to gain insights into E. coli epidemiology and AMR burden in the underlying population. Selective media containing different antibiotic combinations (cefotaxime, ciprofloxacin, cefotaxime + ciprofloxacin + gentamicin) were used to obtain antimicrobial-resistant E. coli from fecal sludge and sewage. Short-read sequencing was performed for the obtained isolates, and a subset of isolates (selected from predominant sequence types (STs)) was also subjected to long-read sequencing. Genomic analysis of the obtained E. coli isolates (n = 181) revealed the prevalence of clonal complex 10, including ST167 (n = 43), ST10 (n = 28), ST1284 (n = 17), and ST617 (n = 4), in both fecal sludge and sewage, irrespective of antibiotics used for selection. We also detected global high-risk clones ST1193 (n = 10) and ST131 (n = 2 clade A, n = 3 subclade C1-M27, and n = 1 subclade C2). Diverse AMR determinants, including extended-spectrum {beta}-lactamase genes (mostly blaCTX-M-15) and mutations in gyrA and parC, were identified. Analysis of the completed genomes revealed that diverse IncF plasmids and chromosomal integration were the major contributors to the spread of AMR genes in the predominant STs. This study showed that a combination of sewage surveillance (or fecal sludge surveillance) and whole-genome sequencing can be a powerful tool for monitoring AMR carriage in the underlying population.

microbiology↗

bioRxiv2022

Novel Escherichia coli genetic markers for tracking domestic wastewater contamination in environmental waters

Escherichia coli has been used as an indicator of fecal pollution in environmental waters. However, its presence in environmental waters does not provide information on the source of water pollution. Identifying the source of water pollution is paramount to be able to effectively reduce contamination. The present study aimed to identify E. coli microbial source tracking (MST) markers that can be used to identify domestic wastewater contamination in environmental waters. We first analyzed wastewater E. coli genomes sequenced by us (n = 50) and RefSeq animal E. coli genomes of fecal origin (n = 82), and identified 144 candidate wastewater-associated marker genes. The sensitivity and specificity of the candidate marker genes were then assessed by screening the genes in 335 RefSeq wastewater E. coli genomes and 3,318 RefSeq animal E. coli genomes. We finally identified two MST markers, namely W_nqrC and W_clsA_2, which could be used for detection of wastewater-associated E. coli isolates. These two markers showed higher performance than the previously developed human wastewater-associated E. coli markers H8 and H12. When used in combination, W_nqrC and W_clsA_2 showed specificity of 98.9% and sensitivity of 25.7%. PCR assays to detect W_nqrC and W_clsA_2 were also developed and validated. The developed PCR assays are potentially useful for detecting E. coli isolates of wastewater origin in environmental waters, though users should keep in mind that the sensitivity of these markers is not high. Further studies are needed to assess the applicability of the developed markers to a culture-independent approach.

microbiology↗

bioRxiv2022

Chromosomal integration of blaCTX-M genes in diverse Escherichia coli isolates recovered from river water in Japan

Occurrence of extended-spectrum {beta}-lactamase (ESBL)-producing Escherichia coli (ESBLEC) in environmental waters is of great concern. However, unlike clinical ESBLEC, their genetic characteristics, in particular the genetic contexts of ESBL genes, are not well understood. In this study, we sequenced and analyzed the genomes of CTX-M-producing E. coli isolates recovered from river water to fully characterize the genetic contexts of blaCTX-M genes. Among the 14 isolates with completed genomes, blaCTX-M genes were detected on the chromosome in nine isolates. All but one chromosomal blaCTX-M genes were associated with ISEcp1 and were carried on different transposition units ranging in size from 2,855 bp to 11,093 bp; the exception, blaCTX-M-2, was associated with ISCR1. The remaining five isolates carried blaCTX-M genes on epidemic IncI1 plasmids of different sequence types (STs) (ST3, ST16, ST113, and ST167) (n = 4) or on an IncB/O/K/Z plasmid (n = 1). This study revealed that environmental E. coli carry blaCTX-M genes in diverse genetic contexts. Apparent high prevalence of chromosomal blaCTX-M potentially indicates that some E. coli can stably maintain blaCTX-M genes in environmental waters, though further studies are needed to confirm this.

microbiology↗

bioRxiv2021

Emergence of rare carbapenemases (FRI, GES-5, IMI, SFC, and SFH-1) in Enterobacterales isolated from surface waters in Japan

ObjectivesCarbapenemase-producing Enterobacterales (CPE) pose serious threats to public health. Compared with clinical CPE, the genetic characteristics of environmental CPE are not well understood. This study aimed to characterize the genetic determinants of carbapenem resistance in CPE isolated from environmental waters in Japan. MethodsEighty-five water samples were collected from rivers and a lake in Japan. CPE were identified using selective media, and genome sequencing was performed for the obtained isolates (n = 21). ResultsVarious rare/novel carbapenemases were identified: GES-5 in Raoultella planticola (n = 1), FRI-8 and FRI-11 in Enterobacter spp. (n = 8), IMI-22 and IMI-23 in Serratia ureilytica (n = 3), and SFC-1, SFC-2 and SFH-1 in Serratia fonticola (n = 9). Genomes of 11 isolates could be closed, allowing the elucidation of the genetic contexts of the carbapenemase genes. The blaGES-5 gene was located within a class 1 integron, In2071 (cassette array, blaGES-5-aacA3-aadA16), on a 33 kb IncP6 plasmid. The blaFRI-8 genes were carried on IncFII(Yp) plasmids ranging in size from 191 kb to 244 kb, and the blaFRI-11 genes were carried on 70 kb and 74 kb IncFII(pECLA)/IncR plasmids. The blaIMI-22 and blaIMI-23 genes were colocated on a 107 kb plasmid. The blaSFC and blaSFH-1 genes were found on putative genomic islands inserted at tRNA-Phe genes in chromosomes. ConclusionsThis study revealed the presence of rare/novel carbapenemases among CPE in aquatic environments, suggesting that the environment may act as a potential reservoir of these minor carbapenemases.

microbiology↗

bioRxiv2020

Detection of plasmid contigs in draft genome assemblies using customized Kraken databases

Plasmids play an important role in bacterial evolution and mediate horizontal transfer of genes including virulence and antimicrobial resistance genes. Although short-read sequencing technologies have enabled large-scale bacterial genomics, the resulting draft genome assemblies are often fragmented into hundreds of discrete contigs. Several tools and approaches have been developed to identify plasmid sequences in such assemblies, but require trade-off between sensitivity and specificity. Here we propose using the Kraken classifier, together with a custom Kraken database comprising known chromosomal and plasmid sequences of Klebsiella pneumoniae species complex (KpSC), to identify plasmid-derived contigs in draft assemblies. We assessed performance using Illumina-based draft genome assemblies for 82 KpSC isolates, for which complete genomes were available to supply ground truth. When benchmarked against five other classifiers (Centrifuge, RFPlasmid, mlplasmids, PlaScope, and Platon), Kraken showed balanced performance in terms of overall sensitivity and specificity (90.8% and 99.4%, respectively for contig count; 96.5% and >99.9%, respectively for cumulative contig length), and the highest accuracy (96.8% vs 91.8%-96.6% for contig count; 99.8% vs 99.0%-99.7% for cumulative contig length), and F1-score (94.5% vs 84.5%-94.1%, for contig count; 98.0% vs 88.9%-96.7% for cumulative contig length). Kraken also achieved consistent performance across our genome collection. Furthermore, we demonstrate that expanding the Kraken database with additional known chromosomal and plasmid sequences can further improve classification performance. Although we have focused here on the KpSC, this methodology could easily be applied to other species with a sufficient number of completed genomes. IMPACT STATEMENTThe assembly of bacterial genomes using short-read data often results in hundreds of discrete contigs due to the presence of repeat sequences in those genomes. Separating plasmid contigs from chromosomal contigs in such assemblies is required, e.g., to assess the mobility of antimicrobial resistance genes. Although several tools have been developed for that purpose, they often suffer from low sensitivity or specificity. Here, we propose that the Kraken classifier coupled with a custom Kraken database comprising plasmid-free chromosomal sequences and complete plasmid sequences can be used for detection of plasmid contigs in draft genome assemblies. We showed that Kraken achieved balanced and higher performance compared with other methods (Centrifuge, RFPlasmid, mlplasmids, PlaScope, and Platon). We therefore consider that the Kraken classifier can be the best option for predicting the origin of contigs for species with a suitable number of completed chromosomal and plasmid sequences. DATA SUMMARYTable S1: Complete chromosomes used for creating the base Kraken database. Plasmid-free chromosomal sequences and complete plasmid sequences used for creating the base Kraken database are also available via Figshare at https://doi.org/10.6084/m9.figshare.13289564. Table S2: Sequence data used for benchmarking. Draft assemblies of these 82 KpSC strains are available via Figshare at https://doi.org/10.6084/m9.figshare.13553432. The corresponding sequence read files and complete genomes were deposited in the NCBI SRA and GenBank under BioProjects PRJEB6891, PRJNA351909, PRJNA486877, and PRJNA646837 (individual BioSample IDs listed in Table S2). Kraken output files are available via Figshare at https://doi.org/10.6084/m9.figshare.13553789.

microbiology↗