Yutaka Miyazaki

Ability of Shiga Toxin-Producing Escherichia coli and Salmonella spp. To Survive in a Desiccation Model System and in Dry Foods

ABSTRACT In order to determine desiccation tolerances of bacterial strains, the survival of 58 diarrheagenic strains (18 salmonellae, 35 Shiga toxin-producing Escherichia coli [STEC], and 5 shigellae) and of 15 nonpathogenic E. coli strains was determined after drying at 35°C for 24 h in paper disks. At an inoculum level of 107 CFU/disk, most of the salmonellae (14/18) and the STEC strains (31/35) survived with a population of 103 to 104 CFU/disk, whereas all of the shigellae (5/5) and the majority of the nonpathogenic E. coli strains (9/15) did not survive (the population was decreased to less than the detection limit of 102 CFU/disk). After 22 to 24 months of subsequent storage at 4°C, all of the selected salmonellae (4/4) and most of the selected STEC strains (12/15) survived, keeping the original populations (103 to 104 CFU/disk). In contrast to the case for storage at 4°C, all of 15 selected strains (5 strains each of Salmonella spp., STEC O157, and STEC O26) died after 35 to 70 days of storage at 25°C and 35°C. The survival rates of all of these 15 strains in paper disks after the 24 h of drying were substantially increased (10 to 79 times) by the presence of sucrose (12% to 36%). All of these 15 desiccated strains in paper disks survived after exposure to 70°C for 5 h. The populations of these 15 strains inoculated in dried foods containing sucrose and/or fat (e.g., chocolate) were 100 times higher than those in the dried paper disks after drying for 24 h at 25°C.

Characterization of Shiga Toxin-Producing Escherichia coli O26 Strains and Establishment of Selective Isolation Media for These Strains

ABSTRACT We characterized the carbohydrate-fermenting ability of 31 strains of Shiga toxin-producing Escherichia coli (STEC) O26 isolated from diarrhea patients in Aichi Prefecture, Japan, in order to establish selective isolation media for these strains. None of the 31 STEC O26 strains (24 O26:H11, 7 O26:H−) fermented rhamnose, whereas all of the other 108 STEC strains (100 O157, 8 O111) and all of the non-STEC strains except one (i.e., 58 of 59) fermented rhamnose. The great majority of the STEC O26 strains (96.8% [30 of 31]) showed very high resistance to potassium tellurite (MIC ≥ 50 μg/ml), whereas the majority of the non-STEC strains (72.9% [43 of 59]) showed very high sensitivity (MIC ≤ 1.56 μg/ml) to this compound. Accordingly, we developed a rhamnose-MacConkey (RMAC) medium in which the lactose in MacConkey medium was replaced by rhamnose, and cefixime-tellurite-RMAC (CT-RMAC) medium in which potassium tellurite (2.5 mg/liter) and cefixime (0.05 mg/liter) were added to RMAC. All of the STEC O26 strains generated colorless (rhamnose-nonfermented) colonies on both media; the vast majority of selected E. coli strains (95.7% [89 of 93; including 26 STEC O157, 8 STEC O111]), other than STEC O26, generated red colonies on RMAC, and most of the non-STEC strains (84.7% [50 of 59]) did not grow on CT-RMAC. We demonstrate that both the RMAC and the CT-RMAC media can be used for the isolation of STEC O26 and that CT-RMAC has better specificity for the routine isolation for STEC O26 in a laboratory.

Continuous removal of middle molecules by hemofiltration in patients with acute liver failure.

In patients with acute liver failure and hepatic coma, an increase in the abnormal “middle molecules” seen on the chromatograms of the sera is suspected of playing an etiologic role in the coma. A pilot study of continuous hemofiltration using a high-performance membrane was conducted in 16 such patients in an attempt to decrease the serum levels of the middle molecules. The procedure was used alternately with plasma exchange. High-performance liquid chromatography showed a notable removal of the substances in the filtrates and a sequential removal from the serum by hemofiltration. Eight (50%) of the 16 patients had amelioration in level of consciousness and were weaned successfully from hemofiltration. Although only three of the 16 patients survived the acute illness, 13 others lived an average of 15 days and five patients survived >3 wk. While the continuous removal of middle molecules from the serum may not reverse liver failure, this procedure used in conjunction with plasma exchange may provide a means of life support, e.g., for patients awaiting a liver transplant.

Gnarled-Trunk Evolutionary Model of Influenza A Virus Hemagglutinin

Human influenza A viruses undergo antigenic changes with gradual accumulation of amino acid substitutions on the hemagglutinin (HA) molecule. A strong antigenic mismatch between vaccine and epidemic strains often requires the replacement of influenza vaccines worldwide. To establish a practical model enabling us to predict the future direction of the influenza virus evolution, relative distances of amino acid sequences among past epidemic strains were analyzed by multidimensional scaling (MDS). We found that human influenza viruses have evolved along a gnarled evolutionary pathway with an approximately constant curvature in the MDS-constructed 3D space. The gnarled pathway indicated that evolution on the trunk favored multiple substitutions at the same amino acid positions on HA. The constant curvature was reasonably explained by assuming that the rate of amino acid substitutions varied from one position to another according to a gamma distribution. Furthermore, we utilized the estimated parameters of the gamma distribution to predict the amino acid substitutions on HA in subsequent years. Retrospective prediction tests for 12 years from 1997 to 2009 showed that 70% of actual amino acid substitutions were correctly predicted, and that 45% of predicted amino acid substitutions have been actually observed. Although it remains unsolved how to predict the exact timing of antigenic changes, the present results suggest that our model may have the potential to recognize emerging epidemic strains.

Profiles of cytokines produced by CD4-positive T lymphocytes stimulated by anti-CD3 antibody in patients with chronic hepatitis C

Abstract: Helper T cells (Th) are classified as type 1 (Th1) and type 2 (Th2) according to the cytokines they produce; interferon-γ is produced by Th1, and interleukin-4 by Th2. We counted the circulating CD4-positive Th cells that produce interferon-γ or interleukin-4 with an enzyme-linked immunospot assay. CD4-positive T cells isolated from patients with chronic hepatitis B (n = 10), chronic hepatitis C (n = 16), and healthy subjects (n = 10) were stimulated with anti-CD3 antibody in vitro. The number of interferon-γ-producing Th cells was significantly lower in patients with chronic hepatitis C than in healthy subjects (P = 0.0024), whereas in patients with chronic hepatitis B, the number was similar to that in healthy subjects (P = 0.8530). The number of interleukin-4-producing Th cells was significantly higher in patients with chronic hepatitis C (P = 0.0010) and chronic hepatitis B (P = 0.0089) than in healthy subjects. In chronic hepatitis C, the number of interferon-γ-producing Th cells was increased after incubation of the cells with interferon-α (P = 0.008) or with recombinant interferon-γla (P = 0.024), but not with interferon-β (P = 0.051). The number of interleukin-4-producing Th cells was decreased after incubation with interferon-α (P = 0.0004), with interferon-β (P = 0.003), and with recombinant interferon-γla (P = 0.0004). Changes in the numbers of interferon-γ- or interleukin-4-producing Th cells in vitro were more evident in sustained responders to interferon therapy than in non-responders. These results suggest that Th2 cells are the predominant cell type in chronic hepatitis C, and that their activity may be suppressed by the administration of interferon.

1(OH) Vitamin D3 Supplementation Improves the Sensitivity of the Immune-Response during Peg-IFN/RBV Therapy in Chronic Hepatitis C Patients-Case Controlled Trial

Objective 1,25(OH)2 vitamin D3 can affect immune cells. However, the mechanism responsible for the favorable effects of 1(OH) vitamin D3, which becomes 1,25(OH)2 vitamin D3 in the liver, is not clear. The aim of this study is to analyze the immunological response of 1(OH) vitamin D3 supplementation in CH-C patients. Design Forty-two CH-C patients were treated with 1(OH) vitamin D3/Peg-IFNα/RBV. Forty-two case-matched controls were treated with Peg-IFNα/RBV. The expression of Interferon-stimulated genes (ISGs)-mRNA in the liver biopsy samples and JFH-1 replicating Huh-7 cells were quantified by real-time PCR. Ten kinds of cytokines in the plasma were quantified during treatment by using a suspension beads array. A trans-well co-culture system with peripheral blood mononuclear cells (PBMCs) and Huh-7 cells was used to analyze the effect of 1(OH) vitamin D3. The activities of the Th1 response were compared between subjects treated with 1(OH) vitamin D3/Peg-IFN/RBV and those treated with Peg-IFN/RBV therapy alone. Results 1(OH) vitamin D3/Peg-IFN/RBV treatment could induce rapid viral reduction, especially in IL28B T/T polymorphism. Several kinds of cytokines including IP-10 were significantly decreased after 4 weeks of 1(OH) vitamin D3 treatment (p<0.05). Th1 responses in the subjects treated with 1(OH) vitamin D3/Peg-IFN/RBV were significantly higher than those treated with Peg-IFN/RBV at 12 weeks after Peg-IFN/RBV therapy (p<0.05). The expression of ISGs in the patient’s liver biopsy samples was significantly lower than in those treated without 1(OH) vitamin D3 (p<0.05). Conclusion 1(OH) vitamin D3 could improve the sensitivity of Peg-IFN/RBV therapy on HCV-infected hepatocytes by reducing the IP-10 production from PBMCs and ISGs expression in the liver.

Relationship between the replication of hepatitis B virus and the localization of virus nucleocapsid antigen (HBcAg) in hepatocytes

According to the localization of hepatitis B virus (HBV) core antigen (HBcAg), detected by the avidin-biotin complex method, infected hepatocytes were classified into three types, i.e. those having nuclear (type I), nuclear and cytoplasmic (type II) or only cytoplasmic (type III) antigen. HBcAg-positive hepatocytes of all specimens (three) from non-specific reactive hepatitis and of most (five of seven) from chronic persistent hepatitis (CPH) patients were only type I; the other two CPH samples and all (seven) chronic active hepatitis samples were composed of a mixture of types I, II and III. Linear correlations between the frequency of type I, as well as that of of all types (I, II and III) of the HBcAg-positive hepatocytes, and the amount of HBV DNA in serum were found. The relative HBV production of HBcAg-positive hepatocytes (serum HBV DNA amount/frequency of HBcAg-positive cells) was 0.11 in type I and 0.07 in all hepatocytes including types I, II and III. HBV core particles and complete HBV particles were found in type I hepatocytes. On the other hand, these particles were not found in a predominantly type III liver specimen. These results suggest that type I hepatocytes are more involved in the propagation of HBV than types II and III.

愛知県における腸炎ビブリオO3: K6血清型による食中毒および散発性下痢症の発生動向 (1988～2001年) と海産魚介類からのO3: K6の検出

Percentage of the outbreaks by O3:K6 Vibrio parahaemolyticus (V. p) in Aichi Prefecture Japan increased from 3% (3/86) for 1988-95 to 75% (33/44) for 1996-2001. The percentage of the sporadic diarrhea cases caused by O3:K6 V. p in a general hospital in Aichi Prefecture also increased from 0% (0/253) to 61% (135/221) during the same periods. Thermostable direct hemolysin (TDH)-positive O3:K6 were isolated from 95% (19/20) of the outbreak incidents and 100% (135/135) of the sporadic cases. Only one TRH (TDH-related hemolysin)-positive O3:K6 was isolated from one outbreak incident. Percentage of the outbreaks by O3:K6 V. p associated with the consumption of boiled shellfishes increased from 5% (6/117) for 1988-95 to 25% (15/59) for 1996-2001, in particular, boiled crabs and squillas associated outbreaks increased from 2% (2/117) to 17% (10/59) and from 2% (2/117) to 10% (6/59), respectively. From 1,548 raw sea foods sampled in the Nagoya Central Wholesale Market in Aichi Prefecture in 1995-99, one TDH-positive O3:K6 was isolated from one live squilla (1/30). Increase in the percentage of outbreaks associated with TDH-positive O3:K6 V. p after 1996 in Aichi Prefecture was revealed to correlate with the increase in the outbreaks associated with consumption of boiled sea foods, especially boiled crabs as well as squillas. Accordingly, it becomes clear that sanitary handling of these boiled foods is important to prevent outbreaks and sporadic cases of diarrhea caused by O3:K6 V. p infection.