Targeted A-to-G base editing in the organellar genomes of Arabidopsis with monomeric programmable deaminases.

Plastids and mitochondria are 2 intracellular organelles containing DNA-encoding partial but essential components for their roles, photosynthesis, and respiration. Precise base editing in both plastid and mitochondrial genomes would benefit their gene functional analysis and crop breeding. Targeted base editing in organellar genomes relies on a protein-based genome-editing system that uses the TALE-DNA recognition motif with deaminases. This is because the efficient delivery of guide RNA for clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 systems into organelles is currently impossible. Since TALE-based base editors used in organellar genomes are usually dimeric types, in this study, we used targeted A-to-G base editing in Arabidopsis (Arabidopsis thaliana) plastid and mitochondrial genomes with monomeric TALE-based deaminase for easier assembling of vectors. As a result, inheritable targeted A-to-G base editing of adenosine triphosphatase subunit 6-2 (atp6-2) in plant mitochondrial genomes and of 16S ribosomal RNA (16S rRNA) in plastid genomes of Arabidopsis was successfully induced by monomeric TALE-based adenine deaminase (AD) without off-target mutations. The monomeric TALE-based adenine deaminases also demonstrated a preference for editing the 8th T on the same strand from the recognition end. Phenotypic analysis showed that A-to-G conversion at 1139A of plastid 16S rRNA conferred substantial spectinomycin resistance in Arabidopsis, but not the other 2 potential-resistant mutations at 1131T and 1137T, predicted from the previous bacterial data. Our study demonstrated the feasibility of monomeric TALE-based ADs in plant organelles and their potential contribution to the functional analyses of plant organelles with easier assembling.

Targeted base editing in the mitochondrial genome of Arabidopsis thaliana.

Beyond their well-known role in respiration, mitochondria of land plants contain biologically essential and/or agriculturally important genes whose function and regulation are not fully understood. Until recently, it has been difficult to analyze these genes or, in the case of crops, to improve their functions, due to a lack of methods for stably modifying plant mitochondrial genomes. In rice, rapeseed, and Arabidopsis thaliana, mitochondria-targeting transcription activator-like effector nucleases (mitoTALENs) have recently been used to disrupt targeted genes in an inheritable and stable manner. However, this technique can also induce large deletions around the targeted sites, as well as cause ectopic homologous recombinations, which can change the sequences and gene order of mitochondrial genomes. Here, we used mitochondria-targeting TALEN-based cytidine deaminase to successfully substitute targeted C:G pairs with T:A pairs in the mitochondrial genomes of plantlets of A. thaliana without causing deletions or changes in genome structure. Expression vectors of the base editor genes were stably introduced into the nuclear genome by the easy-to-use floral dipping method. Some T1 plants had apparent homoplasmic substitutions that were stably inherited by seed progenies, independently of the inheritance of nuclear-introduced genes. As a demonstration of the method, we used it to restore the growth of an organelle transcript processing 87 (otp87) mutant that is defective in the editing of RNA transcripts of the mitochondrial atp1 gene and to identify bases in atp1 that affect the efficiency of RNA editing by OTP87.

Turnover of mammal sex chromosomes in the Sry-deficient Amami spiny rat is due to male-specific upregulation of Sox9.

Mammalian sex chromosomes are highly conserved, and sex is determined by SRY on the Y chromosome. Two exceptional rodent groups in which some species lack a Y chromosome and Sry offer insights into how novel sex genes can arise and replace Sry, leading to sex chromosome turnover. However, intensive study over three decades has failed to reveal the identity of novel sex genes in either of these lineages. We here report our discovery of a male-specific duplication of an enhancer of Sox9 in the Amami spiny rat Tokudaia osimensis, in which males and females have only a single X chromosome (XO/XO) and the Y chromosome and Sry are completely lost. We performed a comprehensive survey to detect sex-specific genomic regions in the spiny rat. Sex-related genomic differences were limited to a male-specific duplication of a 17-kb unit located 430 kb upstream of Sox9 on an autosome. Hi-C analysis using male spiny rat cells showed the duplicated region has potential chromatin interaction with Sox9. The duplicated unit harbored a 1,262-bp element homologous to mouse enhancer 14 (Enh14), a candidate Sox9 enhancer that is functionally redundant in mice. Transgenic reporter mice showed that the spiny rat Enh14 can function as an embryonic testis enhancer in mice. Embryonic gonads of XX mice in which Enh14 was replaced by the duplicated spiny rat Enh14 showed increased Sox9 expression and decreased Foxl2 expression. We propose that male-specific duplication of this Sox9 enhancer substituted for Sry function, defining a novel Y chromosome in the spiny rat.

Characterization and development of a plastid genome base editor, ptpTALECD.

The modification of photosynthesis-related genes in plastid genomes may improve crop yields. Recently, we reported that a plastid-targeting base editor named ptpTALECD, in which a cytidine deaminase DddA functions as the catalytic domain, can homoplasmically substitute a targeted C to T in plastid genomes of Arabidopsis thaliana. However, some target Cs were not substituted. In addition, although ptpTALECD could substitute Cs on the 3' side of T and A, it was unclear whether it could also substitute Cs on the 3' side of G and C. In this study, we identified the preferential positions of the substituted Cs in ptpTALECD-targeting sequences in the Arabidopsis plastid genome. We also found that ptpTALECD could substitute Cs on the 3' side of all four bases in plastid genomes of Arabidopsis. More recently, a base editor containing an improved version of DddA (DddA11) was reported to substitute Cs more efficiently, and to substitute Cs on the 3' side of more varieties of bases in human mitochondrial genomes than a base editor containing DddA. Here, we also show that ptpTALECD_v2, in which a modified version of DddA11 functions as the catalytic domain, more frequently substituted Cs than ptpTALECD in the Arabidopsis plastid genome. We also found that ptpTALECD_v2 tended to substitute Cs at more positions than ptpTALECD. Our results reveal that ptpTALECD can cause a greater variety of codon changes and amino acid substitutions than previously thought, and that ptpTALECD and ptpTALECD_v2 are useful tools for the targeted base editing of plastid genomes.

Loss of One X and the Y Chromosome Changes the Configuration of the X Inactivation Center in the Genus Tokudaia.

INTRODUCTION: X chromosome inactivation (XCI) is an essential mechanism for dosage compensation between females and males in mammals. In females, XCI is controlled by a complex, conserved locus termed the X inactivation center (Xic), in which the lncRNA Xist is the key regulator. However, little is known about the Xic in species with unusual sex chromosomes. The genus Tokudaia includes three rodent species endemic to Japan. Tokudaia osimensis and Tokudaia tokunoshimensis lost the Y chromosome (XO/XO), while Tokudaia muenninki (TMU) acquired a neo-X region by fusion of the X chromosome and an autosome (XX/XY). We compared the gene location and structure in the Xic among Tokudaia species. METHODS: Gene structure of nine genes in Xic was predicted, and the gene location and genome sequences of Xic were compared between mouse and Tokudaia species. The expression level of the gene was confirmed by transcripts per million calculation using RNA-seq data. RESULTS: Compared to mouse, the Xic gene order and location were conserved in Tokudaia species. However, remarkable structure changes were observed in lncRNA genes, Xist and Tsix, in the XO/XO species. In Xist, important functional repeats, B-, C-, D-, and E-repeats, were partially or completely lost due to deletions in these species. RNA-seq data showed that female-specific expression patterns of Xist and Tsix were confirmed in TMU, however, not in the XO/XO species. Additionally, three deletions and one inversion were confirmed in the intergenic region between Jpx and Ftx in the XO/XO species. CONCLUSION: Our findings indicate that even if the Xist and Tsix lncRNAs are expressed, they are incapable of producing a successful and lasting XCI in the XO/XO species. We hypothesized that the significant structure change in the intergenic region of Jpx-Ftx resulted in the inability to perform the XCI, and, as a result, a lack of Xist expression. Our results collectively suggest that structural changes in the Xic occurred in the ancestral lineage of XO/XO species, likely due to the loss of one X chromosome and the Y chromosome as a consequence of the degradation of the XCI system.

Resistance to the herbicide metribuzin conferred to Arabidopsis thaliana by targeted base editing of the chloroplast genome.

The chloroplast genome has considerable potential to enhance crop productivity, but it remains underutilized in breeding because it is difficult to modify. This study elucidates the potential of recently developed chloroplast-targeted C-to-T base editors in facilitating the use of the chloroplast genome for crop breeding. The herbicide metribuzin interferes with photosynthesis by binding to the D1 protein of photosystem II, encoded by the chloroplast genome. Naturally occurring D1 mutants with V219I or A251V substitutions are known to have resistance to some herbicides including metribuzin. Here, using the base editors, we introduced these substitutions and showed that the A251V single mutation and the V219 & A251V double mutations conferred significant metribuzin resistance to Arabidopsis thaliana. The V219I & A251V double mutants exhibited increased metribuzin resistance and grew better than the A251V single mutants. Furthermore, the double mutants grew as well as wild-type plants in the absence of metribuzin. The single and double mutants, which are a challenge to obtain through traditional mutagenesis and crossbreeding methods, can be relatively easily generated using C-to-T base editors. In view of the conservation of V219 and A251 across numerous species, C-to-T base editing can potentially confer metribuzin resistance to a wide range of crops. Compared to nuclear genes, chloroplast genes are also less likely to spread into wild populations. Our findings suggest that chloroplast-targeting C-to-T base editors will find many roles in future crop breeding efforts.

Isolation and whole-genome sequencing analysis of Escherichia fergusonii harboring a heat-labile enterotoxin gene from retail chicken meat in Okinawa, Japan.

This study aimed to reveal the prevalence of heat-labile enterotoxin (LT) gene-positive Escherichia fergusonii in retail chicken meat and genetically characterize these strains. E. fergusonii harboring LT gene was isolated from 6 out of 60 (10%) retail chicken samples in Okinawa, Japan. Whole-genome sequencing analysis revealed that LT gene-positive E. fergusonii from chicken meat and feces contain an IncFII plasmid harboring elt1AB, and suggested to spread clonally to retail chicken through fecal contamination. Additionally, it was found that these strains harbor multidrug-resistant genes on their plasmids. Their pathogenicity and continuous monitoring are required for confirmation.

Escherichia cryptic clade I is an emerging source of human intestinal pathogens.

BACKGROUND: Within the genus Escherichia, several monophyletic clades other than the traditionally defined species have been identified. Of these, cryptic clade I (C-I) appears to represent a subspecies of E. coli, but due to the difficulty in distinguishing it from E. coli sensu stricto, the population structure and virulence potential of C-I are unclear. RESULTS: We defined a set of true C-I strains (n = 465), including a Shiga toxin 2a (Stx2a)-producing isolate from a patient with bloody diarrhoea identified by the retrospective analyses using a C-I-specific detection system. Through genomic analysis of 804 isolates from the cryptic clades, including these C-I strains, we revealed their global population structures and the marked accumulation of virulence genes and antimicrobial resistance genes in C-I. In particular, half of the C-I strains contained hallmark virulence genes of Stx-producing E. coli (STEC) and/or enterotoxigenic E. coli (ETEC). We also found the host-specific distributions of virulence genes, which suggests bovines as the potential source of human infections caused by STEC- and STEC/ETEC hybrid-type C-I strains, as is known in STEC. CONCLUSIONS: Our findings demonstrate the emergence of human intestinal pathogens in C-I lineage. To better understand the features of C-I strains and their infections, extensive surveillance and larger population studies of C-I strains are needed. The C-I-specific detection system developed in this study will be a powerful tool for screening and identifying C-I strains.

Evolutionary History of Sexual Differentiation Mechanism in Insects.

Alternative splicing underpins functional diversity in proteins and the complexity and diversity of eukaryotes. An example is the doublesex gene, the key transcriptional factor in arthropod sexual differentiation. doublesex is controlled by sex-specific splicing and promotes both male and female differentiation in holometabolan insects, whereas in hemimetabolan species, doublesex has sex-specific isoforms but is not required for female differentiation. How doublesex evolved to be essential for female development remains largely unknown. Here, we investigate ancestral states of doublesex using Thermobia domestica belonging to Zygentoma, the sister group of Pterygota, that is, winged insects. We find that, in T. domestica, doublesex expresses sex-specific isoforms but is only necessary for male differentiation of sexual morphology. This result supports the hypothesis that doublesex initially promoted male differentiation during insect evolution. However, T. domestica doublesex has a short female-specific region and upregulates the expression of vitellogenin homologs in females, suggesting that doublesex may already play some role in female morphogenesis of the common ancestor of Pterygota. Reconstruction of the ancestral sequence and prediction of protein structures show that the female-specific isoform of doublesex has an extended C-terminal disordered region in holometabolan insects but not in nonholometabolan species. We propose that doublesex acquired its function in female morphogenesis through a change in the protein motif structure rather than the emergence of the female-specific exon.

Mining RNA-seq data reveals the massive regulon of GcvB small RNA and its physiological significance in maintaining amino acid homeostasis in Escherichia coli.

Bacterial small RNAs regulate the expression of multiple genes through imperfect base-pairing with target mRNAs mediated by RNA chaperone proteins such as Hfq. GcvB is the master sRNA regulator of amino acid metabolism and transport in a wide range of Gram-negative bacteria. Recently, independent RNA-seq approaches identified a plethora of transcripts interacting with GcvB in Escherichia coli. In this study, the compilation of RIL-seq, CLASH, and MAPS data sets allowed us to identify GcvB targets with high accuracy. We validated 21 new GcvB targets repressed at the posttranscriptional level, raising the number of direct targets to >50 genes in E. coli. Among its multiple seed sequences, GcvB utilizes either R1 or R3 to regulate most of these targets. Furthermore, we demonstrated that both R1 and R3 seed sequences are required to fully repress the expression of gdhA, cstA, and sucC genes. In contrast, the ilvLXGMEDA polycistronic mRNA is targeted by GcvB through at least four individual binding sites in the mRNA. Finally, we revealed that GcvB is involved in the susceptibility of peptidase-deficient E. coli strain (Δpeps) to Ala-Gln dipeptide by regulating both Dpp dipeptide importer and YdeE dipeptide exporter via R1 and R3 seed sequences, respectively.

Helicobacter kumamotonensis sp. nov., isolated from human clinical specimens.

A Gram-stain-negative, spiral bacterium (PAGU 1991T) was isolated from the blood of a patient with diffuse large B-cell lymphoma. Phylogenetic analysis based on 16S rRNA gene sequences showed that the isolate was very closely related to Helicobacter equorum LMG 23362T (99.1 % similarity), originally isolated from a faecal sample from a healthy horse. PAGU 1991T was also very closely related to PAGU 1750 in our strain library (=CCUG 41437) with 99.7 % similarity. Additional phylogenetic analyses based on the 23S rRNA gene sequence and GyrA amino acid sequence further supported the close relationship between the two human isolates (PAGU 1991T and PAGU 1750) and the horse strain. However, a phylogenetic analysis based on 16S rRNA showed that the two human isolates formed a lineage that was distinct from the horse strain (less than 99.2 % similarity). In silico whole-genome comparisons based on digital DNA-DNA hybridization, average nucleotide identity based on blast and orthologous average nucleotide identity using usearch between the two human isolates and the type strain of H. equorum showed values of less than 52.40, 93.47, and 93.50 %, respectively, whereas those between the two human isolates were 75.8, 97.2, and 97.2 %, respectively. These data clearly demonstrated that the two human isolates formed a single species, distinct from H. equorum. Morphologically, the human isolates could be distinguished by the type of flagella; the human isolates showed a bipolar sheathed flagellum, whereas that of H. equorum was monopolar. Biochemically, the human isolate was characterized by growth at 42 °C under microaerobic conditions and nitrate reduction unability. We conclude that the two human isolates, obtained from geographically and temporally distinct sources, were a novel species, for which we propose the name Helicobacter kumamotonensis sp. nov., with the type strain PAGU 1991T (=GTC 16810T=CCUG 75774T).

MetaPlatanus: a metagenome assembler that combines long-range sequence links and species-specific features.

De novo metagenome assembly is effective in assembling multiple draft genomes, including those of uncultured organisms. However, heterogeneity in the metagenome hinders assembly and introduces interspecies misassembly deleterious for downstream analysis. For this purpose, we developed a hybrid metagenome assembler, MetaPlatanus. First, as a characteristic function, it assembles the basic contigs from accurate short reads and then iteratively utilizes long-range sequence links, species-specific sequence compositions, and coverage depth. The binning information was also used to improve contiguity. Benchmarking using mock datasets consisting of known bacteria with long reads or mate pairs revealed the high contiguity MetaPlatanus with a few interspecies misassemblies. For published human gut data with nanopore reads from potable sequencers, MetaPlatanus assembled many biologically important elements, such as coding genes, gene clusters, viral sequences, and over-half bacterial genomes. In the benchmark with published human saliva data with high-throughput nanopore reads, the superiority of MetaPlatanus was considerably more evident. We found that some high-abundance bacterial genomes were assembled only by MetaPlatanus as near-complete. Furthermore, MetaPlatanus can circumvent the limitations of highly fragmented assemblies and frequent interspecies misassembles obtained by the other tools. Overall, the study demonstrates that MetaPlatanus could be an effective approach for exploring large-scale structures in metagenomes.

Targeted gene disruption of ATP synthases 6-1 and 6-2 in the mitochondrial genome of Arabidopsis thaliana by mitoTALENs.

We recently achieved targeted disruptions of cytoplasmic male sterility (CMS)-associated genes in the mitochondrial genomes of rice and rapeseed by using mitochondria-targeted transcription activator-like effector nucleases (mitoTALENs). It was the first report of stable and heritable targeted gene modification of plant mitochondrial genomes. Here, we attempted to use mitoTALENs to disrupt two mitochondrial genes in the model plant Arabidopsis thaliana(Arabidopsis) using three different promoters and two types of TALENs. The targets were the two isoforms of the ATP synthase subunit 6 gene, atp6-1 and atp6-2. Each of these genes was successfully deleted and the mitochondrial genomes were recovered in a homoplasmic state. The nuclear genome also has a copy of atp6-1, and we were able to confirm that it was the mitochondrial gene and not the nuclear pseudogene that was knocked out. Among the three mitoTALEN promoters tried, the RPS5A promoter was the most effective. Conventional mitoTALENs were more effective than single-molecule mito-compactTALENs. Targeted mitochondrial gene deletion was achieved by crossing as well as by floral-dip transformation to introduce the mitoTALEN constructs into the nucleus. The gene disruptions were caused by large (kb-size) deletions. The ends of the remaining sequences were connected to distant loci, mostly by illegitimate homologous recombinations between repeats.

Efficient de novo assembly of highly heterozygous genomes from whole-genome shotgun short reads.

Although many de novo genome assembly projects have recently been conducted using high-throughput sequencers, assembling highly heterozygous diploid genomes is a substantial challenge due to the increased complexity of the de Bruijn graph structure predominantly used. To address the increasing demand for sequencing of nonmodel and/or wild-type samples, in most cases inbred lines or fosmid-based hierarchical sequencing methods are used to overcome such problems. However, these methods are costly and time consuming, forfeiting the advantages of massive parallel sequencing. Here, we describe a novel de novo assembler, Platanus, that can effectively manage high-throughput data from heterozygous samples. Platanus assembles DNA fragments (reads) into contigs by constructing de Bruijn graphs with automatically optimized k-mer sizes followed by the scaffolding of contigs based on paired-end information. The complicated graph structures that result from the heterozygosity are simplified during not only the contig assembly step but also the scaffolding step. We evaluated the assembly results on eukaryotic samples with various levels of heterozygosity. Compared with other assemblers, Platanus yields assembly results that have a larger scaffold NG50 length without any accompanying loss of accuracy in both simulated and real data. In addition, Platanus recorded the largest scaffold NG50 values for two of the three low-heterozygosity species used in the de novo assembly contest, Assemblathon 2. Platanus therefore provides a novel and efficient approach for the assembly of gigabase-sized highly heterozygous genomes and is an attractive alternative to the existing assemblers designed for genomes of lower heterozygosity.

Coelacanth genomes reveal signatures for evolutionary transition from water to land.

Coelacanths are known as "living fossils," as they show remarkable morphological resemblance to the fossil record and belong to the most primitive lineage of living Sarcopterygii (lobe-finned fishes and tetrapods). Coelacanths may be key to elucidating the tempo and mode of evolution from fish to tetrapods. Here, we report the genome sequences of five coelacanths, including four Latimeria chalumnae individuals (three specimens from Tanzania and one from Comoros) and one L. menadoensis individual from Indonesia. These sequences cover two African breeding populations and two known extant coelacanth species. The genome is ∼2.74 Gbp and contains a high proportion (∼60%) of repetitive elements. The genetic diversity among the individuals was extremely low, suggesting a small population size and/or a slow rate of evolution. We found a substantial number of genes that encode olfactory and pheromone receptors with features characteristic of tetrapod receptors for the detection of airborne ligands. We also found that limb enhancers of bmp7 and gli3, both of which are essential for limb formation, are conserved between coelacanth and tetrapods, but not ray-finned fishes. We expect that some tetrapod-like genes may have existed early in the evolution of primitive Sarcopterygii and were later co-opted to adapt to terrestrial environments. These coelacanth genomes will provide a cornerstone for studies to elucidate how ancestral aquatic vertebrates evolved into terrestrial animals.

Metabolic engineering with adaptive laboratory evolution for phenylalanine production by Corynebacterium glutamicum.

The mutants resistant to a phenylalanine analog, 4-fluorophenylalanine (4FP), were obtained for metabolic engineering of Corynebacterium glutamicum for producing aromatic amino acids synthesized through the shikimate pathway by adaptive laboratory evolution. Culture experiments of the C. glutamicum strains which carry the mutations found in the open reading frame from the 4FP-resistant mutants revealed that the mutations in the open reading frames of aroG (NCgl2098), pheA (NCgl2799) and aroP (NCgl1062) encoding 3-deoxy-d-arabino-heptulosonate-7-phosphate, prephenate dehydratase, and aromatic amino acid transporter are responsible for 4FP resistance and higher concentration of aromatic amino acids in their culture supernatants in the 4FP-resistant strains. It was expected that aroG and pheA mutations would release feedback inhibition of the enzymes involved in the shikimate pathway by phenylalanine and that aroP mutations would prevent intracellular uptake of aromatic amino acids. Therefore, we conducted metabolic engineering of the C. glutamicum wild-type strain for aromatic amino acid production and found that phenylalanine production at 6.11 ± 0.08 g L-1 was achieved by overexpressing the mutant pheA and aroG genes from the 4FP-resistant mutants and deleting aroP gene. This study demonstrates that adaptive laboratory evolution is an effective way to obtain useful mutant genes related to production of target material and to establish metabolic engineering strategies.

Improved complete genome sequence of Bdellovibrio bacteriovorus 109J, a widely studied laboratory strain of predatory bacteria.

The complete genome sequence of Bdellovibrio bacteriovorus 109J, a well-studied laboratory strain of predatory bacteria, first determined in 2014. Here we report an improved complete genome sequence of B. bacteriovorus 109J, incorporating 16 assembly and 87 nucleotide corrections. This revised genome will be helpful to studies on the predatory bacteria.

Relationship Between Fluoroquinolone Resistance and Mutations in the Quinolone Resistance-Determining Region in Corynebacterium macginleyi.

Purpose: The purpose of this study was to investigate the bacterial composition in the conjunctiva and to determine the relationship between fluoroquinolone resistance and mutations in the quinolone resistance-determining region (QRDR) in Corynebacterium macginleyi (C. macginleyi). Methods: Bacteria isolated from conjunctival swabs of patients awaiting ophthalmic surgery or patients with presumed keratoconjunctivitis were included in this study. For C. macginleyi isolates from 49 samples, the minimum inhibitory concentrations (MICs) of second- to fourth-generation fluoroquinolones were determined by broth microdilution. Additionally, we determined the sequence of the QRDR in the gyrA gene of C. macginleyi-positive isolates by direct sequencing and investigated the relationship between the QRDR mutation and the MICs of fluoroquinolones for C. macginleyi. Results: Among 423 eyes of 296 preoperative patients who underwent conjunctival culture testing, 105 eyes of 89 patients were culture-positive, and among 148 eyes of 147 patients with keratoconjunctivitis, 55 eyes of 54 patients were culture-positive. C. macginleyi accounted for the largest proportion of cultured organisms (34.8%). C. macginleyi-positive isolates were found in 45 eyes of 37 preoperative patients and in 4 eyes of 4 patients with keratoconjunctivitis. Direct sequencing revealed that 91.8% of C. macginleyi-positive isolates had amino acid mutations in the QRDR and 95.5% of mutations were found at Ser-87 and Asp-91. Isolates harboring double mutations at Ser-87 and Asp-91 were resistant to second- to fourth-generation fluoroquinolones. One isolate with double mutations at Ser-87 and Ala-88 but no mutation in Asp-91 showed intermediate susceptibility to moxifloxacin, a fourth-generation fluoroquinolone. Conclusions: C. macginleyi isolated from conjunctiva harboring QRDR amino acid mutations were resistant to second- to fourth-generation fluoroquinolones.

Streptococcus suis subsp. hashimotonensis subsp. nov.: Lancefield group A antigen-positive organisms isolated from human clinical specimens and wild boar oral cavity samples.

Three Streptococcus suis-like strains positive for Lancefield antigen group A were isolated from human boar bite wounds and the oral cavities of boars in Hashimoto City, Wakayama Prefecture, Japan, and their taxonomic positions were investigated. Application of the VITEK2 system identified all three isolates as S. suis with > 94 % probability. The isolates were assigned to S. suis based on the results of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis (Biotyper score of 2.382) but were differentiated according to the characteristic signal peaks (4709 m/z and 9420 m/z) that were not present for S. suis. Sequence analysis of the 16S rRNA and sodA genes determined that the isolates were similar to S. suis; however, these genes appeared on a phylogenetic sub-branch. Phylogenetic analysis of the whole chromosomal DNA showed that the isolate formed a cluster with S. suis but with clear divergence. The average nucleotide index using BLAST between the clinical isolate (PAGU 2482) and a closely related reference strain of S. suis was 94.75 %, which was not clearly conclusive; however, digital DNA-DNA hybridization showed a value of 61.2 %. Biochemical reactions, including those with acid phosphatase, α-chymotrypsin, and tagatose (acidification), distinguished our isolates from S. suis. Thus, based on phylogenetic, genomic, and phenotypic characteristics and MALDI-TOF-MS signal patterns, we propose that the isolate with Lancefield group A positive characteristics be designated as a novel subspecies, Streptococcus suis subsp. hashimotonensis subsp. nov., with the type strain PAGU 2482T (GTC 18290T = CCUG 77434T).

Isolation and Genomic Characterization of a Heat-Labile Enterotoxin 1-Producing Escherichia fergusonii Strain from a Human.

Escherichia fergusonii strains have been isolated from patients with diarrhea, but their virulence determinant has not been well elucidated. Here, we report the first isolation of a heat-labile enterotoxin 1 (LT1)-producing E. fergusonii strain (strain 30038) from a patient in Japan. The complete genome sequence of strain 30038 was determined and subjected to comparative genomics and phylogenetic analyses with 195 publicly available genomes of E. fergusonii. In addition to strain 30038, the elt1 gene was also identified in an E. fergusonii strain that is phylogenetically distinct and which was isolated from poultry in the United Kingdom. Fine genomic comparison revealed that these two strains share comparable elt1-bearing plasmids. However, an intriguing distinction arises in strain 30038, wherein the plasmid has integrated into the chromosome via a recombination process mediated by an insertion sequence. The production of active LT1 toxin by strain 30038 was verified through an in vitro assay using cultured cells. A large plasmid carrying 11 antimicrobial resistance genes was also identified in strain 30038. Our results indicate that extensive surveillance of elt1-positive E. fergusonii strains as diarrheagenic pathogens is needed. IMPORTANCE Escherichia fergusonii, a species closely related to Escherichia coli, is known to cause sporadic conditions in humans, including diarrhea. However, the critical virulence factors in E. fergusonii clinical isolates remain to be identified. This study shows the first isolation of an E. fergusonii strain carrying the elt1 gene, which encodes heat-labile enterotoxin 1, from a patient with diarrhea. Our analysis of public databases also revealed the presence of elt1-positive E. fergusonii strains isolated from poultry in the United Kingdom. Interestingly, while the elt1 gene in the poultry isolate was present on a large plasmid, in the human isolate it was integrated into the chromosome, which may confer stability on the elt1-carrying genetic element. Our findings highlight the need for extensive surveillance of elt1-positive E. fergusonii strains in livestock animals.