Investigation of Amino Acid and Fatty Acid Profiles of Japanese Diets Using the Food Exchange Lists for Diabetes Diet.

Dietary Reference Intakes for Japanese provide target values for proteins, fats, and carbohydrates. However, they do not provide information on reference values for amino acids (AAs) and fatty acids (FAs), which determine the quality of foods in detail. Therefore, we evaluated AAs and FAs using the Food Exchange Lists-Dietary Guidance for Persons with Diabetes (in Japanese) Utilization, Second Edition Sample Menus and Practice (FELD) as an ideal Japanese diet. Based on FELD, 15 different daily meal patterns were employed with combinations of three levels of carbohydrates %energy (high carbohydrate [HC], 60%; middle carbohydrate [MC], 55%; and low carbohydrate [LC], 50%) and five levels of energy (1,200-2,000 kcal). Using the Japanese Food Composition Table 2020 adjusted for 1,000 kcal, 18 AAs, 49 FAs, and calorie densities (CDs, kcal/g) were calculated and compared among the three groups. Dietary AA was rich in glutamic acid, aspartic acid, and leucine; in order, no significant differences were observed among HC, MC, and LC for 18 AAs. Dietary FA was higher for 18:1 total, 16:0, and 18:2 n-6. Moreover, 16:0, 20:0, and 18:1 total in LC and 22:0 and 18:3 n-3 in MC were significantly higher than those in HC. The HC, MC, and LC CD was low at 0.82, 0.84, and 0.93 kcal/g, respectively. No significant differences in 18 AAs and CD were noted among HC, MC, and LC in FELD; however, significant differences were observed in the FA profiles. This study suggests the importance of evaluating diet using AA and FA units.

IncHI2 Plasmid Encoding blaCTX-M-55 and mcr-1.1 in Salmonella enterica SE20-C72-2 and Escherichia coli EC20-C72-1 Isolates from the Edible River Fish Anabas testudineus.

Salmonella enterica SE20-C72-2 and Escherichia coli EC20-C72-1 were isolated from the edible fish Anabas testudineus in Vietnam. The chromosomes and plasmids from both strains were sequenced using Oxford Nanopore and Illumina sequencing. Plasmids approximately 250 kbp long, encoding blaCTX-M-55 and mcr-1.1, were detected in both strains.

Quantitative detection and genetic characterization of thermotolerant Campylobacter spp. in fresh chicken meats at retail in Japan.

Campylobacter jejuni and C. coli are one of the leading causes of gastrointestinal illnesses, and which are considered to be transmitted to humans mainly from chicken meats. Considering the less availability of quantitative contamination data in the retail chicken meats in Japan, 510 fresh chicken meats retailed at five distinct regions in Japan between June 2019 and March 2021 were examined. The quantitative testing resulted that 45.7% of the samples (254/510) were positive at mean ± standard deviation of 1.15 ± 1.03 logCFU/g, whereas 43 samples (8.4%) exceeded 3.0 logCFU/g. Seasonal comparison revealed increased bacterial counts in fall compared with spring and summer. As for the chicken slaughter age, those slaughtered at >75 days old were less contaminated than those at <75 days old. Genome sequencing analyses of 111 representative C. jejuni isolates resulted in the detection of three antimicrobial resistance genes (gyrA substitution T86I, tetO and blaOXA-61) at 25.2, 27.9 and 42.3%, respectively. In silico MLST analysis revealed the predominance of sequence types (ST)-21 clonal complex (CC), followed by ST-45CC and ST-464CC. The single nucleotide polymorphism (SNP)-based phylogenetic tree largely classified the sequenced C. jejuni isolates into two clusters (I and II), where all C. jejuni from highly contaminated samples (STs-21CC, -22CC and -45CC) belonged to cluster I, independent of both season and slaughter age. To our knowledge, this is the first example to study the current status of Campylobacter contamination levels in fresh chicken meats retailed in Japan. Our data would be contributable to future quantitative microbial risk assessment, to establish effective control measures for campylobacteriosis.

Draft Genome Sequences of Two Listeria monocytogenes Strains Isolated from Raccoon Feces in Japan.

Listeria monocytogenes serotype 4b strains RF01 and RF06 were isolated from raccoon feces in Japan. Here, we report the draft genome sequences of the two isolated strains; the genome sizes were 2,918,024 and 2,872,491 bp, with 535× and 510× coverage, for the RF01 and RF06 strains, respectively.

Prevalence and Characterization of Gentamicin Resistance Genes in Escherichia coli Isolates from Beef Cattle Feces in Japan.

Gentamicin is an important antibiotic for the treatment of opportunistic infections in the clinical field. Gentamicin-resistant bacteria have been detected in livestock animals and can be transmitted to humans through the food supply or direct contact. We have previously revealed that gentamicin-resistant Escherichia coli are distributed at a comparatively high rate from beef cattle in Japan, but few studies have focused on the molecular epidemiology of gentamicin-resistant bacteria. To understand these bacteria, this study examined the prevalence of various gentamicin resistance genes in gentamicin-resistant E. coli isolates from beef cattle feces. Of the 239 gentamicin-resistant E. coli isolates, the presence of the aacC2, aadB, or aac(3)-VIa genes was confirmed in 147, 84, and 8 isolates, respectively. All aac(3)-VIa-harboring isolates had an MIC value of 64 µg/mL for gentamicin and exhibited resistance to 11 antibiotic agents. An analysis of the representative aac(3)-VIa-harboring E. coli strain GC1-3-GR-4 revealed that the aac(3)-VIa gene was present on the IncA/C plasmid together with the aadA and blaCMY genes. Furthermore, the upstream region of the aac(3)-VIa gene contained the aadA gene and the class 1 integron-integrase gene (intI1). The aac(3)-VIa gene was detected for the first time in Japan and is expected to be able to transfer between bacteria via the IncA/C plasmid and integron. These results reveal the expansion of the distribution or diversity of gentamicin resistance genes in Japan.

Transcriptional regulation of FRZB in chondrocytes by Osterix and Msx2.

INTRODUCTION: Osteoarthritis is a common joint disease that causes destruction of articular cartilage and severe inflammation surrounding knee and hip joints. However, to date, effective therapeutic reagents for osteoarthritis have not been developed because the underlying molecular mechanisms are complex. Recent genetic findings suggest that a Wnt antagonist, frizzled-related protein B (FRZB), is a potential therapeutic target for osteoarthritis. Therefore, this study aimed to examine the transcriptional regulation of FRZB in chondrocytes. MATERIALS AND METHODS: Frzb/FRZB expression was assessed by RT-qPCR analyses in murine articular chondrocytes and SW1353 chondrocyte cell line. Overexpression and knockdown experiments were performed using adenovirus and lentivirus, respectively. Luciferase-reporter and chromatin immunoprecipitation assays were performed for determining transcriptional regulation. Protein-protein interaction was determined by co-immunoprecipitation analysis. RESULTS: Frzb was highly expressed in cartilages, especially within articular chondrocytes. Interleukin-1α markedly reduced Frzb expression in articular chondrocytes in association with cartilage destruction and increases in ADAM metallopeptidase with thrombospondin type 1 motif (Adamts) 4 and Adamts5 expression. Bone morphogenetic protein 2 (BMP2) increased FRZB expression in SW1353 cells through Smad signaling. Osterix and msh homeobox 2 (Msx2), both of which function as downstream transcription factors of BMP2, induced FRZB expression and upregulated its promoter activity. Co-immunoprecipitation results showed a physical interaction between Osterix and Msx2. Knockdown of either Osterix or Msx2 inhibited BMP2-dependent FRZB expression. Chromatin immunoprecipitation indicated a direct association of Osterix and Msx2 with the FRZB gene promoter. CONCLUSION: These results suggest that BMP2 regulates FRZB expression through Osterix and Msx2.

Regulatory Mechanisms of Prg4 and Gdf5 Expression in Articular Cartilage and Functions in Osteoarthritis.

Owing to the rapid aging of society, the numbers of patients with joint disease continue to increase. Accordingly, a large number of patients require appropriate treatment for osteoarthritis (OA), the most frequent bone and joint disease. Thought to be caused by the degeneration and destruction of articular cartilage following persistent and excessive mechanical stimulation of the joints, OA can significantly impair patient quality of life with symptoms such as knee pain, lower limb muscle weakness, or difficulty walking. Because articular cartilage has a low self-repair ability and an extremely low proliferative capacity, healing of damaged articular cartilage has not been achieved to date. The current pharmaceutical treatment of OA is limited to the slight alleviation of symptoms (e.g., local injection of hyaluronic acid or non-steroidal anti-inflammatory drugs); hence, the development of effective drugs and regenerative therapies for OA is highly desirable. This review article summarizes findings indicating that proteoglycan 4 (Prg4)/lubricin, which is specifically expressed in the superficial zone of articular cartilage and synovium, functions in a protective manner against OA, and covers the transcriptional regulation of Prg4 in articular chondrocytes. We also focused on growth differentiation factor 5 (Gdf5), which is specifically expressed on the surface layer of articular cartilage, particularly in the developmental stage, describing its regulatory mechanisms and functions in joint formation and OA pathogenesis. Because several genetic studies in humans and mice indicate the involvement of these genes in the maintenance of articular cartilage homeostasis and the presentation of OA, molecular targeting of Prg4 and Gdf5 is expected to provide new insights into the aetiology, pathogenesis, and potential treatment of OA.

Genome Sequence of Carbapenemase-Producing Enterobacter cloacae 0102-4P-1 Harboring the IncC-Type Plasmid with a Multidrug Resistance Site Encoding blaNDM-1, Isolated from Commercially Imported Shrimp.

A carbapenem-resistant Enterobacter cloacae 0102-4P-1 strain was isolated from commercially imported shrimp in Japan. Here, we present a draft genome sequence. The complete plasmid sequence was also determined by hybrid assembly sequencing using Oxford Nanopore and Illumina methods. The assembled whole genome and plasmid were 5,164,033 bp and 162,852 bp long, respectively.

Anorexia in a hemodialysis patient due to pneumatosis intestinalis: A case report.

We report a case of pneumatosis intestinalis (PI) in a hemodialysis patient who presented with anorexia and nausea. Anorexia with postprandial nausea can be caused by gastrointestinal diseases, with one of the rare causes being PI. PI may occur in hemodialysis patients, but it is rarely reported. We experienced a case of benign PI in a hemodialysis patient, for whom the conservative treatment with antibiotics improved the patient's clinical symptoms. In patients with PI, it is important to rule out potentially life-threatening complications, such as the presence of hepatic intraportal gas on CT scan.

Bacterial Distribution and Community Structure in Beef Cattle Liver and Bile at Slaughter.

ABSTRACT: In this study, the distribution of hygienic indicator bacteria in cattle livers and bile was examined at slaughterhouses. One hundred twenty-seven cattle livers with gallbladders were carefully eviscerated from carcasses at 10 slaughterhouses. Microbiological examination revealed that nine bile samples (7.1% prevalence) and 19 liver parenchyma samples (15.0% prevalence) were positive for Enterobacteriaceae (EB) with means ± standard deviations of 3.68 ± 4.63 log CFU/mL and 1.59 ± 2.47 log CFU/g, respectively; thus, bacterial contamination was apparent even at the postevisceration stage. Subsequently, 70 cattle livers were obtained at the postprocessing and storage stage from 7 of the 10 slaughterhouses. Microbiological analysis revealed significantly higher levels of EB in the liver parenchyma (3.00 ± 3.89 log CFU/g, P = 0.011) than those at the postevisceration stage, suggesting that bacterial dissemination and/or replication occurred in the liver parenchyma during processing and storage. According to 16S rRNA ion semiconductor sequencing analysis of representative samples from 12 cattle, Proteobacteria, Firmicutes, and Actinobacteria were dominant in both the parenchyma and bile in which EB and Escherichia coli were predominant among livers with higher EB levels. These results suggest that bile plays a role as a vehicle for bacterial transmission to the liver parenchyma. This study is the first to evaluate bacterial distribution and community structure in the liver and biliary microecosystem of cattle at slaughter. Our data support the use of EB testing of bile to screen cattle livers contaminated with high levels of fecal indicator bacteria.

Zfhx4 regulates endochondral ossification as the transcriptional platform of Osterix in mice.

Endochondral ossification is regulated by transcription factors that include SRY-box transcription factor 9, runt-related protein 2 (Runx2), and Osterix. However, the sequential and harmonious regulation of the multiple steps of endochondral ossification is unclear. This study identified zinc finger homeodomain 4 (Zfhx4) as a crucial transcriptional partner of Osterix. We found that Zfhx4 was highly expressed in cartilage and that Zfhx4 deficient mice had reduced expression of matrix metallopeptidase 13 and inhibited calcification of cartilage matrices. These phenotypes were very similar to impaired chondrogenesis in Osterix deficient mice. Coimmunoprecipitation and immunofluorescence indicated a physical interaction between Zfhx4 and Osterix. Notably, Zfhx4 and Osterix double mutant mice showed more severe phenotype than Zfhx4 deficient mice. Additionally, Zfhx4 interacted with Runx2 that functions upstream of Osterix. Our findings suggest that Zfhx4 coordinates the transcriptional network of Osterix and, consequently, endochondral ossification.

Long-Term Grow-Out Affects Campylobacter jejuni Colonization Fitness in Coincidence With Altered Microbiota and Lipid Composition in the Cecum of Laying Hens.

Campylobacter jejuni is one of the leading causes of gastrointestinal illness worldwide and is mainly transmitted from chicken through the food chain. Previous studies have provided increasing evidence that this pathogen can colonize and replicate in broiler chicken during its breeding; however, its temporal kinetics in laying hen are poorly understood. Considering the possible interaction between C. jejuni and gut microbiota, the current study was conducted to address the temporal dynamics of C. jejuni in the cecum of laying hen over 40 weeks, with possible alteration of the gut microbiota and fatty acid (FA) components. Following oral infection with C. jejuni 81-176, inocula were stably recovered from ceca for up to 8 weeks post-infection (p.i.). From 16 weeks p.i., most birds became negative for C. jejuni and remained negative up to 40 weeks p.i. 16S rRNA gene sequencing analyses revealed that most of the altered relative rRNA gene abundances occurred in the order Clostridiales, in which increased relative rRNA gene abundances were observed at >16 weeks p.i. in the families Clostridiaceae, Ruminococcaceae, Lachnospiraceae, and Peptococcaceae. Lipidome analyses revealed increased levels of sterols associated with bile acid metabolisms in the cecum at 16 and/or 24 weeks p.i. compared with those detected at 8 weeks p.i., suggesting that altered microbiota and bile acid metabolism might underlie the decreased colonization fitness of C. jejuni in the gut of laying hens.

Untargeted Phylogenetic Group III of Multi-drug-Resistant Bacillus cereus Isolated Using Fraser Medium from Retail Chickens in Ho Chi Minh City.

The prevalence of food-borne bacteria in developing countries is less well understood than in developed countries. The ISO11290-1 isolation method is commonly used to study Listeria contamination in chicken; however, all isolates are identified as untargeted Bacillus cereus. This study aimed to determine the classification, antibiotic susceptibility, and virulence genes of B. cereus isolated from retail chickens in Vietnam. Bacterial isolation using the ISO11290-1 method yielded 12 strains of B. cereus from seven out of 60 chickens. For determining bacterial diversity, panC and multilocus sequence typing (MLST) analyses were performed. PanC analysis showed that all seven strains belong to the phylogenetic group III, to which the highest risk of foodborne illnesses was associated. MLST analysis showed that most strains contained a ST205 complex; further, all strains were found to be resistant to ampicillin, ciprofloxacin, and tetracycline. Virulence genes were also investigated. ces, a cereulide-related gene, was detected in 50% of the isolated strains, followed by cytK, nheA, and hblA enterotoxins in 41.7%, 16.7%, and 25% of the strains, respectively. In conclusion, B. cereus may be erroneously detected when attempting to detect Listeria in food using the ISO11290-1 method. Further study of the prevalence of B. cereus in Vietnamese food is needed to improve food safety.

Effects of a surface prereacted glass-ionomer filler coating material on biofilm formation and inhibition of dentin demineralization.

OBJECTIVES: This study investigated the ability of a surface prereacted glass-ionomer (S-PRG) coating material to inhibit the biofilm formation and demineralization of dentin. METHODS AND MATERIALS: Dentin specimens were randomly divided into three groups: (1) no coating (control), (2) S-PRG filler-containing coat, and (3) a nonS-PRG filler-containing coat. Streptococcus mutans biofilms were grown on the dentin surfaces in a microcosm for 20 h. Then, the quantity of bacteria and water-insoluble glucan in the retained biofilm on the dentin surface were measured. Regarding demineralization inhibition test, specimens were demineralized for 5 days then sectioned into halves and observed under confocal laser scanning microscope (CLSM). One-way ANOVA and Tukey's HSD were used for statistical analysis. RESULTS: The estimated mean surface roughness for specimens in the S-PRG group was statistically significantly higher than the estimates for both the nonS-PRG and the control group specimens. The quantity of bacteria and water-insoluble glucan/mm2 revealed that the S-PRG group prevented biofilm formation and bacterial adhesion to the dentin surface compared with the control and nonS-PRG groups. The S-PRG group recorded the highest acid-resistance ability with no surface loss. CONCLUSION: Application of S-PRG barrier coat on dentin surfaces can inhibit biofilm formation as well as protecting the dentin surface against demineralization. CLINICAL SIGNIFICANCE: Coating material containing S-PRG fillers might be used for caries prevention, through inhibiting biofilm formation, enhancing mineralization, and reducing acidic attack by cariogenic bacteria.

Draft Genome Sequences of Non-H2S-Producing Strains of Salmonella enterica Serovars Infantis, Enteritidis, Berta, and Kiambu in Japan.

We report the draft genome sequences of six strains of Salmonella enterica serovars Berta, Enteritidis, Infantis, and Kiambu, isolated from humans or chicken meats in Osaka, Japan, that were negative for hydrogen sulfide production. Their genome sizes ranged from 4,460,389 to 4,933,483 bp, with 3 to 9 rRNAs and 64 to 73 tRNAs and with coverages of 95× to 159×.

Plasmid Sequences of Four Large Plasmids Carrying Antimicrobial Resistance Genes in Escherichia coli Strains Isolated from Beef Cattle in Japan.

Escherichia coli is a common reservoir for antimicrobial resistance genes that can be easily transformed to possess multidrug resistance through plasmid transfer. To understand multidrug resistance plasmids, we report the plasmid sequences of four large plasmids carrying a number of genes related to antimicrobial resistance that were found in E. coli strains isolated from beef cattle.

Draft Genome Sequence of Stenotrophomonas maltophilia CRB139-1, Isolated from Poultry Meat in Japan.

Stenotrophomonas maltophilia is a nosocomial pathogen that primarily causes respiratory infection in humans. This pathogen is widely distributed in the environment, including in foods. Here, we report the draft genome sequence of S. maltophilia strain CRB139-1, isolated from poultry meat in Japan. The genome size was 4,619,918 bp at 90× coverage.

Hoxa13 regulates expression of common Hox target genes involved in cartilage development to coordinate the expansion of the autopodal anlage.

To elucidate the role of Hox genes in limb cartilage development, we identified the target genes of HOXA11 and HOXA13 by ChIP-Seq. The ChIP DNA fragment contained evolutionarily conserved sequences and multiple highly conserved HOX binding sites. A substantial portion of the HOXA11 ChIP fragment overlapped with the HOXA13 ChIP fragment indicating that both factors share common targets. Deletion of the target regions neighboring Bmp2 or Tshz2 reduced their expression in the autopod suggesting that they function as the limb bud-specific enhancers. We identified the Hox downstream genes as exhibiting expression changes in the Hoxa13 knock out (KO) and Hoxd11-13 deletion double mutant (Hox13 dKO) autopod by Genechip analysis. The Hox downstream genes neighboring the ChIP fragment were defined as the direct targets of Hox. We analyzed the spatial expression pattern of the Hox target genes that encode two different categories of transcription factors during autopod development and Hox13dKO limb bud. (a) Bcl11a, encoding a repressor of cartilage differentiation, was expressed in the E11.5 autopod and was substantially reduced in the Hox13dKO. (b) The transcription factors Aff3, Bnc2, Nfib and Runx1t1 were expressed in the zeugopodal cartilage but not in the autopod due to the repressive or relatively weak transcriptional activity of Hox13 at E11.5. Interestingly, the expression of these genes was later observed in the autopodal cartilage at E12.5. These results indicate that Hox13 transiently suspends the cartilage differentiation in the autopodal anlage via multiple pathways until establishing the paddle-shaped structure required to generate five digits.

Phylogenetic Diversity and Antimicrobial Resistance of Campylobacter coli from Humans and Animals in Japan.

The phylogenetic diversity and antimicrobial resistance (AMR) of Campylobacter coli from humans and animals in Japan between 2008 and 2014 were investigated. A total of 338 foodborne campylobacterioses were reported in Osaka, and C. coli was isolated from 38 cases (11.2%). In the present study, 119 C. coli strains (42 from humans, 25 each from poultry, cattle, and swine, and 2 from wild mallard) were examined by multilocus sequence typing (MLST). MLST assigned 36 sequence types (STs), including 14 novel STs; all human strains and 91% of animal strains (70/77) were assigned to the ST-828 clonal complex. The predominant human ST was ST-860 (18/42, 43%), followed by ST-1068 (8/42, 19%); these STs were also predominant in poultry (ST-860, 9/25, 36%) and cattle (ST-1068, 18/25, 72%). ST-1562 was only predominant in swine (11/25, 44.0%). Swine strains showed the greatest resistance to erythromycin (EM; 92.0%), while EM resistance was only found in 2 out of the 42 human strains examined (4.8%). All EM-resistant swine strains (n=15) exhibited a common point mutation in the 23S rRNA sequence (A2085G), and the tetO gene was detected in 22 out of the 23 TET-resistant swine strains. A whole genome sequencing analysis of four representative swine ST-1562 strains revealed abundant AMR-associated gene clusters in their genomes, suggesting horizontal gene transfer events during host adaptation. This is the first study to demonstrate the phylogenetic diversity and AMR profiles of C. coli in Japan. The present results suggest that poultry and cattle are major reservoirs, improving our knowledge on the epidemiological and ecological traits of this pathogen.

Investigation of morphological changes for the discrimination of nucleated red blood cells and other leukocytes in Sysmex XN hematology analyzer scattergrams using transmission electron microscopy.

BACKGROUND: The WNR channel of the XN-Series automated hematology analyzer (Sysmex) counts white blood cells (WBCs) and simultaneously performs a differential counting of basophils and nucleated red blood cells (NRBCs). The detection process involves exposing the cells to WNR-specific reagents containing an acidic detergent and a fluorescent dye and measuring the intensity of the forward scattered light (FSC) and side fluorescence light (SFL). METHOD: We treated isolated peripheral WBCs and NRBCs with specific reagents and assessed the morphological changes in NRBCs and each leukocyte type using transmission electron microscopy (TEM). RESULTS: The results from a flow cytometer (FCM) showed that, after exposure to the reagents, basophils appeared on the highest FSC and SFL areas compared to other leukocytes on the WNR scattergram. Owing to the hemolysis of reticulocytes and erythrocytes, NRBCs that survived the reagent treatment could be distinguished by their lower intensity than those of the other leukocytes on the WNR scattergram. We investigated the significance of the relationship between the TEM and FCM results after the reagent treatment. CONCLUSION: We confirmed that the WNR channel differentiates the blood cells on the WNR scattergram based on differences in the amount of residual cytoplasm and nucleic acids.