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IncI1 Plasmid Associated with blaCTX-M-2 Transmission in ESBL producing E. coli Isolated from Healthy Thoroughbred Racehorse, Japan Reviewed

Eddy Sukmawinata, Ryoko Uemura, Wataru Sato, Shuya Mitoma, Takuya Kanda and Masuo Sueyoshi

Antibiotics 2020.4

Language：English Publishing type：Research paper (scientific journal)

DOI： 10.3390/antibiotics9020070

Multidrug-resistant esbl/ampc-producing klebsiella pneumoniae isolated from healthy thoroughbred racehorses in japan Reviewed

Sukmawinata E., Uemura R., Sato W., Htun M.T., Sueyoshi M.

Animals 10 ( 3 ) 2020.3

Language：English Publishing type：Research paper (scientific journal) Publisher：Animals

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. Extended-spectrum β-lactamase (ESBL)- and AmpC β-lactamase (AmpC)-producing Klebsiella spp. have become a major health problem, leading to treatment failure in humans and animals. This study aimed to evaluate the presence of ESBL/AmpC-producing Klebsiella spp. isolated from racehorses in Japan. Feces samples from 212 healthy Thoroughbred racehorses were collected from the Japan Racing Association Training Centers between March 2017 and August 2018. ESBL/AmpC-producing Klebsiella spp. were isolated using selective medium containing 1 μg/mL cefotaxime. All isolates were subjected to bacterial species identification (MALDI-TOF MS), antimicrobial susceptibility test (disk diffusion test), characterization of resistance genes (PCR), conjugation assay, and genetic relatedness (multilocus sequence typing/MLST). Twelve ESBL/AmpC-producing Klebsiella pneumoniae (ESBL/AmpC-KP) were isolated from 3.3% of horse samples. Antimicrobial resistance profiling for 17 antimicrobials showed all ESBL/AmpC-KP were multidrug-resistant (MDR). Only 1 isolate was confirmed as an ESBL producer (blaCTX-M-2-positive), whereas the other 11 isolates were plasmid-mediated AmpC (pAmpC) producers (blaCMY positive). On the basis of MLST analysis, the ESBL-KP isolate was identified as sequence type (ST)-133 and four different STs among AmpC-KP isolates, ST-145, ST-4830, ST-4831, and ST-4832, were found to share six of the seven loci constituting a single-locus variant. This is the first study to show K. pneumoniae carrying MDR pAmpC isolated from a racehorse.

DOI： 10.3390/ani10030369

Upregulation of PD-L1 Expression by Prostaglandin E<inf>2</inf> and the Enhancement of IFN-γ by Anti-PD-L1 Antibody Combined With a COX-2 Inhibitor in Mycoplasma bovis Infection Reviewed

Goto S., Konnai S., Hirano Y., Kohara J., Okagawa T., Maekawa N., Sajiki Y., Watari K., Minato E., Kobayashi A., Gondaira S., Higuchi H., Koiwa M., Tajima M., Taguchi E., Uemura R., Yamada S., Kaneko M.K., Kato Y., Yamamoto K., Toda M., Suzuki Y., Murata S., Ohashi K.

Frontiers in Veterinary Science 7 12 2020.2

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Frontiers in Veterinary Science

© Copyright © 2020 Goto, Konnai, Hirano, Kohara, Okagawa, Maekawa, Sajiki, Watari, Minato, Kobayashi, Gondaira, Higuchi, Koiwa, Tajima, Taguchi, Uemura, Yamada, Kaneko, Kato, Yamamoto, Toda, Suzuki, Murata and Ohashi. Bovine mycoplasmosis caused by Mycoplasma bovis results in pneumonia and mastitis in cattle. We previously demonstrated that the programmed death 1 (PD-1)/PD-ligand 1 (PD-L1) pathway is involved in immune dysfunction during M. bovis infection and that prostaglandin E2 (PGE2) suppressed immune responses and upregulated PD-L1 expression in Johne's disease, a bacterial infection in cattle. In this study, we investigated the role of PGE2 in immune dysfunction and the relationship between PGE2 and the PD-1/PD-L1 pathway in M. bovis infection. In vitro stimulation with M. bovis upregulated the expressions of PGE2 and PD-L1 presumably via Toll-like receptor 2 in bovine peripheral blood mononuclear cells (PBMCs). PGE2 levels of peripheral blood in infected cattle were significantly increased compared with those in uninfected cattle. Remarkably, plasma PGE2 levels were positively correlated with the proportions of PD-L1+ monocytes in M. bovis-infected cattle. Additionally, plasma PGE2 production in infected cattle was negatively correlated with M. bovis-specific interferon (IFN)-γ production from PBMCs. These results suggest that PGE2 could be one of the inducers of PD-L1 expression and could be involved in immunosuppression during M. bovis infection. In vitro blockade assays using anti-bovine PD-L1 antibody and a cyclooxygenase 2 inhibitor significantly upregulated the M. bovis-specific IFN-γ response. Our study findings might contribute to the development of novel therapeutic strategies for bovine mycoplasmosis that target PGE2 and the PD-1/PD-L1 pathway.

DOI： 10.3389/fvets.2020.00012

Clinical efficacy of the combined treatment of anti-PD-L1 rat-bovine chimeric antibody with a COX-2 inhibitor in calves infected with Mycoplasma bovis. Reviewed

Goto, S., Konnai, S., Hirano, Y., Kohara, J., Okagawa, T., Maekawa, N., Sajiki, Y., Watari, K., Minato, E., Kobayashi, A., Gondaira, S., Higuchi, H., Koiwa, M., Tajima, M., Taguchi, E., Ishida, M., Uemura, R., Yamada, S., Kaneko, M., Kato, Y., Yamamoto, K., Toda, M., Suzuki, Y., Murata, S., Ohashi, K.

Japanese Journal of Veterinary Research. 2020.2

Language：English Publishing type：Research paper (scientific journal)

Neuromedin U suppresses prolactin secretion via dopamine neurons of the arcuate nucleus. Reviewed

Nakahara K, Maruyama K, Ensho T, Mori K, Miyazato M, Kangawa K, Uemura R, Sakoda H, Nakazato M, Murakami N

Biochemical and biophysical research communications 521 ( 2 ) 521 - 526 2020.1

Language：English Publishing type：Research paper (scientific journal)

DOI： 10.1016/j.bbrc.2019.10.156

Assessment of reproductive and growth performance of pigs on commercial swine farms in southern Kyushu, Japan. Reviewed

Sasaki, Y., Kawabata, T., Nakatake, S., Kohigashi, T., Toya, R., Uemura, R., Sueyoshi, M.

Animal Science Journal 91 ( 1 ) e13492 2020.1

Language：English Publishing type：Research paper (scientific journal)

Different doses of tannin reflect a double-edged impact on broiler chicken immunity. Reviewed

Ramah A, Yasuda M, Ohashi Y, Urakawa M, Kida T, Yanagita T, Uemura R, Bakry HH, Abdelaleem NM, El-Shewy EA

Veterinary immunology and immunopathology 220 109991 2019.12

Language：English Publishing type：Research paper (scientific journal)

DOI： 10.1016/j.vetimm.2019.109991

Comparison of glucose tolerance between wild-type mice and mice with double knockout of neuromedin U and neuromedin S. Reviewed

Ensho T., Maruyama K., Qattali A., Yasuda M., Uemura R., Murakami N., Nakahara K.

Journal of Veterinary Medical Science 81 ( 9 ) 1305 - 1312 2019.9

Language：English Publishing type：Research paper (scientific journal) Publisher：公益社団法人日本獣医学会

Recently, it has been proposed that neuromedin U (NMU) is "decretin", which suppresses insulin secretion from the pancreas in vitro. Here we examined the possible involvement of NMU in insulin secretion in vivo by comparing the plasma glucose and insulin levels of wild-type mice with those of double knockout (D-KO) of the NMU and neuromedin S (NMS) genes, as NMS binds to the neuromedin U receptor. If NMU is, in fact, "decretin", which inhibits insulin secretion from the pancreas, then NMU-deficient mice might result in higher plasma insulin levels than is the case in wild-type mice, or injection of NMU lead to suppression of plasma insulin level. In this study, we found that the fasting plasma level of insulin was not increased in D-KO mice. Glucose tolerance tests revealed no significant difference in plasma insulin levels between wild-type mice and D-KO mice under non-fasting conditions. After peripheral injection of NMU, plasma glucose and insulin levels did not show any significant changes in either wild-type or D-KO mice. Glucose tolerance testing after 3 weeks of high fat feeding revealed no significant difference in plasma insulin levels during 60 min after glucose injection between wild-type and D-KO mice. These results suggest that even if NMU is a decretin candidate, its physiological involvement in suppression of insulin secretion may be very minor in vivo.

DOI： 10.1292/jvms.19-0320

Total serum protein reference value as a clinical diagnostic index of equine proliferative enteropathy Reviewed

Ueno Y., Uemura R., Niwa H., Higuchi T., Sekiguchi S., Sasaki Y., Sueyoshi M.

Journal of Equine Science 30 ( 3 ) 63 - 67 2019.9

Language：English Publishing type：Research paper (scientific journal) Publisher：Journal of Equine Science

© 2019 Japanese Society of Equine Science. Equine proliferative enteropathy (EPE) caused by Lawsonia intracellularis is characterized by hypoproteinemia. There are currently no reliable reports that provide a reference value for the total serum protein (TP) concentration to clinically diagnose EPE. The objective of this study was to statistically determine the reference value. Feces and sera of 99 foals with EPE-like clinical signs and of 35 healthy foals were obtained. The samples were used for specific-gene detection of L. intracellularis, TP measurement, and specific-antibody detection against L. intracellularis. Based on these results, the optimal reference value for the TP concentration as a clinical diagnostic index of EPE was found to be ≤ 4.8 g/dl. This clinical diagnostic index will provide an effective approach for diagnosing EPE.

DOI： 10.1294/jes.30.63

Extended-spectrum β-lactamase-producing Escherichia coli isolated from healthy thoroughbred racehorses in Japan Reviewed

Sukmawinata E., Sato W., Mitoma S., Kanda T., Kusano K., Kambayashi Y., Sato T., Ishikawa Y., Goto Y., Uemura R., Sueyoshi M.

Journal of Equine Science 30 ( 3 ) 47 - 53 2019.9

Language：English Publishing type：Research paper (scientific journal) Publisher：Journal of Equine Science

© 2019 Japanese Society of Equine Science. Extended-spectrum β-lactamase-producing Escherichia coli (ESBLEC) have become a major health concern in both human and veterinary medicine. These bacteria could become a critical problem in equine medicine due to the limited number of antimicrobial drugs available. However, there are no previous reports of ESBLEC isolated from horses in Japan. The objectives of this study were to investigate the occurrence of ESBLEC isolated from feces in healthy Thoroughbred racehorses in Japan. Feces samples were collected from 147 healthy Thoroughbred racehorses by equine veterinarians at the Japan Racing Association (103 from Miho Training Center and 44 from Ritto Training Center) between March 2017 and April 2018. Samples were screened for ESBLECs using MacConkey agar supplemented with 1 µg/ml cefotaxime. Detection of ESBL genes was performed by PCR and confirmed by DNA sequencing. Horizontal transmission was demonstrated by conjugation assay. In this study, 24 ESBLECs were isolated from twelve horse feces samples (8.2%). All ESBLECs harbored blaCTX-M-2, and both blaTEM-1 and blaCTX-M-2 were detected in nine isolates (37.5%). ESBLECs showed resistance to all β-lactam antibiotics (100%) tested, followed by trimethoprim (66.7%), streptomycin (62.5%), tetracycline (25.0%), and oxytetracycline (25.0%). Horizontal transmission was successfully demonstrated by conjugation assay in eight of 13 isolates, and blaCTX-M-2 was detected by PCR in all transconjugants. This study showed that racehorses in Japan are potential reservoirs of ESBLECs.

DOI： 10.1294/jes.30.47

Bovine Endocarditis Associated with Mycoplasma bovis Reviewed

Kanda T., Tanaka S., Suwanruengsri M., Sukmawinata E., Uemura R., Yamaguchi R., Sueyoshi M.

Journal of Comparative Pathology 171 53 - 58 2019.8

Language：English Publishing type：Research paper (scientific journal) Publisher：Journal of Comparative Pathology

© 2019 Elsevier Ltd Mycoplasma bovis is a microorganism associated with pneumonia, mastitis, arthritis and otitis media of cattle; however, there are no reports of this organism causing bovine endocarditis. Five adult cattle with endocarditis characterized by caseated lesions (diameter 5–12 cm) of the endocardial surface of the left atrium, but without lesions in heart valves or affecting the right side of the heart, were identified in slaughterhouses in Japan. M. bovis was successfully isolated from the lesions and M. bovis antigen was detected immunohistochemically within the lesions. The results suggest that the lesions may have been associated with M. bovis alone. To our knowledge, this is the first demonstration of bovine endocarditis associated with M. bovis.

DOI： 10.1016/j.jcpa.2019.07.003

A FIELD STUDY ON ANTIBIOTIC-RESISTANT ESCHERICHIA COLI ISOLATED FROM LIVESTOCK AND SMALL WILD ANIMAL IN A STOCK FARM Reviewed

HIROKI Hayate, KUROYANAGI Aakira, KOBAYASHI Ikuo, UEMURA Ryoko, NUKAZAWA Kei, SUZUKI Yoshihiro

Journal of Japan Society of Civil Engineers, Ser. G (Environmental Research) 74 ( 7 ) III_231 - III_238 2018.12

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Japan Society of Civil Engineers

Livestock that have been administered an antibiotic treatment are highly likely to carry antibiotic-resistant bacteria. Because small animals (mice) live alongside livestock in stock farms and consume their feed and feces, it is possible that these antibiotic-resistant bacteria are transmitted to them. In this study, cattle and mice feces collected from the Sumiyoshi Livestock Science Station, University of Miyazaki, Japan, were tested for the presence of antibiotic-resistant Escherichica coli. Susceptibility of the E. coli isolates from the fecal samples to 11 antibiotics was evaluated using the disk diffusion method. The resistance profiles of E. coli isolates to various antibiotics were evaluated and compared between cattle and mice feces. Antibiotic-resistant E. coli were detected in 50% of cattle (5/10 animals) and 41% of mice (7/17 animals). In addition, all isolates from both cattle and mice feces had high detection rates of antibiotic resistance for ampicillin and tetracycline, and one isolate from the mice feces contained multidrug-resistant E. coli. Examination of the distribution of mice with antibiotic-resistant E. coli suggested that these bacteria are transmitted by mice and can spread to the stock farm.

DOI： 10.2208/jscejer.74.III_231

Antimicrobial Resistant Enterococcus faecium, Enterococcus faecalis, and Other Enterococcus Species Isolated From Foal Feces in Japan Reviewed

Sukmawinata E., Sato W., Uemura R., Sueyoshi M.

Journal of Equine Veterinary Science 63 51 - 54 2018.4

Language：English Publishing type：Research paper (scientific journal) Publisher：Journal of Equine Veterinary Science

© 2018 Elsevier Inc. Antimicrobial resistant (AMR) Enterococci are an emerging problem for human and animal health. Few studies have focused on the detection of AMR in foals; therefore, we conducted this study to investigate the occurrence of AMR Enterococci in foal feces in Japan. A total of 72 fecal samples from healthy foals were collected from seven horse stables, in Hokkaido Prefecture, from June 2015 to January 2016. Enterococci were isolated and identified using EF Agar Base selective media, and the species confirmed using MALDI Biotyper systems. The minimum inhibitory concentration of nine antimicrobial agents for the isolates was determined based on the Clinical Laboratory Standard Institute guidelines. A total of 183 isolates of Enterococci were identified as Enterococcus faecium 54.1% (99/183), Enterococcus faecalis 16.4% (30/183), and other species 29.5% (54/183). The highest occurrence of E. faecium antibiotic resistant was to erythromycin at 55.6% (55/99), followed by enrofloxacin, kanamycin, and oxytetracycline, at 30.3%, 7.1%, and 4.0%, respectively. In contrast, E. faecalis isolates showed higher resistant to oxytetracycline 76.7%, kanamycin 46.7%, gentamycin 30.0%, chloramphenicol 16.7%, lincomycin 30.0%, and tylosin 30.0%. E. faecium highly resistant to erythromycin and enrofloxacin but lowly resistant to gentamycin and tylosin, and E. faecalis was highly resistant to kanamycin, oxytetracycline, and lincomycin. Antimicrobial resistant E. faecalis (30.0%) and E. faecium (4.0%) isolates show multidrug resistant to at least three different classes of antibiotics. Our study indicated that foal feces may act as a source of AMR genes for Enterococci that may be transmitted to other animals, humans, and the environment.

DOI： 10.1016/j.jevs.2018.01.005

Effects of zinc supplementation on Shiga toxin 2e-producing Escherichia coli in vitro. Reviewed

Uemura, R., Katsuge T., Sasaki, Y., Goto, S., Sueyoshi, M.

Journal of Veterinary Medical Science 79 ( 10 ) 1637 - 1643 2017.10

Language：English Publishing type：Research paper (scientific journal) Publisher：公益社団法人日本獣医学会

Swine edema disease is caused by Shiga toxin (Stx) 2e–producing Escherichia coli (STEC). Addition of highly concentrated zinc formulations to feed has been used to treat and prevent the disease, but the mechanism of the beneficial effect is unknown. The purpose of the present study was to investigate the effects of highly concentrated zinc formulations on bacterial growth, hemolysin production, and an Stx2e release by STEC in vitro. STEC strain MVH269 isolated from a piglet with edema disease was cultured with zinc oxide (ZnO) or with zinc carbonate (ZnCO3), each at up to 3,000 ppm. There was no effect of zinc addition on bacterial growth. Nonetheless, the cytotoxic activity of Stx2e released into the supernatant was significantly attenuated in the zinc-supplemented media compared to that in the control, with the 50% cytotoxic dose values of 163.2 ± 12.7, 211.6 ± 33.1 and 659.9 ± 84.2 after 24 hr of growth in the presence of ZnO, ZnCO3, or no supplemental zinc, respectively. The hemolytic zones around colonies grown on sheep blood agar supplemented with zinc were significantly smaller than those of colonies grown on control agar. Similarly, hemoglobin absorbance after exposure to the supernatants of STEC cultures incubated in sheep blood broth supplemented with zinc was significantly lower than that resulting from exposure to the control supernatant. These in vitro findings indicated that zinc formulations directly impair the factors associated with the virulence of STEC, suggesting a mechanism by which zinc supplementation prevents swine edema disease.

DOI： 10.1292/jvms.16-0471

Assessment of the Campylobacter jejuni and C. coli in broiler chicken ceca by conventional culture and loop-mediated isothermal amplification method. Reviewed

Sabike, I., Uemura, R., Kirino, Y., Mekata, H., Sekiguchi, S., Farid, A., Goto, Y., Horii, Y., Yamazaki, W.

Food Control 74 107 - 111 2017.4

Language：English Publishing type：Research paper (scientific journal) Publisher：Food Control

© 2016 Elsevier Ltd In a two-year survey of the 24 Japanese broiler chicken flocks at 9 farms from 2013 to 2014, C. jejuni/C. coli prevalence was assessed in a total of 131 slaughtered broiler chicken cecal samples by conventional culture methods and loop-mediated isothermal amplification (LAMP) assay. While 93 samples were C. jejuni/C. coli-negative, 38 (29.0%) showed Campylobacter loads of between 6.4 and 9.0 log CFU/g of ceca in conventional culture methods. The performance of LAMP assay was 100% accurate in terms of diagnostic sensitivity (38/38), specificity (93/93). Furthermore, LAMP assay enabled direct screening of C. jejuni and C. coli in cecal samples from broiler chicken chickens as rapid and cost-effective detection within 90 min and less than 1 US dollar, which can help monitor release of Campylobacter-contaminated chicken into the food chain, thereby reducing the incidence and public health risk of campylobacteriosis. Seasonal changes in C. jejuni and C. coli prevalence in broiler chicken ceca were significantly correlated with the frequency of food poisoning incidents caused by these bacteria in Japan.

DOI： 10.1016/j.foodcont.2016.11.037

Use of direct LAMP screening of broiler fecal samples for Campylobacter jejuni and Campylobacter coli in the positive flock identification strategy. Reviewed

Sabike, I., Uemura, R., Kirino, Y., Mekata, H., Sekiguchi, S., Okabayashi, T., Goto, Y., Yamazaki, W.

Frontiers in Microbiology 7 1582 2016.9

Language：English Publishing type：Research paper (scientific journal) Publisher：Frontiers in Microbiology

© 2016 Sabike, Uemura, Kirino, Mekata, Sekiguchi, Okabayashi, Goto and Yamazaki. Rapid identification of Campylobacter-positive flocks before slaughter, following freezing and heat treatment for the Campylobacter-positive carcasses at the slaughterhouses is an effective control strategy against foodborne campylobacteriosis. We evaluated a loop-mediated isothermal amplification (LAMP) assay for the direct screening of naturally contaminated chicken cloacal swabs for C. jejuni/C. coli to compare this assay with conventional qu antitative culture methods. In a comparison study of 165 broilers, the LAMP assay showed 82.8% (48/58 by conventional culture) sensitivity, 100% (107/107) specificity, 100% (48/48) positive predictive value (PPV), and 91.5% (107/117) negative predictive value (NPV). In a comparison of 55 flocks, LAMP showed 90.5% (19/21) sensitivity, 100% (34/34) specificity, 100% (19/19) PPV, and 94.4% (34/36) NPV. In the cumulative total of 28 farm-level comparisons, LAMP showed 100% (12/12) sensitivity, 100% (16/16) specificity, 100% (12/12) PPV, and 100% (16/16) NPV. The LAMP assay required less than 90 min from the arrival of the fecal samples to final results in the laboratory. This suggests that the LAMP assay will facilitate the identification of C. jejuni/C. coli-positive broiler flocks at the farm level or in slaughterhouses before slaughtering, which would make it an effective tool in preventing the spread of Campylobacter contamination.

DOI： 10.3389/fmicb.2016.01582