Unique B-1 cells specific for both N-pyrrolated proteins and DNA evolve with apolipoprotein E deficiency.

Lysine N-pyrrolation, a posttranslational modification, which converts lysine residues to Nε-pyrrole-L-lysine, imparts electronegative properties to proteins, causing them to mimic DNA. Apolipoprotein E (apoE) has been identified as a soluble receptor for pyrrolated proteins (pyrP), and accelerated lysine N-pyrrolation has been observed in apoE-deficient (apoE-/-) hyperlipidemic mice. However, the impact of pyrP accumulation consequent to apoE deficiency on the innate immune response remains unclear. Here, we investigated B-1a cells known to produce germline-encoded immunoglobulin M (IgM) from mice deficient in apoE and identified a particular cell population that specifically produces IgM antibodies against pyrP and DNA. We demonstrated an expansion of B-1a cells involved in IgM production in the peritoneal cavity of apoE-/- mice compared with wild-type mice, consistent with a progressive increase of IgM response in the mouse sera. We found that pyrP exhibited preferential binding to B-1a cells and facilitated the production of IgM. B cell receptor analysis of pyrP-specific B-1a cells showed restricted usage of gene segments selected from the germline gene set; most sequences contained high levels of non-templated-nucleotide additions (N-additions) that could contribute to junctional diversity of B cell receptors. Finally, we report that a subset of monoclonal IgM antibodies against pyrP/DNA established from the apoE-/- mice also contained abundant N-additions. These results suggest that the accumulation of pyrP due to apoE deficiency may influence clonal diversity in the pyrP-specific B cell repertoire. The discovery of these unique B-1a cells for pyrP/DNA provides a key link connecting covalent protein modification, lipoprotein metabolism, and innate immunity.

Analysis of disease model iPSCs derived from patients with a novel Fanconi anemia-like IBMFS ADH5/ALDH2 deficiency.

We have recently discovered Japanese children with a novel Fanconi anemia-like inherited bone marrow failure syndrome (IBMFS). This disorder is likely caused by the loss of a catabolic system directed toward endogenous formaldehyde due to biallelic variants in ADH5 combined with a heterozygous ALDH2*2 dominant-negative allele (rs671), which is associated with alcohol-induced Asian flushing. Phytohemagglutinin-stimulated lymphocytes from these patients displayed highly increased numbers of spontaneous sister chromatid exchanges (SCEs), reflecting homologous recombination repair of formaldehyde damage. Here, we report that, in contrast, patient-derived fibroblasts showed normal levels of SCEs, suggesting that different cell types or conditions generate various amounts of formaldehyde. To obtain insights about endogenous formaldehyde production and how defects in ADH5/ALDH2 affect human hematopoiesis, we constructed disease model cell lines, including induced pluripotent stem cells (iPSCs). We found that ADH5 is the primary defense against formaldehyde, and ALDH2 provides a backup. DNA repair capacity in the ADH5/ALDH2-deficient cell lines can be overwhelmed by exogenous low-dose formaldehyde, as indicated by higher levels of DNA damage than in FANCD2-deficient cells. Although ADH5/ALDH2-deficient cell lines were healthy and showed stable growth, disease model iPSCs displayed drastically defective cell expansion when stimulated into hematopoietic differentiation in vitro, displaying increased levels of DNA damage. The expansion defect was partially reversed by treatment with a new small molecule termed C1, which is an agonist of ALDH2, thus identifying a potential therapeutic strategy for the patients. We propose that hematopoiesis or lymphocyte blastogenesis may entail formaldehyde generation that necessitates elimination by ADH5/ALDH2 enzymes.

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

OBJECTIVES: Carbapenemase-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. METHODS: Eighty-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). RESULTS: Various 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 co-located on a 107 kb plasmid. The blaSFC and blaSFH-1 genes were found on putative genomic islands inserted at tRNA-Phe genes in chromosomes. CONCLUSIONS: This 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.

Protective effects of Alda-1, an ALDH2 activator, on alcohol-derived DNA damage in the esophagus of human ALDH2*2 (Glu504Lys) knock-in mice.

Alcohol consumption is the key risk factor for the development of esophageal squamous cell carcinoma (ESCC), and acetaldehyde, a metabolite of alcohol, is an alcohol-derived major carcinogen that causes DNA damage. Aldehyde dehydrogenase2 (ALDH2) is an enzyme that detoxifies acetaldehyde, and its activity is reduced by ALDH2 gene polymorphism. Reduction in ALDH2 activity increases blood, salivary and breath acetaldehyde levels after alcohol intake, and it is deeply associated with the development of ESCC. Heavy alcohol consumption in individuals with ALDH2 gene polymorphism significantly elevates the risk of ESCC; however, effective prevention has not been established yet. In this study, we investigated the protective effects of Alda-1, a small molecule ALDH2 activator, on alcohol-mediated esophageal DNA damage. Here, we generated novel genetically engineered knock-in mice that express the human ALDH2*1 (wild-type allele) or ALDH2*2 gene (mutant allele). Those mice were crossed, and human ALDH2*1/*1, ALDH2*1/*2 and ALDH2*2/*2 knock-in mice were established. They were given 10% ethanol for 7 days in the presence or absence of Alda-1, and we measured the levels of esophageal DNA damage, represented by DNA adduct (N2-ethylidene-2'-deoxyguanosine). Alda-1 significantly increased hepatic ALDH2 activity both in human ALDH2*1/*2 and/or ALDH2*2/*2 knock-in mice and reduced esophageal DNA damage levels after alcohol drinking. Conversely, cyanamide, an ALDH2-inhibitor, significantly exacerbated esophageal DNA adduct level in C57BL/6N mice induced by alcohol drinking. These results indicate the protective effects of ALDH2 activation by Alda-1 on esophageal DNA damage levels in individuals with ALDH2 gene polymorphism, providing a new insight into acetaldehyde-mediated esophageal carcinogenesis and prevention.

DNA Adductome Analysis Identifies N-Nitrosopiperidine Involved in the Etiology of Esophageal Cancer in Cixian, China.

Esophageal cancer is prevalent in Cixian, China, but the etiology of this disease remains largely unknown. Therefore, we explored this by conducting a DNA adductome analysis. Both tumorous and nontumorous tissues were collected from patients who underwent surgical procedures at Cixian Cancer Hospital and the Fourth Hospital of Hebei Medical University, which is in a low-incidence area. N2-(3,4,5,6-Tetrahydro-2H-pyran-2-yl)deoxyguanosine (THP-dG) was the major adduct detected in samples from esophageal cancer patients in Cixian. The precursor of THP-dG, N-nitrosopiperidine (NPIP), exhibited a strong mutagenic activity under metabolic activation in the Ames test and a significant dose-dependent increase in mutation frequency during an in vivo mutagenicity test with guanine phosphoribosyltransferase (gpt) delta rats. The NPIP-induced mutation was dominated by A:T to C:G transversions, followed by G:C to A:T and A:T to G:C transitions, in the liver and esophagus of animal samples. A similar mutational pattern was observed in the mutational signature of esophageal cancer patients that demonstrated weak correlation with THP-dG levels. These findings suggested that NPIP exposure is partly involved in the development of esophageal cancer in Cixian residents.

A decision tree-based integrated testing strategy for tailor-made carcinogenicity evaluation of test substances using genotoxicity test results and chemical spaces.

Genotoxicity evaluation has been widely used to estimate the carcinogenicity of test substances during safety evaluation. However, the latest strategies using genotoxicity tests give more weight to sensitivity; therefore, their accuracy has been very low. For precise carcinogenicity evaluation, we attempted to establish an integrated testing strategy for the tailor-made carcinogenicity evaluation of test materials, considering the relationships among genotoxicity test results (Ames, in vitro mammalian genotoxicity and in vivo micronucleus), carcinogenicity test results and chemical properties (molecular weight, logKow and 179 organic functional groups). By analyzing the toxicological information and chemical properties of 230 chemicals, including 184 carcinogens in the Carcinogenicity Genotoxicity eXperience database, a decision tree for carcinogenicity evaluation was optimised statistically. A decision forest model was generated using a machine-learning method-random forest-which comprises thousands of decision trees. As a result, balanced accuracies in cross-validation of the optimised decision tree and decision forest model, considering chemical space (71.5% and 75.5%, respectively), were higher than balanced accuracy of an example regulatory decision tree (54.1%). Moreover, the statistical optimisation of tree-based models revealed significant organic functional groups that would cause false prediction in standard genotoxicity tests and non-genotoxic carcinogenicity (e.g., organic amide and thioamide, saturated heterocyclic fragment and aryl halide). In vitro genotoxicity tests were the most important parameters in all models, even when in silico parameters were integrated. Although external validation is required, the findings of the integrated testing strategies established herein will contribute to precise carcinogenicity evaluation and to determine new mechanistic hypotheses of carcinogenicity.

Characteristics of Carbapenemase-Producing Enterobacteriaceae in Wastewater Revealed by Genomic Analysis.

Wastewater is considered a major source of antibiotic-resistant bacteria released into the environment. Here, we characterized carbapenemase-producing Enterobacteriaceae (CPE) in wastewater by whole-genome analysis. Wastewater samples (n = 40) were collected from municipal wastewater treatment plants and hospital wastewater in Japan and Taiwan. Samples were screened for CPE using selective media, and the obtained isolates were sequenced using an Illumina MiSeq. The isolates (n = 45) included the following microorganisms: Klebsiella quasipneumoniae (n = 12), Escherichia coli (n = 10), Enterobacter cloacae complex (n = 10), Klebsiella pneumoniae (n = 8), Klebsiella variicola (n = 2), Raoultella ornithinolytica (n = 1), Citrobacter freundii (n = 1), and Citrobacter amalonaticus (n = 1). Among the 45 isolates, 38 harbored at least one carbapenemase-encoding gene. Of these, the blaGES (blaGES-5, blaGES-6, and blaGES-24) genes were found in 29 isolates. The genes were situated in novel class 1 integrons, but the integron structures were different between the Japanese (In1439 with blaGES-24 and In1440 with blaGES-5) and Taiwanese (In1441 with blaGES-5 and In1442 with blaGES-6) isolates. Other carbapenemase-encoding genes (blaVIM-1, blaNDM-5, blaIMP-8, blaIMP-19, and blaKPC-2) were found in one to three isolates. Notably, class 1 integrons previously reported among clinical isolates obtained in the same regions as the present study, namely, In477 with blaIMP-19 and In73 with blaIMP-8, were found among the Japanese and Taiwanese isolates, respectively. The results indicate that CPE with various carbapenemase-encoding genes in different genetic contexts were present in biologically treated wastewater, highlighting the need to monitor for antibiotic resistance in wastewater.

Molecular Analysis of a blaIMP-1-Harboring Class 3 Integron in Multidrug-Resistant Pseudomonas fulva.

A multidrug-resistant (MDR) Pseudomonas fulva strain was isolated in 2006 from a urine sample. The isolate harbored the blaIMP-1 gene, which was located in a chromosomal Tn402-like class 3 integron as a gene cassette array of aacA31-fosE-blaIMP-1 Two mutations in gyrA and one mutation in parC were detected in quinolone-resistance-determining regions (QRDRs). We report a full-length, novel, blaIMP-1-carrying class 3 integron. This integron, together with mutations in QRDRs, could have influenced the MDR phenotype.

Nuclear pyruvate kinase M2 complex serves as a transcriptional coactivator of arylhydrocarbon receptor.

Pyruvate kinase M2 (PKM2) and pyruvate dehydrogenase complex (PDC) regulate production of acetyl-CoA, which functions as an acetyl donor in diverse enzymatic reactions, including histone acetylation. However, the mechanism by which the acetyl-CoA required for histone acetylation is ensured in a gene context-dependent manner is not clear. Here we show that PKM2, the E2 subunit of PDC and histone acetyltransferase p300 constitute a complex on chromatin with arylhydrocarbon receptor (AhR), a transcription factor associated with xenobiotic metabolism. All of these factors are recruited to the enhancer of AhR-target genes, in an AhR-dependent manner. PKM2 contributes to enhancement of transcription of cytochrome P450 1A1 (CYP1A1), an AhR-target gene, acetylation at lysine 9 of histone H3 at the CYP1A1 enhancer. Site-directed mutagenesis of PKM2 indicates that this enhancement of histone acetylation requires the pyruvate kinase activity of the enzyme. Furthermore, we reveal that PDC activity is present in nuclei. Based on these findings, we propose a local acetyl-CoA production system in which PKM2 and PDC locally supply acetyl-CoA to p300 from abundant PEP for histone acetylation at the gene enhancer, and our data suggest that PKM2 sensitizes AhR-mediated detoxification in actively proliferating cells such as cancer and fetal cells.

Occurrence of Clinically Important Lineages, Including the Sequence Type 131 C1-M27 Subclone, among Extended-Spectrum-ß-Lactamase-Producing Escherichia coli in Wastewater.

Contamination of environmental waters by extended-spectrum-ß-lactamase (ESBL)-producing Escherichia coli (ESBLEC) is of great concern. Wastewater treatment plants (WWTPs) and hospitals release large amounts of ESBLEC into the environment. In the present study, we isolated ESBLEC strains from wastewater collected from a WWTP and a hospital in Japan and performed whole-genome sequencing to characterize these strains. Genomic analysis of 54 strains (32 from the WWTP and 22 from hospital wastewater) revealed the occurrence of clinically important clonal groups with extraintestinal pathogenic E. coli status in the WWTP and hospital wastewater. Fine-scale phylogenetic analysis was performed to further characterize 15 sequence type 131 (ST131) complex strains (11 from the WWTP and 4 from hospital wastewater). These ST131 complex strains were comprised of the following different subgroups: clade A (n = 2), C1-M27 (n = 8), and C1 (non-C1-M27) (n = 1) for strains from the WWTP and clade A (n = 2), C1-M27 (n = 1), and C1 (non-C1-M27) (n = 1) for strains from hospital wastewater. The results indicate that ESBLEC strains belonging to clinically important lineages, including the C1-M27 clade, may disseminate into the environment through wastewater, highlighting the need to monitor for antibiotic resistance in wastewater.

Rapid Identification of Different Escherichia coli Sequence Type 131 Clades.

Escherichia coli sequence type 131 (ST131) is a pandemic clonal lineage that is responsible for the global increase in fluoroquinolone resistance and extended-spectrum-ß-lactamase (ESBL) producers. The members of ST131 clade C, especially subclades C2 and C1-M27, are associated with ESBLs. We developed a multiplex conventional PCR assay with the ability to detect all ST131 clades (A, B, and C), as well as C subclades (C1-M27, C1-nM27 [C1-non-M27], and C2). To validate the assay, we used 80 ST131 global isolates that had been fully sequenced. We then used the assay to define the prevalence of each clade in two Japanese collections consisting of 460 ESBL-producing E. coli ST131 (2001-12) and 329 E. coli isolates from extraintestinal sites (ExPEC) (2014). The assay correctly identified the different clades in all 80 global isolates: clades A (n = 12), B (n = 12), and C, including subclades C1-M27 (n = 16), C1-nM27 (n = 20), C2 (n = 17), and other C (n = 3). The assay also detected all 565 ST131 isolates in both collections without any false positives. Isolates from clades A (n = 54), B (n = 23), and C (n = 483) corresponded to the O serotypes and the fimH types of O16-H41, O25b-H22, and O25b-H30, respectively. Of the 483 clade C isolates, C1-M27 was the most common subclade (36%), followed by C1-nM27 (32%) and C2 (15%). The C1-M27 subclade with blaCTX-M-27 became especially prominent after 2009. Our novel multiplex PCR assay revealed the predominance of the C1-M27 subclade in recent Japanese ESBL-producing E. coli isolates and is a promising tool for epidemiological studies of ST131.

Alcohol Consumption and Multiple Dysplastic Lesions Increase Risk of Squamous Cell Carcinoma in the Esophagus, Head, and Neck.

BACKGROUND & AIMS: Some patients develop multiple squamous cell carcinomas (SCCs) in the upper aerodigestive tract, attributed to field cancerization; alcohol consumption has been associated with this process. We examined the association between multiple areas of dysplastic squamous epithelium with the development of SCC of the esophagus or head and neck cancer, as well as alcohol consumption and smoking. METHODS: We examined 331 patients with early stage esophageal SCC using Lugol chromoendoscopy to evaluate the dysplastic squamous epithelium in the esophagus. Patients then were assigned to 3 groups, based on the number of Lugol-voiding lesions: A, no lesion; B, 1-9 lesions; or C, 10 or more lesions. Participants completed lifestyle surveys on their history of drinking, smoking, and diet. All participants were evaluated by laryngopharyngoscopy before registration; only those without head and neck cancer were included, except for patients with superficial SCC limited to the subepithelial layer. Lesions detected in the esophagus and head and neck by surveillance were considered to be metachronous. The study end point was the cumulative incidence of metachronous SCCs in the esophagus and head and neck after endoscopic resection of esophageal SCC, according to the grade of Lugol-voiding lesions. At study entry, all patients were instructed to abstain from alcohol and smoking. RESULTS: Over the 2-year study period, metachronous SCCs of the esophagus were detected in 4% of patients in group A, in 9.4% of patients in group B, and in 24.7% of patients in group C (P < .0001 for patients in group A vs B or B vs C). Head and neck SCCs were detected in none of the patients in group A, in 1.7% of the patients in group B, and in 8.6% of the patients in group C (P = .016 for patients in group A vs C and P = .008 for patients in group B vs C). SCC of the esophagus or head and neck developed in 4.0% of patients in group A, in 10.0% of patients in group B, and in 31.4% of patients in group C (P < .0001 for group A vs B or A vs C). Alcohol abstinence decreased the risk of multiple SCCs of the esophagus (adjusted hazard ratio, 0.47, 95% confidence interval, 0.25-0.91; P = .025), whereas smoking abstinence did not. CONCLUSIONS: Multiple dysplastic lesions in the esophagus increase the risk of multiple SCCs. Alcohol abstinence reduces the risk of metachronous SCCs. Clinical Trials registry: UMIN000001676 and UMIN000005466.

Whole-Genome Analysis of Antimicrobial-Resistant and Extraintestinal Pathogenic Escherichia coli in River Water.

Contamination of surface waters by antimicrobial-resistant bacteria and pathogenic bacteria is a great concern. In this study, 531 Escherichia coli isolates obtained from the Yamato River in Japan were evaluated phenotypically for resistance to 25 antimicrobials. Seventy-six isolates (14.3%) were multidrug resistant (MDR), 66 (12.4%) were nonsusceptible to one or two classes of agents, and 389 (73.3%) were susceptible. We performed whole-genome sequencing of selected strains by using Illumina technology. In total, the genome sequences of 155 strains were analyzed for antibiotic resistance determinants and phylogenetic characteristics. More than 50 different resistance determinants, including acquired resistance genes and chromosomal resistance mutations, were detected. Among the sequenced MDR strains (n = 66), sequence type 155 (ST155) complex (n = 9), ST10 complex (n = 9), and ST69 complex (n = 7) were prevalent. Among extraintestinal pathogenic E. coli (ExPEC) strains (n = 58), clinically important clonal groups, namely, ST95 complex (n = 18), ST127 complex (n = 8), ST12 complex (n = 6), ST14 complex (n = 6), and ST131 complex (n = 6), were prevalent, demonstrating the clonal distribution of environmental ExPEC strains. Typing of the fimH (type 1 fimbrial adhesin) gene revealed that ST131 complex strains carried fimH22 or fimH41, and no strains belonging to the fimH30 subgroup were detected. Fine-scale phylogenetic analysis and virulence gene content analysis of strains belonging to the ST95 complex (one of the major clonal ExPEC groups causing community-onset infections) revealed no significant differences between environmental and clinical strains. The results indicate contamination of surface waters by E. coli strains belonging to clinically important clonal groups.IMPORTANCE The prevalence of antimicrobial-resistant and pathogenic E. coli strains in surface waters is a concern because surface waters are used as sources for drinking water, irrigation, and recreational purposes. In this study, MDR and ExPEC strains in river water were characterized by genomic sequencing and analysis. We detected more than 50 resistance determinants and identified clonal groups specific to MDR and ExPEC strains. This study showed contamination of surface waters by E. coli strains belonging to clinically important clonal groups. Overall, this study advances our understanding of environmental MDR and ExPEC strains.

Molecular Mechanisms of Acetaldehyde-Mediated Carcinogenesis in Squamous Epithelium.

Acetaldehyde is a highly reactive compound that causes various forms of damage to DNA, including DNA adducts, single- and/or double-strand breaks (DSBs), point mutations, sister chromatid exchanges (SCEs), and DNA-DNA cross-links. Among these, DNA adducts such as N²-ethylidene-2'-deoxyguanosine, N²-ethyl-2'-deoxyguanosine, N²-propano-2'-deoxyguanosine, and N²-etheno-2'-deoxyguanosine are central to acetaldehyde-mediated DNA damage because they are associated with the induction of DNA mutations, DNA-DNA cross-links, DSBs, and SCEs. Acetaldehyde is produced endogenously by alcohol metabolism and is catalyzed by aldehyde dehydrogenase 2 (ALDH2). Alcohol consumption increases blood and salivary acetaldehyde levels, especially in individuals with ALDH2 polymorphisms, which are highly associated with the risk of squamous cell carcinomas in the upper aerodigestive tract. Based on extensive epidemiological evidence, the International Agency for Research on Cancer defined acetaldehyde associated with the consumption of alcoholic beverages as a "group 1 carcinogen" (definite carcinogen) for the esophagus and/or head and neck. In this article, we review recent advances from studies of acetaldehyde-mediated carcinogenesis in the squamous epithelium, focusing especially on acetaldehyde-mediated DNA adducts. We also give attention to research on acetaldehyde-mediated DNA repair pathways such as the Fanconi anemia pathway and refer to our studies on the prevention of acetaldehyde-mediated DNA damage.

Hypermutation and unique mutational signatures of occupational cholangiocarcinoma in printing workers exposed to haloalkanes.

Cholangiocarcinoma is a relatively rare cancer, but its incidence is increasing worldwide. Although several risk factors have been suggested, the etiology and pathogenesis of the majority of cholangiocarcinomas remain unclear. Recently, a high incidence of early-onset cholangiocarcinoma was reported among the workers of a printing company in Osaka, Japan. These workers underwent high exposure to organic solvents, mainly haloalkanes such as 1,2-dichloropropane (1,2-DCP) and/or dichloromethane. We performed whole-exome analysis on four cases of cholangiocarcinoma among the printing workers. An average of 44.8 somatic mutations was detected per Mb in the genome of the printing workers' cholangiocarcinoma tissues, approximately 30-fold higher than that found in control common cholangiocarcinoma tissues. Furthermore, C:G-to-T:A transitions with substantial strand bias as well as unique trinucleotide mutational changes of GpCpY to GpTpY and NpCpY to NpTpY or NpApY were predominant in all of the printing workers' cholangiocarcinoma genomes. These results were consistent with the epidemiological observation that they had been exposed to high concentrations of chemical compounds. Whole-genome analysis of Salmonella typhimurium strain TA100 exposed to 1,2-DCP revealed a partial recapitulation of the mutational signature in the printing workers' cholangiocarcinoma. Although our results provide mutational signatures unique to occupational cholangiocarcinoma, the underlying mechanisms of the disease should be further investigated by using appropriate model systems and by comparison with genomic data from other cancers.

Global Escherichia coli Sequence Type 131 Clade with blaCTX-M-27 Gene.

The Escherichia coli sequence type (ST) 131 C2/H30Rx clade with the blaCTX-M-15 gene had been most responsible for the global dissemination of extended-spectrum ß-lactamase (ESBL)-producing E. coli. ST131 C1/H30R with blaCTX-M-27 emerged among ESBL-producing E. coli in Japan during the late 2000s. To investigate the possible expansion of a single clade, we performed whole-genome sequencing for 43 Japan and 10 global ST131 isolates with blaCTX-M-27 (n = 16), blaCTX-M-14 (n = 16), blaCTX-M-15 (n = 13), and others (n = 8). We also included 8 ST131 genomes available in public databases. Core genome-based analysis of 61 isolates showed that ST131 with blaCTX-M-27 from 5 countries formed a distinct cluster within the C1/H30R clade, named C1-M27 clade. Accessory genome analysis identified a unique prophage-like region, supporting C1-M27 as a distinct clade. Our findings indicate that the increase of ESBL-producing E. coli in Japan is due mainly to emergence of the C1-M27 clade.

Interspecies Dissemination of a Mobilizable Plasmid Harboring blaIMP-19 and the Possibility of Horizontal Gene Transfer in a Single Patient.

Carbapenemase-producing Gram-negative bacilli have been a global concern over the past 2 decades because these organisms can cause severe infections with high mortality rates. Carbapenemase genes are often carried by mobile genetic elements, and resistance plasmids can be transferred through conjugation. We conducted whole-genome sequencing (WGS) to demonstrate that the same plasmid harboring a metallo-ß-lactamase gene was detected in two different species isolated from a single patient. Metallo-ß-lactamase-producing Achromobacter xylosoxidans (KUN4507), non-metallo-ß-lactamase-producing Klebsiella pneumoniae (KUN4843), and metallo-ß-lactamase-producing K. pneumoniae (KUN5033) were sequentially isolated from a single patient and then analyzed in this study. Antimicrobial susceptibility testing, molecular typing (pulsed-field gel electrophoresis and multilocus sequence typing), and conjugation analyses were performed by conventional methods. Phylogenetic and molecular clock analysis of K. pneumoniae isolates were performed with WGS, and the nucleotide sequences of plasmids detected from these isolates were determined using WGS. Conventional molecular typing revealed that KUN4843 and KUN5033 were identical, whereas the phylogenetic tree analysis revealed a slight difference. These two isolates were separated from the most recent common ancestor 0.74 years before they were isolated. The same resistance plasmid harboring blaIMP-19 was detected in metallo-ß-lactamase-producing A. xylosoxidans and K. pneumoniae Although this plasmid was not self-transferable, the conjugation of this plasmid from A. xylosoxidans to non-metallo-ß-lactamase-producing K. pneumoniae was successfully performed. The susceptibility patterns for metallo-ß-lactamase-producing K. pneumoniae and the transconjugant were similar. These findings supported the possibility of the horizontal transfer of plasmid-borne blaIMP-19 from A. xylosoxidans to K. pneumoniae in a single patient.

hCAS/CSE1L regulates RAD51 distribution and focus formation for homologous recombinational repair.

Homologous recombinational repair (HR) is one of the major repair systems for DNA double-strand breaks. RAD51 is a key molecule in HR, and the RAD51 concentration in the cell nucleus increases after DNA damage induction. However, the mechanism that regulates the intracellular distribution of RAD51 is still unclear. Here, we show that hCAS/CSE1L associates with RAD51 in human cells. We found that hCAS/CSE1L negatively regulates the nuclear protein level of RAD51 under normal conditions. hCAS/CSE1L is also required to repress the DNA damage-induced focus formation of RAD51. Moreover, we show that hCAS/CSE1L plays roles in the regulation of the HR activity and in chromosome stability. These findings suggest that hCAS/CSE1L is responsible for controlling the HR activity by directly interacting with RAD51.

Benzotriazole ultraviolet stabilizers show potent activities as human aryl hydrocarbon receptor ligands.

Benzotriazole ultraviolet stabilizers (BUVSs) used in consumer products are raising concerns as new pollutants in the aquatic environment. We determined the agonistic activities of eight BUVSs and a chemically distinct UV absorber (4-methylbenzylidinecamphor) toward the human aryl hydrocarbon receptor (AhR) and thyroid hormone receptors alpha and beta. Although none of the BUVSs showed ligand activity against the thyroid hormone receptors, four of them (UV-P, UV-9, UV-326, and UV-090) showed significant AhR ligand activity. Their half-maximal effective concentrations (EC50) were 130 nM for UV-P, 460 nM for UV-9, and 5.1 µM for UV-090 (a value for UV-326 could not be determined). Of the numerous AhR ligands, it is well-known that those considered nontoxic are quickly metabolized by enzymes such as CYP1A1, which destroys their ability to function as ligands. Accordingly, we established a new yeast assay for simultaneous monitoring of both the strength of AhR ligand activity and ligand degradation by CYP1A1. We found the AhR ligand activities of the above four BUVSs to be stable in the presence of CYP1A1; therefore, they have the potential to accumulate and exert potent physiological effects in humans, analogous to polycyclic aromatic hydrocarbons and dioxins, which are known stable and toxic ligands.

Characterization of Pathogenic Escherichia coli in River Water by Simultaneous Detection and Sequencing of 14 Virulence Genes.

The occurrence of pathogenic Escherichia coli in environmental waters increases the risk of waterborne disease. In this study, 14 virulence genes in 669 E. coli isolates (549 isolates from the Yamato River in Japan, and 30 isolates from each of the following hosts: humans, cows, pigs, and chickens) were simultaneously quantified by multiplex PCR and dual index sequencing to determine the prevalence of potentially pathogenic E. coli. Among the 549 environmental isolates, 64 (12%) were classified as extraintestinal pathogenic E. coli (ExPEC) while eight (1.5%) were classified as intestinal pathogenic E. coli (InPEC). Only ExPEC-associated genes were detected in human isolates and pig isolates, and 11 (37%) and five (17%) isolates were classified as ExPEC, respectively. A high proportion (63%) of cow isolates possessed Shiga-toxin genes (stx1 or stx2) and they were classified as Shiga toxin-producing E. coli (STEC) or enterohemorrhagic E. coli (EHEC). Among the chicken isolates, 14 (47%) possessed iutA, which is an ExPEC-associated gene. This method can determine the sequences as well as the presence/absence of virulence genes. By comparing the sequences of virulence genes, we determined that sequences of iutA were different among sources and may be useful for discriminating isolates, although further studies including larger numbers of isolates are needed. Results indicate that humans are a likely source of ExPEC strains in the river.