Akira Hishinuma

Complete Sequencing of the blaNDM-1-Positive IncA/C Plasmid from Escherichia coli ST38 Isolate Suggests a Possible Origin from Plant Pathogens

The complete sequence of the plasmid pNDM-1_Dok01 carrying New Delhi metallo-β-lactamase (NDM-1) was determined by whole genome shotgun sequencing using Escherichia coli strain NDM-1_Dok01 (multilocus sequence typing type: ST38) and the transconjugant E. coli DH10B. The plasmid is an IncA/C incompatibility type composed of 225 predicted coding sequences in 195.5 kb and partially shares a sequence with bla CMY-2-positive IncA/C plasmids such as E. coli AR060302 pAR060302 (166.5 kb) and Salmonella enterica serovar Newport pSN254 (176.4 kb). The bla NDM-1 gene in pNDM-1_Dok01 is terminally flanked by two IS903 elements that are distinct from those of the other characterized NDM-1 plasmids, suggesting that the bla NDM-1 gene has been broadly transposed, together with various mobile elements, as a cassette gene. The chaperonin groES and groEL genes were identified in the bla NDM-1-related composite transposon, and phylogenetic analysis and guanine-cytosine content (GC) percentage showed similarities to the homologs of plant pathogens such as Pseudoxanthomonas and Xanthomonas spp., implying that plant pathogens are the potential source of the bla NDM-1 gene. The complete sequence of pNDM-1_Dok01 suggests that the bla NDM-1 gene was acquired by a novel composite transposon on an extensively disseminated IncA/C plasmid and transferred to the E. coli ST38 isolate.

First Case of New Delhi Metallo-β-Lactamase 1–Producing Escherichia coli Infection in Japan

To the Editor—Emergence of multidrug-resistant organisms is a threat to public health, because there is limited effort devoted to the development of new antibiotics [1]. Multiple new mechanisms of resistance have been described recently, including a new type of carbapenem resistance gene, blaNDM-1, reported in 2009 [2]. Infections due to strains with blaNDM-1 have been reported in India, Pakistan, the United Kingdom, and the United States [2–4]. We report the first case, to our knowledge, of New Delhi metallo-blactamase 1 (NDM-1)–producing Escherichia coli infection in Japan. A 54-year-old Japanese man traveled to India for business in March 2009. During his trip, he developed dysarthria, blurring of his left eye, and tingling in his right upper extremity. He was admitted to a local hospital in India on 25 March 2009 with a diagnosis of Guillain–Barre syndrome, and he subsequently developed bradycardia, hypotension, and respiratory failure. He was intubated and received mechanical ventilation. Once he was deemed stable enough to travel with mechanical ventilation, he was transferred to the intensive care unit at Dokkyo Medical University Hospital in Japan on 4 April 2009. One month after the transfer, the patient developed fever, and 2 sets of blood cultures showed 2 types of E. coli simultaneously. One isolate was an extended-spectrum b lactamase (ESBL)

A Novel Homozygous Missense Mutation of the Dual Oxidase 2 (DUOX2) Gene in an Adult Patient with Large Goiter

OBJECTIVE To describe the first adult case of large goiter associated with a novel R1110Q mutation in the dual oxidase 2 (DUOX2) gene. She was initially euthyroid, and developed hypothyroidism later in her forties. DUOX2 is an essential enzyme in iodine organification of thyroid hormone biosynthesis. Only infant cases of congenital hypothyroidism due to mutations of the DUOX2 gene have been reported. Biallelic mutation of DUOX2 is thought to lead to total iodine organification defect. PATIENTS AND MEASUREMENT: This 57-year-old woman became first aware of goiter around the age of 20 years. Since the goiter had enlarged gradually, she consulted us at the age of 32 years. Goiter was soft, and thyroid function was normal. Antithyroid antibodies were negative. Both physical and mental development was normal. Three of her nine siblings and her mother had large goiters. At the age of 44 years, thyroid function demonstrated subclinical hypothyroidism. She started to take levo-thyroxine at a dose of 100 mug/day to reduce goiter. At the age of 56 years, goiter size remained the same. The perchlorate discharge rate was 72.8%, suggesting partial iodine organification defect. Thus, thyroid peroxidase (TPO) gene and DUOX2 gene were analyzed. RESULTS There was no mutation in the TPO gene, but a novel homozygous mutation (R1110Q) in the DUOX2 gene was identified. The same heterozygous mutation was detected in her two sons and two grandchildren. This mutation was not detected in 104 control alleles and was located at a site differing from any other reported mutations in the DUOX2 gene. CONCLUSIONS This homozygous missense mutation can be associated with thyroid dysfunction and goiter formation of an enlarged thyroid gland.

Partial iodide organification defect caused by a novel mutation of the thyroid peroxidase gene in three siblings

background Three siblings with goitre and latent to mild hypothyroidism were suspected of having thyroid peroxidase (TPO) abnormality. Direct sequencing of their genomic DNAs showed two novel mutations of the TPO gene, one of which was G1687T (Gly533Cys; exon 9) and the other 1808–13del (Asp574/Leu575del; exon 10). The two mutations were compound heterozygous, as the former was found in their fathers DNA as heterozygous, and the latter was found in DNA from their mother, also as heterozygous. As Gly533 and Asp574/Leu575 were well‐conserved amino acids in the peroxidase superfamily, Gly533Cys‐ and Asp574/Leu575del‐TPOs were thought to be affected structurally or functionally. In expression studies using CHO‐Kl cells and mRNAs introduced with individual mutations, both mutated TPO proteins were expressed at the same molecular size as wild‐type TPO and had enzyme activity, although Gly533Cys‐TPO was slightly lower in efficiency of expression and more degenerative than wild‐type TPO.

A novel compound heterozygous mutation in the thyroglobulin gene resulting in congenital goitrous hypothyroidism with high serum triiodothyronine levels

AbstractThyroglobulin abnormality is a rare cause of congenital hypothyroidism and only a limited number of mutations in the thyroglobulin gene have been reported. We analyzed the thyroglobulin gene in a patient with congenital goitrous hypothyroidism. This girl was identified with hyperthyrotropinemia in a neonatal mass-screening test. The patient had goiter, and her body weight gain was poor. Distal femoral epiphysis was absent on roentgenography. Her serum thyroxine level was low; however, her triiodothyronine level was high. Autoantibodies against triiodothyronine, thyroid peroxidase, and thyroglobulin were all negative. Her serum thyroglobulin level was undetectable. The thyroglobulin gene from the genomic DNA of the patient was analyzed by direct sequencing. Two novel heterozygous missense mutations, Cys1897Tyr (exon 31) and Arg2336Gln (exon 40), were found in the patient. The former mutation was derived from her mother, suggesting a compound heterozygous state. Normal triiodothyronine and low thyroxine concentrations are often observed in patients with thyroglobulin gene mutations. We considered that some patients with thyroglobulin abnormality might have high triiodothyronine levels. In cases of congenital goitrous hypothyroidism with normal-to-high triiodothyronine levels and low serum thyroglobulin levels, thyroglobulin abnormality should be considered.

Genetic modification of cold-preserved renal grafts using HSP70 or bcl-2 HVJ-liposome method.

BACKGROUND We tested the hypothesis that the best time for genetic modification is while the cell viability of the graft is reduced for long-term preservation. The hemagglutinating virus of Japan (HVJ)-liposome method, a nonviral gene transfer technique, was used with a luciferase gene to test the efficacy of protein induction under the critical preservation time. Furthermore, we tested this genetic modification with heat shock protein (HSP) 70 or bcl-2 genes to prevent primary nonfunction (PNF) after long-term preservation. METHODS Orthotopic rat renal transplantation (RT) was performed using the cuff technique in the syngeneic combination of Lew (major histocompatible complex, haplotype: RT1(l)). Rat kidney grafts were preserved for 24 or 48 h in University of Wisconsin (UW) or Ringers lactate solution using HVJ method with the luciferase gene. Rats with gene-transfected kidneys were re-laparotomized 48 h after transplantation to estimate the lack of arterial flow in the graft and killed for histological evaluation of the degree of PNF luciferase intensity assay. Then, two functional genes (HSP70 or bcl-2) were tested for the occurrence of PNF and histological and immunohistochemical analysis of the grafted kidneys preserved for 48 h in the UW solution. RESULTS In the kidneys preserved for 24 h, 50% of the Ringers lactate group had PNF; but all of the UW group had sufficient blood flow. The graft viability was well corrected by the degree of luciferase intensity. The PNF rate was significantly suppressed in the bcl-2 gene-transfer group, and tended to be reduced in the HSP70 group. CONCLUSIONS The HVJ-liposome method effectively induced the foreign gene for kidney grafts even in the cold-preservation solution. Induction of bcl-2 or the HSP70 gene reduced the occurrence of PNF in the rat renal graft. The results suggest that gene transfer not only maintains graft viability, but also graft activation.

Complete sequencing of an IncFII NDM-1 plasmid in Klebsiella pneumoniae shows structural features shared with other multidrug resistance plasmids

Sir, Since the first report of New Delhi metallo-b-lactamase 1 (NDM-1) in 2009, it has spread worldwide in Europe, America, Africa, Asia andAustralia. ThefirstcasewasaSwedishpatientwhohadpreviously been treated in India. Retrospective analysis of carbapenemaseproducing Enterobacteriaceaetraced the blaNDM-1 geneto Escherichia coli collected from an Indian hospital in 2006. Recently, blaNDM-1producing bacteria were identified from the environment in New Delhi and Vietnam. Also, the blaNDM-1 gene has been found on a mobile plasmid in an Acinetobacter lwoffii isolate from chicken meat. We reported the first case of NDM-1-producing E. coli in Japan from a patient who had been treated in India in 2009. Subsequently, we determined the complete sequence of the IncA/C blaNDM-1positive plasmid (pNDM-1_Dok01), which suggested a possible origin from plant pathogens such as Pseudoxanthomonas and Xanthomonas spp. Here, we report another NDM-1 plasmid, in Klebsiella pneumoniae isolated from a second Japanese patient, who acquired the blaNDM-1-producing organism locally in Japan. From 15 September 2010 to 28 December 2010 the Ministry of Health, Labour and Welfare of the Japanese government conducted nationwide surveillance of multidrug-resistant Enterobacteriaceae that showed resistance to three classes of antibiotics: carbapenems, fluoroquinolones and aminoglycosides. A woman aged over 90 years, who resided in a nursing home and had not travelled overseas, was admitted to a local hospital due to aspiration pneumonia. Multidrug-resistant K. pneumoniae NDM-1-Saitama, which is susceptible only to aztreonam, was isolated from her urine, and subsequent PCR analysis revealed the presence of the blaNDM-1 gene. Multilocus sequence typing (http://www.pasteur.fr/ recherche/genotype/PF8/mlst/Kpneumoniae.html) indicated that NDM-1-Saitama belongs to sequence type ST42, which had not been reported previously. Complete sequencing of the plasmid from the clinical isolate K. pneumoniae NDM-1-Saitama by Illumina Hiseq (San Diego, USA) and gap closing by PCR demonstrated a 49441 bp IncFII plasmid (DDBJ/EMBL/GenBank accession number AB759690) (Figure 1 and Table S1, available as Supplementary data at JAC Online) that bears 53 genes predicted by EMBOSS GetORF software (http://emboss.sourceforge.net/). This plasmid did not possess any transfer operon and was not transferable by conjugation. Replicon typing showed IncFII (K1:A-:B-). The repA and repB genes were located from nucleotide 7776 to nucleotide 13111, which showed 98% identity with the IncFII (K1:A-:B-) pKPN4 plasmid in K. pneumoniae MGH 78578 (accession number CP000649). The plasmid pNDM-1-Saitama consists of a 22 kb backbone and a 27 kb multidrug resistance region. Half of the backbone (nucleotides 1–10815) showed a 100% perfect match with the sequence of K. pneumoniae plasmid pKP048 that contains blaKPC-2. 8 The second half of the backbone (nucleotides 10816–22344) showed only partial similarity to the plasmids containing blaNDM-1 (pNDM-OM, pNDM-HK, pNDM-CIT, pKOX_R1 and pMR0211) and blaCTX-M-3 (pCTX-M3) (Figure S1, available as Supplementary data at JAC Online). The multidrug resistance region of pNDM-1-Saitama (nucleotides 22345–49441) contains 13 drug resistance elements, including blaNDM-1, bleMBL, DblaDHA-1, sul1, armA, mel and mph2 (Figure 1). The complete sequence of an ISAba125 insertion sequence upstream of the blaNDM-1 gene suggests horizontal transfer of the blaNDM-1 gene between Enterobacteriaceae and Acinetobacter. The 235 sequences and 210 sequences also provide a promoter for NDM-1 expression. Interestingly, a part of the multidrug resistance region (nucleotides 30585–49333) was bracketed by insertion sequences ISAba125 and IS26 at both ends. This region is 100% identical to IncL/M plasmids pNMD-HK (accession number HQ451074) and pNDM-OM (accession number JX988621), except that the ISAba125 and IS26 insertion sequences are complete with inverted repeats in pNDM-1-Saitama. This composite transposon structure suggests that recombination events took place between two different transposons for the acquisition of these multidrug resistance genes. The blaNDM-1 gene followed by the bleomycin resistance protein gene bleMBL was acquired from transposon Tn125 with the ISAba125 insertion sequence, while truncated class C b-lactamase DblaDHA-1, dihydropteroate synthase sul1, 16S rRNA methylase armA, macrolide efflux protein mel and macrolide 2′-phophotransferase mph2 were provided by another transposon, Tn1548, with the IS26 insertion sequence. The blaNDM-1-bleMBL region is also found in another IncFII plasmid, pGUE-NDM, (accession number JQ364967) and IncN2 plasmids p271A (accession number JF785549) and pTR3 (accession number JQ349086). The DblaDHA-1-sul1-armA-mel-mph2 region was provided by the Tn1548 transposon found in KPC-bearing

Subclinical hypothyroidism caused by a mutation of the thyrotropin receptor gene

The propositus (a girl) was the first child of non-consanguineous Japanese parents. She was born at 39 weeks of gestation following an uncomplicated pregnancy and delivery, with a weight of 2682 g. She was referred because of a TSH level of 15 mU/L on neonatal screening at day 5 of life. The family history revealed no thyroid disease. At recall examination at day 46 of life, a serum TSH concentration of 12.0 mU/L in conjunction with normal serum concentrations of T 4 (10.4 μ g/dL) and T 3 (181 ng/dL) were detected. She did not have a goiter nor abnormal physical findings. Though her serum thyroid hormone levels were within normal limits, she was followed up for hyperthyrotropinemia. At the age of 3 years, an excessive TSH response to intravenous administration of thyrotropin releasing hormone (TRH) was considered diagnostic of congenital primary hypothyroidism, and subsequently, she was treated with levothyroxine (L-T 4 ). Figure 1 shows the clinical course of the propositus until 13 years of age. Before treatment with L-T 4 , the serum TSH level fluctuated between 10 and 30 mU/L. It declined to within normal limits after treatment with L-T 4 . The serum T 3 and T 4 levels were almost within normal limits throughout the course. At the age of 8 years, she underwent 123 I scintigraphy and TRH testing after the discontinuation of L-T 4 treatment. 123 I scintigraphy showed a normal-sized eutopic thyroid gland. Her 24 h 123 I uptake value was 17%. The perchlorate discharge test was negative. After the intravenous administration of 300 μ g/m 2 of TRH, her serum TSH concentration increased from 18.8 mU/L to a peak of 93.7 mU/L, while serum T 3 did not increase from the basal level of 111 ng/dL to 100 ng/dL at 120 min. This study revealed unresponsiveness to TSH. In addition, baseline and 120, and 180 min thyroglobulin levels after TRH administration were 19, 19 and 18 ng/mL, respectively. Consequently, L-T 4 therapy was reinstalled and has not been discontinued since then. During the following 20 years, the physical and intellectual development of the patient was normal.

TTF-2 stimulates expression of 17 genes, including one novel thyroid-specific gene which might be involved in thyroid development

Thyroid dysgenesis is the most frequent cause of congenital hypothyroidism, but its molecular pathophysiology is largely unknown. Our hypothesis that some genes downstream to thyroid transcription factor-2 (TTF-2) might be responsible for development of the thyroid prompted us to identify genes whose expression is stimulated by TTF-2. PCR products of cDNA clones obtained by a subtraction PCR method in TTF-2 expressing cell lines were screened with labeled cDNA by microarray analysis. We isolated 17 genes up-regulated by TTF-2, which were subsequently confirmed by quantitative reverse transcription-polymerase chain reaction (RT-PCR). One of them is a novel gene designated T1560 that showed a highly thyroid-specific expression pattern. Luciferase reporter assays showed that expression of all of the 14 genes tested was stimulated by both TTF-2 and TTF-1, another thyroid-specific transcription factor. Our results have important implications for understanding normal thyroid development as well as the molecular defects underlying thyroid dysgenesis.

Thyroglobulin Gene Mutation with Cold Nodule on Thyroid Scintigraphy

Thyroglobulin gene mutation is a rare cause of congenital hypothyroidism, but thyroglobulin gene mutations are thought to be associated with thyroid cancer development. A 21-year-old Japanese man treated with levothyroxine for congenital hypothyroidism had an enlarged thyroid gland with undetectable serum thyroglobulin despite elevated serum TSH level. The patient was diagnosed with thyroglobulin gene mutation, with compound heterozygosity for Gly304Cys missense mutation and Arg432X nonsense mutation. Ultrasonography showed a hypovascular large tumor in the left lobe that appeared as a cold nodule on thyroid scintigraphy. He underwent total thyroidectomy, but pathological study did not reveal findings of thyroid carcinoma, but rather a hyperplastic nodule with hemorrhage. Strong cytoplasmic thyroglobulin immunostaining was observed, but sodium iodide symporter immunostaining was hardly detected in the hyperplastic nodule. The clinical characteristics of patients with thyroglobulin gene mutations are diverse, and some patients are diagnosed by chance on examination of goiter in adults. The presence of thyroid tumors that appear as cold nodules on thyroid scintigraphy should consider the potential for thyroid carcinoma, if the patient has relatively low serum thyroglobulin concentration in relation to the degree of TSH without thyroglobulin autoantibody.