Shigeko Ueda

Detection of seg, seh, and sei genes in Staphylococcus aureus Isolates and Determination of the Enterotoxin Productivities of S. aureus Isolates Harboring seg, seh, or sei Genes

ABSTRACT To investigate the distribution of staphylococcal enterotoxin (SE) A to I (SEA to SEI) genes (sea to sei) in Staphylococcus aureus, 146 isolates obtained in Japan from humans involved in and samples from food poisoning outbreaks, healthy humans, cows with mastitis, and bovine raw milk were analyzed by multiplex PCR. One hundred thirteen (77.4%) S. aureus isolates were found to be positive for one or more se genes. The se genotype was classified into 14 genotypes. seg and sei coexisted in the same S. aureus strain. The newly developed sandwich enzyme-linked immunosorbent assay showed that most seh-harboring S. aureus isolates were able to produce a significant amount of SEH. However, most of the S. aureus isolates harboring seg and about 60% of the isolates harboring sei did not produce a detectable level of SEG or SEI, while reverse transcription-PCR analysis proved that the mRNAs of SEG and SEI were transcribed in S. aureus strains harboring seg and sei genes. These results suggest the importance of quantitative assessment of SEG and SEI production in foods in order to clarify the relationship between these new SEs and food poisoning.

Characterization of novel antimicrobial compounds from mango (Mangifera indica L.) kernel seeds

Abstract The antimicrobial properties of mango seed kernel ethanol extract (MKE) were investigated. Minimum inhibitory concentrations (MICs) of the MKE against 18 species, of 43 strains, containing food-borne pathogenic bacteria were determined using the agar dilution method. The MKE had a broad antimicrobial spectrum, and was more active against gram-positive than gram-negative bacteria with a few exceptions. The antimicrobial activity of the MKE was stable against heat (121°C, 15 min), freezing (−20°C, 16 h) and pH treatment (pH 3–9) normally used in food processing. Chemical analysis showed that the MKE was composed of 79.5% polyphenol and 21.7% carbohydrate. The MKE was separated by reverse-phase HPLC with 10–30% acetonitrile linear gradient to characterize antimicrobial compounds. There were two fractions having antimicrobial activity; both peaks had maximum absorbance at 275 nm. These results indicated that the active component of the MKE was a type of polyphenol.

A PCR assay based on a sequence-characterized amplified region marker for detection of emetic Bacillus cereus.

A PCR assay for the detection of Bacillus cereus strains able to produce an emetic toxin (cereulide) was developed in this study based on a sequence-characterized amplified region (SCAR) derived from a random amplified polymorphic DNA (RAPD) fragment. One of the RAPD fragments generated was selected, cloned, and sequenced. A set of PCR primers was newly designed from the SCAR obtained (the sequence of the cloned RAPD fragment) and used in this assay. To determine the specificity of the assay, 30 different B. cereus strains, 8 other Bacillus strains (of six species), and 16 other non-Bacillus strains (from 16 genera) were tested. Results were positive for every emetic B. cereus strain and for only one nonemetic B. cereus strain. For all other bacterial strains, results were negative. Bacterial DNA for PCR was prepared by a simple procedure using Chelex 100 resin from the bacterial colony on the agar plate or from culture after growth in brain heart infusion medium. This PCR assay enabled us to detect the bacteria of emetic B. cereus grown on agar plates but not the bacteria of nonemetic B. cereus. To test this PCR assay for the monitoring of the emetic bacteria, 10 to 70 CFU of B. cereus DSM 4312 (emetic) per g of food was inoculated into several foods as an indicator, followed by a 7-h enrichment culture step. Because this PCR assay based on the SCAR derived from the RAPD fragment was able to detect bacterial cells, this assay should be useful for rapid and specific detection of emetic B. cereus.

Occurrence of Cronobacter spp. in Dried Foods, Fresh Vegetables and Soil

　The present study surveyed the occurrence of Cronobacter spp. in dried foods including milk powder, spices and herbs and others, and fresh vegetables commercially available in markets, and ground soil materials for the agriculture. Cronobacter spp. were isolated from 15% of 33 spice and herb samples and 3% of 36 taste foods, and these were C. turicensis, C. malonaticus, C. sakazakii and C. dubliensis. Cronobacter spp. from fresh vegetables were detected in 12% of field vegetables and 13% of hydroponic vegetables. C. turicensis was prevalent in field vegetables, and C. malonaticus was in hydroponic ones. And, Cronobacter spp. in shredded vegetables were detected from 44% of 9 samples, and these were C. dubliensis, C. turicensis and C. sakazakii. Also, Cronobacter spp. in soil from rice field, vegetable field and sandpits were predominantly C. sakazakii and C. malonaticus.

Cytokine profile in a premature infant with systemic Bacillus cereus infection

Bacillus cereus is a large aerobic, Gram-positive, spore-forming rod that can be isolated from any substance in the environment, and has been identified as a cause of food-borne disease. B. cereus does not usually develop severe and systemic infection in immunocompetent hosts. In contrast, systemic infection can arise in immunocompromised hosts. Systemic infection of B. cereus has a high mortality rate, especially in premature infants. We encountered an extremely preterm infant with fulminant systemic B. cereus infection. As in previous reports, the present patient developed systemic B. cereus infection and died within 1 week of life. We investigated the serum cytokine profile in order to identify a defective immune response in the patient, but we found a strong response involving cytokine production in the patient. We report herein the bacteriological and immunological findings in the premature infant who developed systemic fulminant infection of B. cereus.

Identification of Cereulide-Producing Bacillus cereus by Nucleic Acid Chromatography and Reverse Transcription Real-Time PCR

RNA extracts were analyzed with a nucleic acid sequence-based amplification (NASBA) - nucleic acid chromatography and a reverse transcription-quantitative PCR assay (RT-qPCR) based on the TaqMan probe for identification of cereulide-producing Bacillus cereus. All 100 emetic B. cereus strains were found to give positive results, but 50 diarrheal B. cereus strains and other bacterial species showed negative results in the NASBA-chromatography. That is, the assay could selectively identify the emetic strains among B. cereus strains. Also, the B. cereus contents of more than 10(7) cfu/ml were required for the identification of the cereulide-producing strains in this assay. In qRT-PCR assays, all 100 emetic type strains of B. cereus produced 10(2) - 10(4) copy numbers per ng of the RNA preparation, and the strains produced 10(4) copies including ones which had the high vacuolation activities of HEp-2 cells.

The Effects of Temperature on the Growth and Heat Resistance of Cronobacter spp.

　The growth and survival of Cronobacter isolates were examined under incubation at different temperatures, and their thermal death behavior was investigated at high temperature conditions of above 50℃. Seventy three strains isolated from fresh vegetables, dried foods and soil were tested: 28 of Cronobacter sakazakii, 5 of C. dublienensis, 27 of C. malonaticus and 13 of C. turicensis. All Cronobacter strains grew and multiplied predominantly at 35 and 44℃ until 16 hours of incubation, but showed poor growth at 15℃, and no growth at 5℃. At 48℃, the bacteria grew slightly during 6 to 8 h-incubation but decreased or were inactivated after 16 h-incubation. The heat resistance of Cronobacter spp. was measured under the conditions of 50, 55, 60, 65 and 70℃. Cronobacter strains survived almost without decrement for 30 min at 50℃, but decreased suddenly and perished completely within 10 to 20 min at 55℃ and within 2 - 5 min at above 60℃. Some food materials should be stored below 5℃ until the preparation, and dried food including powdered infant milk formula should be utilized immediately after reconstitution and preparation.

Evaluation of the Immunochromatographic Device for the Detection of Verotoxins in Cultures of Food Materials

The immunochromatographic assay, which targets Shiga toxin 1/verotoxin 1 (VT1) and/or Shiga toxin 2/verotoxin 2 (VT2) independently with same test device, was used for easily, rapidly and specifically detecting verotoxin-producing Escherichia coli among E. coli strains from food and fecal materials. All 10 strains of VT 1 and/or VT 2- producing E. coli among E. coli isolates from various sources showed a positive reaction to VT1- or VT2- antibodies, but other gram-negative and positive bacterial species had a negative reaction. Bacterial counts of 10(8) cfu/ml in enrichment broth and food suspension were required for the detection of VT-producing E. coli. The IC assay described here could detect easily and specifically the verotoxin-producing E. coli within 20 min by pure culture.

Exposure of Workers to TPN during Application Using Different Spraying Methods.

アスパラガスに対してTPN (クロロタロニール: chlorothaloni1) 製剤を動力噴霧機による歩行散布とスピードスプレーヤにより散布した時の散布作業者の被曝状況を比較・検討した。その結果, 動力噴務機による散布者の身体各部位への農薬付着量は, 同一量の薬液をスピードスプレーヤにより散布した場合よりも推定全身被曝量は10倍以上多く, 散布作業時間も数倍必要とした。さらに, 散布後の圃場内のTPNの消長について測定し, 再入園時の農薬被曝の可能性について考察を行うために, スピードスプレーヤによる散布圃場内のTPN残留量を測定した。アスパラガス葉の農薬は, 散布1時間後から1日後にかけて1/3に急減したが, その後の減少は非常に緩やかであった。また, 圃場内の気中農薬はTPN濃度は, 散布4時間後には散布1時間後の1/3以下になり, 測定箇所によってはTPNは検出されなかった。このことから散布後1日以降の再入園にさいしては, 特に茎葉との接触を避けるように作業すべきものと考えられた。

Exposure of Spraying and Reentering Workers in Apple Orchard to Dichlorvos.

30aのリンゴ園にスピードスプレイヤーによりDDVP乳剤を有効成分として500g散布した時の散布作業者の農薬曝露状況を知るとともに, 散布後の圃場内への立入り者の農薬曝露状況を検討した。散布作業者の農薬被曝は頭部と上肢に多く認められ, 推定全身被曝量は散布量の0.0009%に相当する4.4mg (56μg/kg) であり, 適切な防除衣を着用して作業すればほとんど健康影響はないものと思われた。さらに, 散布終了1時間, 6時間, 1日, 2日および4日後に2名がこの圃場内に30分間立入り巡回した。その結果, 散布1時間後の再入園にさいしては全身的に若干の農薬曝露が認められ, それらの推定全身暴露量は105～120μg (1.8～2.3μg/kg) であった。しかし, 6時間以降の立入りにさいしては身体への曝露は認められなかった。再入園時の気中DDVP濃度は1時間後には平均13.9μg/m3であったが, 6時間後には9分の1程度に減少し, 1日後には検出されなかった。また, 葉面DDVP残留量は1時間後で331ng/cm2であったが, その減衰は速く, 6時間後には7分の1に減少し, 2日後には検出されなかった。これらのことから1時間後の再入園にさいしては, 若干の呼吸器曝露も示唆されるが, 主に葉との接触による曝露が重視される。DDVPは揮発しやすく, 分解されやすいことから, 6時間以降には接触によるDDVPの作業者への曝露は認められず, また呼吸器曝露もほとんどなくなるものと考えられた。以上のことから, DDVPに関しては散布1日後の圃場へ再入園してもほとんど安全上の問題はないものと考えられた。