Publications by authors named "Yukiko Asano"

8 Publications

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Subtype screening of bla genes using bipartite primers for DNA sequencing.

Jpn J Infect Dis 2021 Mar 31. Epub 2021 Mar 31.

Department of Microbiology, Ehime Prefectural Institute of Public Health and Environmental Science, Japan.

Genes conferring carbapenem resistance have spread worldwide among gram-negative bacteria. Subtyping of these genes has epidemiological value due to the global cross-border movement of people. Subtyping of bla genes that frequently detected in Japan appears to be important in public health settings; however, there are few useful tools for this purpose. We developed a subtyping screening tool based on PCR direct sequencing, which targets the internal sequences of almost all bla genes. The tool used bipartite multiplex primers with M13 universal sequences at the 5'-end. According to in silico analysis, among the 78 known IMP-type genes, except for bla, 77 detected genes were estimated to be differentiated. In vitro evaluation indicated that sequences of amplicons of IMP-1, IMP-6, IMP-7, and IMP-20 templates were identical to their respective subtypes. Even if the amplicons were small or undetectable through the first PCR, sufficient amplicons for DNA sequencing were obtained through a second PCR using the M13 universal primers. In conclusion, our tool can be possibly used for subtype screening of bla, which is useful for the surveillance of bacteria with bla in clinical and public health settings or environmental fields.
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http://dx.doi.org/10.7883/yoken.JJID.2020.926DOI Listing
March 2021

Evaluating Methods for Detecting Escherichia albertii in Chicken Meat.

J Food Prot 2021 Apr;84(4):553-562

Division of Microbiology, National Institute of Health Sciences, 3-25-26, Tonomachi, Kawasaki-ku, Kawasaki, Kanagawa 210-9501, Japan.

Abstract: Escherichia albertii is an emerging foodborne pathogen. The source of the E. albertii infection in most foodborne outbreaks is unknown because E. albertii is difficult to isolate from suspected food or water. E. albertii has a broad host range among birds and can be isolated from chicken meat. In this study, PCR assay, enrichment, and isolation conditions for detecting E. albertii in chicken meat were evaluated. The growth of 47 E. albertii strains isolated in Japan between 1994 and 2018 and a type strain was evaluated in modified EC broth (mEC) and mEC supplemented with novobiocin (NmEC) and on media containing carbohydrates. The enzyme used for the nested PCR, the enrichment conditions, the most-probable-number (MPN) method, and agar media were also evaluated with chicken meat. To distinguish E. albertii from presumptive non-E. albertii bacteria, desoxycholate hydrogen sulfide lactose agar (DHL), MacConkey agar (MAC), and these agars supplemented with rhamnose and xylose (RX-DHL and RX-MAC, respectively) were used. All E. albertii strains grew in mEC and NmEC at both 36 and 42°C and did not utilize rhamnose, sucrose, or xylose. Both the first and nested PCRs with TaKaRa Ex Taq, which was 10 to 100 times more active than the other enzymes, produced positive results in enrichment culture of 25 g of chicken meat inoculated with >20 CFU of E. albertii and incubated in mEC and NmEC at 42°C for 22 ± 2 h. Thus, the first PCR was sensitive enough to detect E. albertii in chicken meat. The MPN values in mEC and NmEC were 0.5- and 2.3-fold higher than the original inoculated bacterial levels, respectively. E. albertii in chicken meat was more efficiently isolated with enrichment in NmEC (70.1 to 100%) and plating onto RX-DHL (85.4%) and RX-MAC (100%) compared with enrichment in mEC (53.5 to 83.3%) and plating onto DHL (70.1%) and MAC (92.4%). Thus, optimized conditions for the surveillance of E. albertii contamination in food and investigations of E. albertii outbreaks, including the infectious dose, were clarified.

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http://dx.doi.org/10.4315/JFP-20-206DOI Listing
April 2021

Correlation between the pronucleus size and the potential for human single pronucleus zygotes to develop into blastocysts : 1PN zygotes with large pronuclei can expect an embryo development to the blastocyst stage that is similar to the development of 2PN zygotes.

J Assist Reprod Genet 2018 May 26;35(5):817-823. Epub 2018 Feb 26.

Department of Infertility, Green Bell Clinic, Kitamachi 2-160, Toyota, Aichi, 471-0027, Japan.

Purpose: In this study, we examined the correlation between pronucleus size and the potential for human single pronucleus (1PN) zygotes to develop into blastocysts after IVF and ICSI.

Methods: This study included 112 patients who underwent a total of 112 cycles of IVF/ICSI. To evaluate embryo development, 1PN zygotes were compared with 2PN zygotes in the same IVF/ICSI cycle (control cycles) using time-lapse live embryo imaging. To assess the potential for blastocyst formation, cutoff values for pronuclear area and diameter were established through receiver operating characteristic curve analysis, after which 1PN zygotes were classified based on those cutoff values.

Results: Among 1PN zygotes cultured to day 5/6, the rate of embryo development was significantly lower than from 2PN zygotes. However, the rates of blastocyst formation and good quality blastocysts from 1PN zygotes with large pronuclear areas (≥ 710 μm) or diameters (≥ 31 μm) were significantly higher than from 1PN zygotes with smaller pronuclear areas (≤ 509, 510-609, and 610-709 μm) or diameters (≤ 24, 25-27,and 28-30 μm) (P < 0.01). Moreover, the results for 1PN zygotes with large pronuclei were similar to those for 2PN zygotes.

Conclusions: The developmental potential of 1PN zygotes with large pronuclear areas (≥ 710 μm) or diameters (31 μm) appears to be similar to that of 2PN zygotes, and measurement of pronuclear area or diameter in 1PN zygotes is a simple, potentially useful, clinical method.
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http://dx.doi.org/10.1007/s10815-018-1137-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5984884PMC
May 2018

Clinical outcomes after IVF or ICSI using human blastocysts derived from oocytes containing aggregates of smooth endoplasmic reticulum.

Reprod Biomed Online 2017 Apr 25;34(4):337-344. Epub 2017 Jan 25.

Department of Infertility, Angel Bell Hospital, Nishiki-machi 5-1, Okazaki, Aichi 444-0067, Japan.

In this study the clinical and neo-natal outcomes after transfer of blastocysts derived from oocytes containing aggregates of smooth endoplasmic reticulum (SER) were compared between IVF and intracytoplasmic sperm injection (ICSI) cycles. Clinical and neo-natal outcomes of blastocysts in cycles with at least one SER metaphase II oocyte (SER + MII; SER + cycles) did not significantly differ between the two insemination methods. When SER + MII were cultured to day 5/6, fertilization, embryo cleavage and blastocyst rates were not significantly different between IVF and ICSI cycles. In vitrified-warmed blastocyst transfer cycles, the clinical pregnancy rates from SER + MII in IVF and ICSI did not significantly differ. In this study, 52 blastocysts (27 IVF and 25 ICSI) derived from SER + MII were transferred, yielding 15 newborns (5 IVF and 10 ICSI) and no malformations. Moreover, 300 blastocysts (175 IVF and 125 ICSI) derived from SER-MII were transferred, yielding 55 newborns (24 IVF and 31 ICSI cycles). Thus, blastocysts derived from SER + cycles exhibited an acceptable ongoing pregnancy rate after IVF (n = 125) or ICSI (n = 117) cycles. In conclusion, blastocysts from SER + MII in both IVF and ICSI cycles yield adequate ongoing pregnancy rates with neo-natal outcomes that do not differ from SER-MII.
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http://dx.doi.org/10.1016/j.rbmo.2017.01.007DOI Listing
April 2017

Embryological outcomes in cycles with human oocytes containing large tubular smooth endoplasmic reticulum clusters after conventional in vitro fertilization.

Gynecol Endocrinol 2016 25;32(4):315-8. Epub 2015 Nov 25.

b Department of Infertility , Angel Bell Hospital , Okazaki , Japan .

There have been no studies analyzing the effect of large aggregates of tubular smooth endoplasmic reticulum (aSERT) after conventional in vitro fertilization (cIVF). The aim of this study was to investigate whether aSERT can be identified after cIVF and the association between the embryological outcomes of oocytes in cycles with aSERT. This is a retrospective study examining embryological data from cIVF cycles showing the presence of aSERT in oocytes 5-6 h after cIVF. To evaluate embryo quality, cIVF cycles with at least one aSERT-metaphase II (MII) oocyte observed (cycles with aSERT) were compared to cycles with normal-MII oocytes (control cycles). Among the 4098 MII oocytes observed in 579 cycles, aSERT was detected in 100 MII oocytes in 51 cycles (8.8%). The fertilization rate, the rate of embryo development on day 3 and day 5-6 did not significantly differ between cycles with aSERT and control group. However, aSERT-MII oocytes had lower rates for both blastocysts and good quality blastocysts (p < 0.05). aSERT can be detected in the cytoplasm by removing the cumulus cell 5 h after cIVF. However, aSERT-MII oocytes do not affect other normal-MII oocytes in cycles with aSERT.
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http://dx.doi.org/10.3109/09513590.2015.1115831DOI Listing
January 2017

Birth of nine normal healthy babies following transfer of blastocysts derived from human single-pronucleate zygotes.

J Assist Reprod Genet 2015 Sep 26;32(9):1401-7. Epub 2015 Jun 26.

Department of Infertility, Angel Bell Hospital, Nishiki-cho 5-1, Okazaki, Aichi, 444-0067, Japan.

Purpose: The purpose of this study is to examine the clinical outcomes of blastocysts derived from human single-pronucleate (1PN) embryos after conventional in vitro fertilization (cIVF) and intracytoplasmic sperm injection (ICSI) cycles.

Methods: This was a retrospective study at a reproductive center of a hospital. To evaluate embryo quality and clinical outcomes, cIVF or ICSI cycles with one or more 1PN embryos were compared with same cycles with 2PN embryos (control cycles).

Results: A total of 623 cycles (426 cIVF cycles and 197 ICSI cycles) were treated with cIVF or ICSI. The single pronuclear status rate was similar between cIVF (22.1 %) and ICSI (25.1 %) cycles. Although the development rates of 1PN embryos on day 3 and day 5/6 in cIVF were significantly higher than those in ICSI, those of 1PN embryos in cIVF were significantly lower compared to 2PN embryos (p < 0.01). Nonetheless, the ongoing pregnancy rates achieved with 1PN blastocysts in 1PN embryos did not significantly differ from the control group. Thirty-three transfer cycles with 33 blastocysts derived from 1PN embryos in cIVF resulted in nine deliveries with no newborn malformations; however, no implantation was observed in three ICSI cycles.

Conclusion: Although the blastocyst formation rate of 1PN embryos was significantly lower than 2PN embryos in cIVF and ICSI cycles, 1PN blastocysts in cIVF, and not from ICSI, demonstrated an adequate ongoing pregnancy rate. These results suggested that 1PN blastocysts in cIVF are available for clinical use and may lead to an increase in the chance of pregnancy in patients receiving assisted reproductive technology with 1PN embryos.
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http://dx.doi.org/10.1007/s10815-015-0518-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4595390PMC
September 2015

Phylogenetic Clades 6 and 8 of Enterohemorrhagic Escherichia coli O157:H7 With Particular stx Subtypes are More Frequently Found in Isolates From Hemolytic Uremic Syndrome Patients Than From Asymptomatic Carriers.

Open Forum Infect Dis 2014 Sep 12;1(2):ofu061. Epub 2014 Aug 12.

Department of Bacteriology I , National Institute of Infectious Diseases , Tokyo , Japan.

Background: Enterohemorrhagic Escherichia coli (EHEC) O157:H7 infection causes severe diseases such as bloody diarrhea and hemolytic uremic syndrome (HUS). Although EHEC O157:H7 strains have exhibited high genetic variability, their abilities to cause human diseases have not been fully examined.

Methods: Clade typing and stx subtyping of EHEC O157:H7 strains, which were isolated in Japan during 1999-2011 from 269 HUS patients and 387 asymptomatic carriers (ACs) and showed distinct pulsed-field gel electrophoresis patterns, were performed to determine relationships between specific lineages and clinical presentation.

Results: Clades 6 and 8 strains were more frequently found among the isolates from HUS cases than those from ACs (P = .00062 for clade 6, P < .0001 for clade 8). All clade 6 strains isolated from HUS patients harbored stx2a and/or stx2c, whereas all clade 8 strains harbored either stx2a or stx2a/stx2c. However, clade 7 strains were predominantly found among the AC isolates but less frequently found among the HUS isolates, suggesting a significant association between clade 7 and AC (P < .0001). Logistic regression analysis revealed that 0-9 year old age is a significant predictor of the association between clade 8 and HUS. We also found an intact norV gene, which encodes for a nitric oxide reductase that inhibits Shiga toxin activity under anaerobic condition, in all clades 1-3 isolates but not in clades 4-8 isolates.

Conclusions: Early detection of EHEC O157:H7 strains that belonged to clades 6/8 and harbored specific stx subtypes may be important for defining the risk of disease progression in EHEC-infected 0- to 9-year-old children.
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http://dx.doi.org/10.1093/ofid/ofu061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4281788PMC
September 2014

Characterization of the Escherichia coli O157:H7 outbreak strain whose Shiga toxin 2 gene is inactivated by IS1203v insertion.

Jpn J Infect Dis 2013 ;66(3):201-6

Ehime Prefectural Institute of Public Health and Environmental Science, Ehime, Japan.

A total of 12 enterohemorrhagic Escherichia coli (EHEC) O157:H7 strains were isolated during a recent outbreak in a nursery school in Ehime Prefecture, Japan. These isolates were considered to be derived from a common strain when analyzed using an IS-printing method and pulsed-field gel electrophoresis. PCR analysis revealed that the isolates harbor stx1, stx2, eae, and hlyA. However, assessment of the production of the Stx proteins revealed that these isolates produced Stx1 but not Stx2. We determined their stx2 variants such as stx2c and found that the size of the PCR product was much larger than the expected size. Sequencing of the entire stx2 gene revealed that a 1310-bp fragment was inserted into the coding region of the Stx2A subunit and that the sequences of the insert were identical to those of IS1203v. According to the sequences around the insertion site, additional amino acid residues should be attached at the C-terminus of the A subunit, which may hamper the Stx2 complex formation. Finally, this study also suggested that such an insertion may lead to the misinterpretation of results when screening EHEC isolates for virulence genes by PCR.
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http://dx.doi.org/10.7883/yoken.66.201DOI Listing
December 2013