ĐẠI HỌC QUỐC GIA HÀ NỘI TRƢỜNG ĐẠI HỌC KHOA HỌC TƢ̣ NHIÊN - DƢƠNG THU HƢƠNG SỰ HIỆN DIỆN CỦA CÁC VI KHUẨN KỊ KHÍ Ở CÁC BỆNH NHÂN BỊ CHẢY MÁU DẠ DÀY Ở VIỆT NAM LUẬN VĂN THẠC SĨ SINH HỌC Hà Nội - 2014 ĐẠI HỌC QUỐC GIA HÀ NỘI TRƢỜNG ĐẠI HỌC KHOA HỌC TƢ̣ NHIÊN DƢƠNG THU HƢƠNG SỰ HIỆN DIỆN CỦA CÁC VI KHUẨN KỊ KHÍ Ở CÁC BỆNH NHÂN BỊ CHẢY MÁU DẠ DÀY Ở VIỆT NAM Chuyên ngành: Sinh học thực nghiệm Mã số: 60 42 30 LUẬN VĂN THẠC SĨ SINH HỌC Ngƣời hƣớng dẫn khoa học: PGS.TS NGUYỄN THỊ HỒNG HẠNH Hà Nội - 2014 LỜI CẢM ƠN Để hoàn thành chƣơng trình cao học viết luận văn này, nỗ lực học tập, nghiên cứu, tìm tịi thân, tơi đã nhận đƣợc hƣớng dẫn, góp ý nhiệt tình Q thầy cô, ngƣời làm công tác khoa học, nhƣ bạn bè, đồng nghiệp, ngƣời thân Trƣớc tiên, tơi xin bày tỏ lịng kính trọng biết ơn sâu sắc tới PGS.TS Nguyễn Thị Hồng Hạnh – ngƣời thầy đã tận tâm, dành nhiều thời gian tâm huyết nhiệt tình bảo, hƣớng dẫn tơi suốt trình thực luận văn thạc sĩ này Qua đây, xin chân thành cảm ơn PGS.TS Guillermo I Perez-Perez, trƣờng Đại học Tổng hợp New York nhiệt tình hỗ trợ ơng suốt thời gian thực nghiên cứu Mỹ Xin chân thành cảm ơn Ban Giám hiệu trƣờng Đại học Khoa học tự nhiên, Phòng Sau đại học, thầy Khoa Sinh học đã tận tình truyền dạy cho suốt thời gian học tập trƣờng Xin chân thành cảm ơn cô chú, anh chị em đồng nghiệp đã hỗ trợ, tạo điều kiện để tơi hồn thành tốt khóa học Xin gửi lời tri ân đến gia đình, bạn bè ngƣời thân u đã ln động viên, khích lệ, giúp đỡ tơi trình học tập nghiên cứu Luận văn này đánh dấu bƣớc trƣởng thành riêng thân tơi Mặc dù tơi đã có nhiều cố gắng hồn thiện luận văn nhiệt tình và lực mình, nhiên khơng thể tránh khỏi thiếu sót, mong nhận đƣợc đóng góp quý báu quý thầy cô bạn Hà Nội, ngày 10 tháng 01 năm 2014 Học viên Dương Thu Hương LỜI CAM ĐOAN Tôi xin cam đoan toàn nội dung số liệu luận văn này tự nghiên cứu, khảo sát thực hiện, bên cạnh giúp đỡ, hỗ trợ thành viên nhóm nghiên cứu thuộc đề tài Nghị định thƣ hợp tác với Mỹ, và đã nhận đƣợc đồng ý cho phép sử dụng số liệu chung nhóm nghiên cứu Học viên thực Luận văn Dương Thu Hương MỤC LỤC MỞ ĐẦU TỔNG QUAN TÀI LIỆU HỆ VI SINH VẬT MICROBIOTA CỦA NGƢỜI - 1.1.1 Các Microbiota địa 1.1.2 Các Microbiota bệnh - 1.2 CÁC PHƢƠNG PHÁP NGHIÊN CỨU MICROBIOTA 1.2.1 Phƣơng pháp cổ điển - 1.2.2 Phƣơng pháp sinh học phân tử 1.2.3 Kết hợp phƣơng pháp nuôi cấy cổ điển và phƣơng pháp đại - 1.3 MICROBIOTA DẠ DÀY NGƢỜI - 1.3.1 Microbiota địa dày ngƣời khỏe mạnh 10 1.3.2 Microbiota dày ngƣời bệnh 11 1.4 HỘI CHỨNG CHẢY MÁU DẠ DÀY 12 1.4.1 Các dấu hiệu nhận biết chảy máu dày - 12 1.4.2 Nguyên nhân yếu tố nguy gây chảy máu dày 13 ĐỐI TƢỢNG VÀ PHƢƠNG PHÁP NGHIÊN CỨU 15 2.1 ĐỐI TƢỢNG NGHIÊN CỨU 16 2.2 PHƢƠNG TIỆN NGHIÊN CỨU 16 2.2.2 Hóa chất, thiết bị máy móc - 16 2.2.3 Địa điểm thực nghiên cứu 17 2.3 PHƢƠNG PHÁP NGHIÊN CỨU 18 2.3.1 Khám lâm sàng - 19 2.3.2 Nội soi lấy bệnh phẩm - 19 2.3.3 Đo pH dịch vị dày 19 2.3.4 Nuôi cấy phân lập chủng vi khuẩn - 20 2.3.5 Nhuộm Gram và quan sát dƣới kính hiển vi thƣờng 21 2.3.6 Test Helicotest 21 2.3.7 Tách chiết ADN từ vi khuẩn từ sinh thiết - 21 2.3.8 Xác định HPstatus bệnh nhân 21 2.3.9 PCR nhân gen 23S rARN H pylori - 22 2.3.10 PCR nhân gen 16S rARN vi khuẩn 23 2.3.11 Giải trình tự - 23 2.3.12 Định tên vi khuẩn - 24 KẾT QUẢ VÀ BÀN LUẬN 25 3.1 ĐẶC ĐIỂM CÁC BỆNH NHÂN 26 3.1.1 Đặc điểm tuổi, giới, độ pH dày - 26 3.1.2 Hình ảnh nội soi dày bệnh nhân 27 3.2 XÁC ĐỊNH HP STATUS CỦA CÁC BỆNH NHÂN - 28 3.3 PHÂN LẬP CÁC VI KHUẨN Ở ĐIỀU KIỆN KỊ KHÍ 31 3.4 NGHIÊN CỨU CÁC VI KHUẨN - 35 3.4.1 Xác định Gram chủng phân lập 35 3.4.2 Định tên vi khuẩn 35 KẾT LUẬN 44 KIẾN NGHỊ - 45 TÀI LIỆU THAM KHẢO 46 DANH MỤC CÁC BẢNG Bảng Phân bố bệnh nhân nghiên cứu theo nhóm tuổi giới tính ……………… 26 Bảng Kết phân tích gen thị vi khuẩn H pylori 27 bệnh nhân ………………………………………………… 30 Bảng Tỷ lệ bệnh nhân nhiễm vi khuẩn …………………………………………… 31 Bảng Tên loài vi khuẩn phân lập từ bệnh phẩm …………………………… 39 Bảng Một số đặc điểm vi khuẩn phân lập điều kiện kị khí từ Microbiota dày ngƣời bệnh bị viêm dày cấp tính chảy máu …………………………… 42 DANH MỤC CÁC HÌNH Hình Sự khác biệt Microbiota ngƣời trẻ và ngƣời già ……………… Hình Cấu trúc bậc hai 16S rARN ……………………………………………… Hình Microbiota đƣờng tiêu hóa ngƣời ……………………………………… 10 Hình Hình ảnh nội soi niêm mạc dày xuất huyết ………………………… 13 Hình Đĩa mơi trƣờng thạch máu dùng để ni cấy vi khuẩn …………………… 17 Hình Hộp giấy đo pH 0~14 (EMD-Mỹ) ………………………………………… 19 Hình Hệ thống bình ni cấy kị khí …………………………………………… 20 Hình Xác định độ pH dịch dày 27 bệnh nhân bị chảy máu dày ……… 27 Hình Ảnh chụp dày xuất huyết 27 bệnh nhân …………………………… 28 Hình 10 Hình ảnh khuẩn lạc vi khuẩn đĩa thạch máu số bệnh nhân……………………………………………………………………………………31 Hình 11 Hình ảnh khuẩn lạc đƣợc nhận định ban đầu giống với H pylori … .32 Hình 12 H pylori sau nhuộm Gram dƣới kính hiển vi quang học ………………33 Hình 13 Kết thử hoạt tính Urease Helicotest …………………………… 33 Hình 14 Sản phẩm PCR khuếch đại gen 23S rARN xác định H pylori …………… 33 Hình 15 Các chủng H pylori phân lập điều kiện kị khí ………………………… 34 Hình 16 Một số chủng vi khuẩn đƣợc phân lập làm điều kiện kị khí ….….34 Hình 17 Ảnh chụp số chủng vi khuẩn dƣới kính hiển vi Olympia …………… 35 Hình 18 Sản phẩm PCR khuếch đại từ gen 16S rARN ………………………………36 MỞ ĐẦU Biến đổi khí hậu Tồn cầu kèm với suy thối tài ngun, ô nhiễm môi trƣờng làm thay đổi toàn diện, sâu sắc hệ sinh thái tự nhiên, dẫn đến thay đổi nhiều bệnh đã biết nhƣ xuất nhiều bệnh Đây thách thức lớn nhân loại kỷ 21 Viêm dày cấp tính chảy máu Hội chứng hay gặp ngƣời trƣởng thành - lứa tuổi phải chịu nhiều áp lực từ công việc lao động, xã hội và gia đình nhƣ dễ nhiễm thói quen sinh hoạt có hại cho sức khỏe nhƣ hút thuốc lá, nhậu nhẹt rƣợu chè Ở Việt Nam, năm gần đây, ngƣời 20 tuổi bị chảy máu dày Nguyên nhân dịch chuyển tuổi tác Hội chứng chƣa đƣợc xác định Viêm dày cấp tính chảy máu là đợt tiến triển cấp tính viêm dày mạn tính, biểu bất ngờ gây ảnh hƣởng nặng nề đến khả lao động nhƣ chất lƣợng sống ngƣời bệnh Hội chứng có tỷ lệ tái phát cao sau điều trị Nguyên nhân gây viêm dày cấp tính chảy máu là vấn đề tranh cãi [6], [30] Mối liên quan bệnh lý viêm loét dày biến chứng chảy máu dày với vi khuẩn Helicobacter pylori đã đƣợc thừa nhận Các bệnh nhân bị viêm dày cấp tính chảy máu đƣợc điều trị khỏi nhờ liệu pháp kháng sinh Tuy nhiên thực tế y học lâm sàng đã xác nhận, có nhiều trƣờng hợp bệnh nhân bị viêm dày cấp tính chảy máu không nhiễm H pylori, nhƣng phác đồ điều trị có kháng sinh mang lại kết tốt Vấn đề đặt là, H pylori dày ngƣời cịn có lồi vi khuẩn khác liên quan mà khoa học chƣa biết? Hiện tại, giới nhƣ Việt Nam chƣa có nhiều cơng trình nghiên cứu viêm dày cấp tính chảy máu có thơng tin thành phần Microbiota dày ngƣời bệnh Sự hạn chế này ảnh hƣởng nhiều đến hiệu điều trị – theo dõi diễn biến bệnh cho bệnh nhân Xuất phát từ nhu cầu hiểu biết phục vụ công tác chẩn trị bệnh nhân bị viêm dày cấp tính chảy máu, tiến hành đề tài nghiên cứu “Sự diện vi khuẩn kị khí bệnh nhân bị chảy máu dày Việt Nam", với mục tiêu góp phần tìm hiểu thêm vi khuẩn có mặt Microbiota dày nhóm bệnh nhân Nội dung nghiên cứu luận văn bao gồm: Phân lập loại vi khuẩn từ bệnh phẩm dày chảy máu điều kiện kị khí Xác định tỷ lệ nhiễm H pylori bệnh nhân Định tên vi khuẩn phƣơng pháp giám định gen 16S rARN Tham khảo đặc tính vi khuẩn đƣợc phân lập qua liệu Internet >20B1 Bacillus megaterium TTGTTACGAC TCCACCGACT GGAACGTATT CGAGTTGCAG TCTTGCAGCC GATGATTTGA CCAACTTAAT TCTCACGACA ACGCTCTATC TCGAATTAAA GTCTTGCGAC GGAAACCCTC CTGTTTGCTC CCACTGGTGT TCTTCTGCAC ACATCAGACT GCCACCTACG CCGTCAAGGC CCCGAAAGCC ATTCCCTACT CACCCTCTCA AATGCACCGC AAGGAGAAGA CAGGTTGCCC CAGTTCGCTC TTCACCCCAA TCGGGTGTTA CACCGCGGCA CCTACAATCC CTTTGTACCA CGTCATCCCC GCTGGCAACT CGAGCTGACG TCTAGAGTTG CCACATGCTC CGTACTCCCC TAACACTTAG CCCACGCTTT TCCTCCACAT TCAAGTTCCC TAAGAAACCG TATTACCGCG ACGAGCAGTT TTCATCACTC GCTGCCTCCC GGTCGGCTAT GGGCCCATCT TCCTATCCGG ACGTGTTACT GACTTGCATG TCATCTGTCC CAAACTCTCG TGCTGATCCG GAACTGAGAA TCCATTGTAG ACCTTCCTCC AAGATCAAGG ACAACCATGC TCAGAGGATG CACCGCTTGT AGGCGGAGTG CACTCATCGT CGCGCCTCAG CTCTACGCAT CAGTTTCCAA CCTGCGCGCG GCTGCTGGCA ACTCTCGTAC ACGCGGCGTT GTAGGAGTCT GCATCGTTGC GTAAGTGATA TATTAGCTTC CACCCGTCCG TATTAGGCAC CACCTTAGGC TGGTGTGACG CGATTACTAG TGGTTTTATG CACGTGTGTA GGTTTGTCAC GTTGCGCTCG ACCACCTGTC TCAAGACCTG GCGGGCCCCC CTTAATGCGT TTACGGCGTG CGTCAGTTAC TTCACCGCTA TGACCCTCCA CTTTACGCCC CGTAGTTAGC TTGTTCTTCC GCTCCGTCAG GGGCCGTGTC CTTGGTGAGC GCCGAAACCA GGTTTCCCGA CCGCTAACGT GCCGCCAGCG GGCTAGCTCC GGCGGTGTGT CGATTCCAGC GGATTGGCTT GCCCAGGTCA CGGCAGTCAC TTGCGGGACT ACTCTGTCCC GTAAGGTTCT GTCAATTCCT TAGCTGCAGC GACTACCAGG AGACCAAAAA CACGTGGAAT CGGTTGAGCC AATAATTCCG CGTGGCTTTC CTAACAACAG ACTTTCGTCC TCAGTCCCAG CGTTACCTCA TCTTTCAATC AGTTATCCCA CATAGAAGCA TTCATC TTACGGTTAC ACAAGGCCCG TTCATGTAGG GACCTCGCGG TAAGGGGCAT CTTAGAGTGC TAACCCAACA CCGAAGGGGA TCGCGTTGCT TTGAGTTTCA ACTAAAGGGC GTATCTAATC GCCGCCTGCG TCCGCTTTTC GTGGGCTTTC GATAACGCTT TGGTTAGGTA AGTTTTACGA ATTGCGGAAG TGTGGCCGAT CCAACTAGCT ATCTCCCATG GTCTTACAGG AGCTTCTAAT 60 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 >8B1, 9B2 Streptococcus rubneri CTATCCCACC ACTCTCGTGG TGATCCGCGA CTGAGACTGG ATTGTAGCAC TTCCTCCGGT AATAGGGGTT ACCATGCACC GGATGTCAAG CTTGTGCGGG GAGTGCTTAA ATCGTTTACG CTCAGCGTCA CGCATTTCAC TTCCAAAGCG GCTCGCTTTA TGGCACGTAG TCACACTCGT GGCGTTGCTC GAGTCTGGGC CGTCGCCTTG GTGGTGCATT CTATCGTTTC TTCGCAACTC G TTAGGCGGCT TGTGACGGGC TTACTAGCGA CTTTAAGAGA GTGTGTAGCC TTATTACCGG GCGCTCGTTG ACCTGTCACC ACCTGGTAAG CCCCCGTCAA TGCGTTAGCT GCGTGGACTA GTTACAGACC CGCTACACAT TACTATGGTT CGCCCAATAA TTAGCCGTCC TCTTCTCTTA GGTCAGACTT CGTGTCTCAG GTGAGCCGTT TGCACCTTTC CAATAGTTAT ATCCGGAAAG GGCTCCTAAA GGTGTGTACA TTCCGACTTC TTAGCTTGTC CAGGTCATAA CAGTCTCGCT CGGGACTTAA TCTGTCCCGA GTTCTTCGCG TTCCTTTGAG GCGGCACTGA CCAGGGTATC AGAGAGCCGC GGAATTCCAC AAGCCACAGC ATCCGGATAA CTTTCTGGTA CAACAGAGCT CCGTCCATTG TCCCAGTGTG ACCTCACCAA AAGTAAATGT CCCCCGCTAC AGCAAGCTCC AGGTTACCTC AGGCCCGGGA ATGTAGGCGA GTCACCGACT GGGGCATGAT AGAGTGCCCA CCCAACATCT AGGAAAACTC TTGCTTCGAA TTTCAACCTT GTCCCGGAAA TAATCCTGTT TTTCGCCACC TCTCCCCCTT CTTTAACTTC CGCTCGGGAC AGATACCGTC TTACGATCCG CCGAAGATTC GCCGATCACC CTAGCTAATA CATGCAACAT CAGGCAGGTT TTCCTTCAGC ACCGACTTCG ACGTATTCAC GTTGCAGCCT TGCGACTCGT GATTTGACGT ACTGAATGAT CACGACACGA TATCTCTAGA TTAAACCACA GCGGTCGTAC GGACCCAACA TGCTCCCCAC GGTGTTCCTC CTGCACTCAA AGACTTATCT CTACGTATTA ACAGTGTGAA AAAACCTTCT CCTACTGCTG CTCTCAGGTC CAACGCAGGT CTACTGTTAT ACCTACGCGT GTTCTACTTG GGTGTTACAA CGCGGCGTGC ACAATCCGAA TGTACCAGCC CATCCCCACC GGCAACTAAC GCTGACGACA GCGGTCAGAG TGCTCCACCG TCCCCAGGCG CCTAGCACTC GCTTTCGAGC CATATATCTA GTTAAACAGT AACCGCCTGC CCGCGGCTGC CTTTCCACTC TCACTCACGC CCTCCCGTAG GGCTATGTAT CCATCTGGTA GCGGTATTAG TACTCACCCG CATGTATTAG 60 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 >5B4 Actinomyces odontolyticus CTTACACAT GCAGTCGAAC GCTGAAGCCT AGCTTGCTGG GTGGATGAGT GGCGAACGGG 60 TGAGTAACAC GTGAGTAACC TGCCCCCTTC TTTGGGATAA CGCCCGGAAA CGGGTGCTAA 120 TACTGGATAT TCACTGATCT TCGCATGGGG GTTGGTGGAA AGGTTTTTTC TGGTGGGGGA 180 TGGGCTCGCG GCCTATCAGC TTGTTGGTGG GGTGATGGCC TACCAAGGCT TTGACGGGTA 240 GCCGGCCTGA GAGGGTGACC GGTCACATTG GGACTGAGAT ACGGCCCAGA CTCCTACGGG 300 AGGCAGCAGT GGGGAATATT GCACAATGGG CGAAAGCCTG ATGCAGCGAC GCCGCGTGAG 360 GGATGGAGGC CTTCGGGTTG TAAACCTCTT TCGCTCATGG TCAAGCCGCA ACGTGTGGTT 420 GTGGTGAGGG TAGTGGGTAA AGAAGCGCCG GCTAACTACG TGCCAGCAGC CGCGGTAATA 480 CGTAGGGCGC GAGCGTTGTC CGGAATATTG GCGTAAAGGG CTGTACGGGG GTTGGTCGCG 540 TCTGCCGTGA AATCCTCTGG CTTAACTGGG GGCGTGCGGT GGGTACGGGC TGACTTGAGT 600 GCGGTAGGGG AGACTGGAAC TCCTGGTGTA GCGGTGGAAT GCGCAGATAT CAGGAAGAAC 660 ACCGGTGGCG AAGGCGGGTC TCTGGGCCGT TACTGACGCT GAGGAGCGAA AGCGTGGGGA 720 GCGAACAGGA TTAGATACCC TGGTAGTCCA CGCTGTAAAC GTTGGGCACT AGGTGTGGGG 780 GCCACCCGTG GTTTCTGCGC CGTAGCTAAC GCTTTAAGTG CCCCGCCTGG GGAGTACGGC 840 CGCAAGGCTA AAACTCAAAG GAATTGACGG GGGCCCGCAC AAGCGGCGGA GCATGCGGAT 900 TAATTCGATG CAACGCGAAG AACCTTACCA AGGCTTGACA TGCACGGCGG CACTGCAGAG 960 ATGTGGTGGC ATTTAGTTGG TCGTGTGCAG GTGGTGCATG GTTGTCGTCA GCTCGTGTCG 1020 TGAGATGTTG GGTTAAGTCC CGCAACGAGC GCAACCCTTG CCCTATGTTG CCAGCACGTG 1080 ATGGTGGGGA CTCGTGGGGG ACTGCCGGGG TTAACTCGGA GGAAGGTGGG GATGACGTCA 1140 AATCATCATG CCCCTTATGT CTTGGGCTTC ACGCATGCTA CAATGGTTGG TACAGAGGGT 1200 TGCGATACTG TGAGGTGGAG CGAATCCCTT AAAGCCAGTC TCAGTTCGGA TTGGGGTCTG 1260 CAACTCGACC CCATGAAGGT GGAGTCGCTA GTAATCGCAG ATCAGCAACG CTGCGGTGAA 1320 TACGTTCTCG GGCCTTGTAC ACACCGCCCG TCACGTCACG AAAGTTGGTA ACACCCGAAG 1380 CCCATGGCCT AACCGCGTTG CGGGGGGAGT TGTCGAAA Phụ lục IV: Trình tự gen 23S rARN 15 chủng H pylori >1B-HP strain CCAAAGCCCC CTAAGTTGTA ACCTCCACTA CATGCAGGTC GCCGCCGTTT CCTTCGAGCA TACTTCAAAG GTAAAGGTCC CAATTTCACT GGTATTTAAC ACTCGGGCTT CCGGGCAGGC CCTCCCACCT ACGGGGTCTT GAATCTCTGG CGACAAGGAA CAATTCAACG GTCACACCTT ATCCTGCGCA TCCGTCTTGC TTGAGACAGC TTTCGCTACC CTTCATCTTG ATACTTCCTC TGATATTCCC CGCGGGTAGG TCCCATCTCG TTAGGACCGT CGACTGACGC TTACGAGTTG GTTAGCAGTG AGGAATTTTC TTACGCCATT TATAGTTACG ATCCTCTTAA GCAAAGTGCT 60 120 180 240 300 360 >3B-HP strain CAAAGCCCTT TAAGTTGTAG CCTCCACTAC ATGCAGGTCG CCGCCGTTTA CTTCGAGCAC ACTTCAAAGC TAAAGGTCCA AATTTCACTG GTATTTAACC CTCGGGCTTC CGGGCAGGCG CTCCCACCTA CGGGGTCTAT AATCTCTGGT GACAAGGAAT AATTCAACGC TCACACCTTA TCCTGCGCAT CCGTCTTGCC TGAGACAGCT TTCGCTACCT TTCATCTTGC TACTTCCTCT GATATTCCCG GCGGGTAGGA CCCATCTCGT TAGGACCGTT GACTGACGCA TACGAGTTGG TTAGCAGTGC GGAATTTTCA TACGCCATTC ATAGTTACGG TCCTCTTAAC CAAAGTGCTG 60 120 180 240 300 360 >4B-HP strain CCAAAGCCCT CTAAGTTGTA ACCTCCACTA CATGCAGGTC GCCGCCGTTT CCTTCGAGCA GTG TACTTCAAAG GTAAAGGTCC CAATTTCACT GGTATTTAAC ACTCGGGCTT CCGGGCAGGC CCTCCCACCT ACGGGGTCTT GAATCTCTGG CGACAAGGAA CAATTCAACG GTCACACCTT ATCCTGCGCA TCCGTCTTGC TTGAGACAGC TTTCGCTACC CTTCATCTTG ATACTTCCTC TGATATTCCC CGCGGGTAGG TCCCATCTCG TTAGGACCGT CGACTGACGC TTACGAGTTG GTTAGCAGTG AGGAATTTTC TTACGCCATT TATAGTTACG ATCCTCTTAA GCAAAGTGCT 60 120 180 240 300 360 >5B-HP strain CCAAAGCCCT CTAAGTTGTA ACCTCCACTA CATGCAGGTC GCCGCCGTTT CCTTCGAGCA GTG TACTTCAAAG GTAAAGGTCC CAATTTCACT GGTATTTAAC ACTCGGGCTT CCGGGCAGGC CCTCCCACCT ACGGGGTCTT GAATCTCTGG CGACAAGGAA CAATTCAACG GTCACACCTT ATCCTGCGCA TCCGTCTTGC TTGAGACAGC TTTCGCTACC CTTCATCTTG ATACTTCCTC TGATATTCCC CGCGGGTAGG TCCCATCTCG TTAGGACCGT CGACTGACGC TTACGAGTTG ATTAGCAGTG AGGAATTTTC TTACGCCATT TATAGTTACG ATCCTCTTAA GCAAAGTGCT 60 120 180 240 300 360 TACTTCAAAG GTAAAGGTCC CAATTTCACT GGTATTTAAC ACTCGGGCTT CCGGGCAGGC CCTCCCACCT ACGGGGTCTT GAATCTCTGG CGACAAGGAA CAATTCAACG GTCACACCTT ATCCTGCGCA TCCGTCTTGC TTGAGACAGC TTTCGCTACC CTTCATCTTG ATACTTCCTC TGATATTCCC CGCGGGTAGG TCCCATCTCG TTAGGACCGT CGACTGACGC TTACGAGTTG ATTAGCAGTG AGGAATTTTC TTACGCCATT TATAGTTACG ATCCTCTTAA GCAAAGTGCT 60 120 180 240 300 360 >7B-HP strain CCAAAGCCCC CTAAGTTGTA ACCTCCACTA CATGCAGGTC GCCGCCGTTT CCTTCGAGCA GTG >8B-HP strain CCAAAGCCCT CTAAGTTGTA ACCTCCACTA CATGCAGGTC GCCGCCGTTT CCTTCGAGCA GTG TACTTCAAAG GTAAAGGTCC CAATTTCACT GGTATTTAAC ACTCGGGCTT CCGGGCAGGC CCTCCCACCT ACGGGGTCTT GAATCTCTGG CGACAAGGAA CAATTCAACG GTCACACCTT ATCCTGCGCA TCCGTCTTGC TTGAGACAGC TTTCGCTACC CTTCATCTTG ATACTTCCTC TGATATTCCC CGCGGGTAGG TCCCATCTCG TTAGGACCGT CGACTGACGC TTACGAGTTG GTTAGCAGTG AGGAATTTTC TTACGCCATT TATAGTTACG ATCCTCTTAA GCAAAGTGCT 60 120 180 240 300 360 TACTTCAAAG GTAAAGGTCC CAATTTCACT GGTATTTAAC ACTCGGGCTT CCGGGCAGGC CCTCCCACCT ACGGGGTCTT GAATCTCTGG CGACAAGGAA CAATTCAACG GTCACACCTT ATCCTGCGCA TCCGTCTTGC TTGAGACAGC TTTCGCTACC CTTCATCTTG ATACTTCCTC TGATATTCCC CGCGGGTAGG TCCCATCTCG TTAGGACCGT CGACTGACGC TTACGAGTTG GTTAGCAGTG AGGAATTTTC TTACGCCATT TATAGTTACG ATCCTCTTAA GCAAAGTGCT 60 120 180 240 300 360 >17B-HP strain CCAAAGCCCC TACTTCAAAG CTAAGTTGTA GTAAAGGTCC ACCTCCACTA CAATTTCACT CATGCAGGTC GGTATTTAAC GCCGCCGTTT ACTCGGGCTT CCTTCGAGCA CCGGGCAGGC CCTCCCACCT ACGGGGTCTT GAATCTCTGG CGACAAGGAA CAATTCAACG GTCACACCTT ATCCTGCGCA TCCGTCTTGC TTGAGACAGC TTTCGCTACC CTTCATCTTG ATACTTCCTC TGATATTCCC CGCGGGTAGG TCCCATCTCG TTAGGACCGT CGACTGACGC TTACGAGTTG GTTAGCAGTG AGGAATTTTC TTACGCCATT TATAGTTACG ATCCTCTTAA GCAAAGTGCT 60 120 180 240 300 360 >19B-HP strain CAAAGCCCTT ACTTCAAAGC TAAGTTGTAG TAAAGGTCCA CCTCCACTAC AATTTCACTG ATGCAGGTCG GTATTTAACC CCGCCGTTTA CTCGGGCTTC CTTCGAGCAC CGGGCAGGCG CTCCCACCTA CGGGGTCTAT AATCTCTGGT GACAAGGAAT AATTCAACGC TCACACCTTA TCCTGCGCAT CCGTCTTGCC TGAGACAGCT TTCGCTACCT TTCATCTTGC TACTTCCTCT GATATTCCCG GCGGGTAGGA CCCATCTCGT TAGGACCGTT GACTGACGCA TACGAGTTGG TTAGCAGTGC GGAATTTTCA TACGCCATTC ATAGTTACGG TCCTCTTAAC CAAAGTGCTG 60 120 180 240 300 360 >20B-HP strain CCAAAGCCCT TACTTCAAAG CTAAGTTGTA GTAAAGGTCC ACCTCCACTA CAATTTCACT CATGCAGGTC GGTATTTAAC GCCGCCGTTT ACTCGGGCTT CCTTCGAGCA CCGGGCAGGC GTG CCTCCCACCT ACGGGGTCTT GAATCTCTGG CGACAAGGAA CAATTCAACG GTCACACCTT ATCCTGCGCA TCCGTCTTGC TTGAGACAGC TTTCGCTACC CTTCATCTTG ATACTTCCTC TGATATTCCC CGCGGGTAGG TCCCATCTCG TTAGGACCGT CGACTGACGC TTACGAGTTG GTTAGCAGTG AGGAATTTTC TTACGCCATT TATAGTTACG ATCCTCTTAA GCAAAGTGCT 60 120 180 240 300 360 >21B-HP strain CCAAAGCCCT TACTTCAAAG CTAAGTTGTA GTAAAGGTCC ACCTCCACTA CAATTTCACT CATGCAGGTC GGTATTTAAC GCCGCCGTTT ACTCGGGCTT CCTTCGAGCA CCGGGCAGGC GT CCTCCCACCT ACGGGGTCTT GAATCTCTGG CGACAAGGAA CAATTCAACG GTCACACCTT ATCCTGCGCA TCCGTCTTGC TTGAGACAGC TTTCGCTACC CTTCATCTTG ATACTTCCTC TGATATTCCC CGCGGGTAGG TCCCATCTCG TTAGGACCGT CGACTGACGC TTACGAGTTG ATTAGCAGTG AGGAATTTTC TTACGCCATT TATAGTTACG ATCCTCTTAA GCAAAGTGCT 60 120 180 240 300 360 >12B-HP strain CCAAAGCCCT CTAAGTTGTA ACCTCCACTA CATGCAGGTC GCCGCCGTTT CCTTCGAGCA GTG >24B-HP strain CCAAAGCCCC TACTTCAAAG CTAAGTTGTA GTAAAGGTCC ACCTCCACTA CAATTTCACT CATGCAGGTC GGTATTTAAC GCCGCCGTTT ACTCGGGCTT CCTTCGAGCA CCGGGCAGGC GTG CCTCCCACCT ACGGGGTCTT GAATCTCTGG CGACAAGGAA CAATTCAACG GTCACACCTT ATCCTGCGCA TCCGTCTTGC TTGAGACAGC TTTCGCTACC CTTCATCTTG ATACTTCCTC TGATATTCCC CGCGGGTAGG TCCCATCTCG TTAGGACCGT CGACTGACGC TTACGAGTTG ATTAGCAGTG AGGAATTTTC TTACGCCATT TATAGTTACG ATCCTCTTAA GCAAAGTGCT 60 120 180 240 300 360 >25B-HP strain CCAAAGCCCT TACTTCAAAG CTAAGTTGTA GTAAAGGTCC ACCTCCACTA CAATTTCACT CATGCAGGTC GGTATTTAAC GCCGCCGTTT ACTCGGGCTT CCTTCGAGCA CCGGGCAGGC GTG CCTCCCACCT ACGGGGTCTT GAATCTCTGG CGACAAGGAA CAATTCAACG GTCACACCTT ATCCTGCGCA TCCGTCTTGC TTGAGACAGC TTTCGCTACC CTTCATCTTG ATACTTCCTC TGATATTCCC CGCGGGTAGG TCCCATCTCG TTAGGACCGT CGACTGACGC TTACGAGTTG GTTAGCAGTG AGGAATTTTC TTACGCCATT TATAGTTACG ATCCTCTTAA GCAAAGTGCT 60 120 180 240 300 360 >26B-HP strain CCAAAGCCCT TACTTCAAAG CTAAGTTGTA GTAAAGGTCC ACCTCCACTA CAATTTCACT CATGCAGGTC GGTATTTAAC GCCGCCGTTT ACTCGGGCTT CCTTCGAGCA CCGGGCAGGC GTG CCTCCCACCT ACGGGGTCTT GAATCTCTGG CGACAAGGAA CAATTCAACG GTCACACCTT ATCCTGCGCA TCCGTCTTGC TTGAGACAGC TTTCGCTACC CTTCATCTTG ATACTTCCTC TGATATTCCC CGCGGGTAGG TCCCATCTCG TTAGGACCGT CGACTGACGC TTACGAGTTG GTTAGCAGTG AGGAATTTTC TTACGCCATT TATAGTTACG ATCCTCTTAA GCAAAGTGCT 60 120 180 240 300 360 >27B-HP strain CCAAAGCCCC TACTTCAAAG CTAAGTTGTA GTAAAGGTCC ACCTCCACTA CAATTTCACT CATGCAGGTC GGTATTTAAC GCCGCCGTTT ACTCGGGCTT CCTTCGAGCA CCGGGCAGGC G CCTCCCACCT ACGGGGTCTT GAATCTCTGG CGACAAGGAA CAATTCAACG GTCACACCTT ATCCTGCGCA TCCGTCTTGC TTGAGACAGC TTTCGCTACC CTTCATCTTG ATACTTCCTC TGATATTCCC CGCGGGTAGG TCCCATCTCG TTAGGACCGT CGACTGACGC TTACGAGTTG GTTAGCAGTG AGGAATTTTC TTACGCCATT TATAGTTACG ATCCTCTTAA GCAAAGTGCT 60 120 180 240 300 360 Phụ lục V: Kết PCR thị sinh học H pylori Gen glmM 1B 11B 2B 3B 12B 4B 13B 19B 20B 21B 5B 14B 6B 7B 15B 22B 23B 24B 8B 9B 16B 17B 25B 26B 10B 18B 27B (-) (+) (-) (+) (-) (+) Gen cagA 1B 3B 4B 5B 22B 23B 24B 6B 25B (+) (-) 26B 27B 7B 8B 9B 11B 12B 14B 15B 17B 18B 19B 20B 21B Gen hspA 1B 2B 11B 3B 4B 12B (+) (+) (-) 5B 6B 7B 13B 14B 15B 16B (-) 21B 22B 23B 8B 9B 10B (+) 17B 18B 19B 20B 24B 25B 26B 27B (-) Gen 23S rARN 1B 15B 3B 17B 4B 5B 18B 19B 6B 20B 7B 8B 21B 22B 9B 11B 23B 24B 12B 25B 14B (-) (+) 26B 27B Phụ lục VI Giấy cho phép bảo vệ Guillermo I Perez-Perez PhD Associate Professor of Medicine and Microbiology 6027W VAMC 423 East 23d Street New York NY 10010 Telephone: (212) 263-4101 Facsimile: (212) 263-4108 E-mail: perezg02@med.nyu.edu New York, December 30, 2013 To whom it may concern, We, the group leaders of American-Vietnam Cooperation project entitled: Study methods for diagnosing, treating and preventing microbial infection in Helicobacter pylori-infected patients agree to permit to student Duong Thu Huong to use the results associated with the isolation of Helicobacter pylori from gastric biopsies of 27 patients with gastric bleeding, as well as the nucleotide sequences of the 16S rRNA gene of the bacteria that provide information for her Master thesis The student has the duty to inform this and to provide the appropriate credit of our group Dr Guillermo I Perez Perez Associate Professor of Medicine and Microbiology Dr Nguyen Thi Hong Hanh Associate Professor of Molecular Biology and Cellular Immunology Institute of Biotechnology-VAST Phụ lục VII Bài báo công bố liên quan Medical Mycology November 2013, 51, 884–887 Candida krusei colonization in patients with gastrointestinal diseases Med Mycol Downloaded from informahealthcare.com by University New South Wales on 11/03/13 For personal use only DUONG THU HUONG∗,YANAN ZHAO†, NGUYEN THI NGUYET∗, TA THI LOAN∗, NGUYEN THI THANH BINH∗, NGUYEN VAN THINH‡, NGUYEN THI HONG HANH∗, GUILLERMO I PEREZ-PEREZ§ & DAVID S PERLIN† ∗Institute of Biotechnology, Vietnam Academy of Sciences and Technology, Viet Nam, †Public Health Research Institute, UMDNJ, Newark, NJ, USA, ‡Hospital Buu Dien, Hà Nô.i,Viet Nam, and §New York University School of Medicine, New York, NY, USA A total of 135 stomach samples from patients with gastrointestinal diseases and normal controls were examined for Helicobacter pylori infection and Candida colonization Candida krusei was found in specimens from 20% bleeding, 52% ulcer, and 100% gastritis patients, whereas H pylori infection rates were 82%, 35% and 30%, respectively, for the same groups of patients C krusei was not detected in stomach samples from normal controls Keywords Candida krusei, colonization, Helicobacter pylori, gastrointestinal diseases Introduction At least 50% of the world population harbor Helicobacter pylori in their upper gastrointestinal (GI) tract [1] The incidence of its infection is mostly associated with childhood, socioeconomic and sanitary conditions and plays an important role in the pathogenesis of upper GI tract disorders Meanwhile, Candida colonization of the gastric mucosa has been considered to be involved with multiple diseases of GI tract, including chronic gastritis and ulcerous diseases [2] As such, Candida colonization may be of clinical significance, not only because it creates a vicious cycle in which inflammation and fungal colonization are mutually promoted, but also because it raises the risk of invasive candidiasis or candidemia, in which there may be excessive morbidity and mortality [3,4] This study was initially carried out to evaluate the status of H pylori infection in Vietnamese patients with GI diseases Molecular detection methods were performed later to explore Received October 2012; Received in final revised form April 2013; Accepted May 2013 Duong Thu Huong and Yanan Zhao contributed equally to this paper Correspondence: David S Perlin, Public Health Research Institute, UMDNJ-New Jersey Medical School, 225 Warren Street, Newark, NJ 07103, USA Tel.: ϩ 973 854 3200; fax: ϩ 973 854 3101; E-mail: perlinds@umdnj.edu © 2013 ISHAM Candida colonization in these patients and its relationship with H pylori infection Materials and methods Patients and sampling A total of 125 stomach biopsies were collected from December 2010 to April 2011 from outpatients with gastrointestinal diseases in BuuDien Hospital, Hanoi These included 27 antrum and 26 corpus biopsies from 27 patients with gastric bleeding, 10 pairs of antrum and corpus samples from 10 gastritis patients, and 52 antrum biopsies from 52 patients with duodenal ulcers Ten stomach biopsies were also collected from 10 healthy volunteers to serve as controls After collection, biopsies were immediately snap-frozen in liquid nitrogen and stored at Ϫ80°C until processed The study protocol was approved by local institutional review boards and all subjects provided written informed consent Culture isolation and H pylori identification Frozen gastric biopsies were homogenized in 300 μl sterile ϫ PBS, from which an aliquot of 30 μl of homogenate was inoculated onto non-selective blood agar TSA, and incubated under anaerobic condition (BBL GasPak™ DOI: 10.3109/13693786.2013.804215 C krusei colonization in gastrointestinal tract Table Primers and probes used for species-specific Candida identification Species C albicans C glabrata C krusei C tropicalis C parapsilosis Med Mycol Downloaded from informahealthcare.com by University New South Wales on 11/03/13 For personal use only 885 Primer/probe Sequences (5′–3′) Forward primer Reverse primer MB Forward primer Reverse primer MB Forward primer Reverse primer MB Forward primer Reverse primer MB Forward primer Reverse primer MB CGAGTGTTTGGGTGTAAAAC CCGAACACATCAGGATCG CGAGCTACGAAGGTGGGGGCCCATTAGGAGCTCG ACGCGTAATGAAAGTGAACG ATCCATCCGAAGACATCAG CGCATCGGTTGGGGCCCTCCACCTGGGATGCG GCGTAATGAAAGTGAACGTAGG GCTCCTACTCAAATCCTTCC CGGTCACGGGGAGGGGAGCACGATCGATGACCG CGAGTGTTTGGGTGTAAAAC CCGAAGACATCAGGATCG CGAGCTACGTAGGTGGGGGCCCGTATGGAGCTCG AGTGAACGTAGGTAGGACCTC ATCCATCCGAAGACATCAG CGAGCTCTTTAGGAGTGCACTATCGAAGCTCG Anaerobic System) at 37°C for 3–5 days Single colonies were selected from the agar surface and purified by subculturing several times prior to DNA extraction and 16S rRNA sequencing DNA was extracted using genomic DNA purification Kit (Fermentas K0722) H pylori identification methods included culture, rapid urease test, PCRs to detect H pylori biomarkers CagA [5], HspA [6], 23S rRNA [7] and GlmM [8], detection by serum ELISA [9], or PCR amplification of VacA isotype [10] or EPIYA motif of the cagA gene [11] Positive H pylori infection status was indicated by positive culture or in the case of negative-culture samples, by positive results from at least two of the tests indicated above Molecular detection of Candida colonization Molecular beacon (MB) real-time PCR assays, including pan-Candida PCR and species-specific PCRs for Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis and Candida tropicalis, were used for Candida detection All probes targeted the 28S ribosomal RNA gene The pan-Candida assay has been described previously [12], with the species-specific probes listed in Table Probes were purchased from Biosearch Technologies, Inc., (Novato, CA, USA) and labeled with 5-carboxyfluorescein (FAM) at the 5′ end and a quencher, benzoic acid succinimidyl ester (Dabcyl), at the 3′ end Real-time PCR amplification was performed using the Stratagene Mx3005P real-time PCR system (Agilent Technologies, Santa Clara, CA, USA) Results The demographic characteristics of patients are listed on Table H pylori infection (Table 3) was detected in 81.5% (22/27) of patients with gastric bleeding, which is © 2013 ISHAM, Medical Mycology, 51, 884–887 significantly higher than that noted in 34.6% (18/52; P Ͻ 0.001) of duodenal ulcer patients and 30% (3/10; P Ͻ 0.001) of gastritis patients In contrast, all samples from controls were negative for H pylori infectionspositive The most common species among 32 nonH pylori bacteria strains recovered from gastric bleeding patients, were Streptococcus spp (S australis, S infantis) and Bacillus spp (B licheniformis, B subtilis, B cereus, B megaterium) The remaining non-H pylori bacterial isolates were identified as Ochrobactrum intermedium, Rothia mucilaginosa and R dentocariosa Only H pylori isolates were recovered from gastritis patients Non-H pylori bacteria isolated from specimens collected from controls included Acinetobacter spp., Mycobacterium spp and Sphingobacterium spp Using pan-Candida real-time PCR, 63% (17/27) antrum and 50% (13/26) corpus samples from gastric bleeding patients, 100% (10/10) antrum and 100% (10/10) corpus samples from gastritis patients, and 77% (40/52) of the antrum specimens from duodenal ulcer patients, were found to be colonized by Candida spp In contrast, only 30% (3/10) of the samples from the control patients were positive for Candida spp Unexpectedly, C krusei was the only Candida species found in the specimens in Table Demographic characteristics of the patients Characteristic Bleedinga (n ϭ 27) Gastritisb (n ϭ 10) Duodenal ulcers (n ϭ 52) Controls (n ϭ 10) Sex: male (%) 15 (55.6) (20.0) 23 (44.2) (30.0) Age: mean 36.6 (25–57) 30.5 (21–36) 46.5 (18–71) 32.8 (20–49) (range), years Mean pH 2.5 3.1 NT NT aIn bleeding group, one patient had a stomach pH Ͼ bIn gastric group, three patients had a stomach pH Ͼ NT, not tested 886 Huong et al Table Helicobacter pylori infection status by patient group H pylori infection statusa Positive Group Bleeding Duodenal ulcers Gastritis Control By culture By non-culture detections Total Negative 15 2 16 22 18 34 10 aPositive Med Mycol Downloaded from informahealthcare.com by University New South Wales on 11/03/13 For personal use only H pylori infection status was determined by positive culture or by at least two positive molecular detection results in the case of culturenegative samples which 58% (52/90) of pan-Candida positive samples from the three GI diseases groups were positive for this species and the remaining samples were negative for all species tested The three pan-Candida positive samples from control group did not elicit any detectable signal in species-specific detection tests The presence of C krusei was found in 22% (6/27) of gastric bleeding patients, 52% (27/52) of patients with duodenal ulcers, and 100% (10/10) of gastritis patients, with lower colonization rates for corpus samples as compared to antrum specimens The level of C krusei colonization was also lower in gastric bleeding patients, who had the highest H pylori infection rate, compared to those with ulcer and gastritis (Figure 1) Discussion The stomach microbiota is in a continual state of flux determined by a variety of factors, such as age, diet, hormonal state, health, and personal hygiene Many microorganisms are washed from the mouth into the stomach, where most microorganisms are killed as a result of the low pH (2 ∼ 3) environment In adults with normal acid secretion, the only organisms present in this microbiota are acid-tolerant bacteria, such as the lactic acid producing bacteria Lactobacillus, Streptococcus and H pylori Fig Candida krusei real-time PCR threshold cycle (Ct) value distribution by of disease category and anatomic position Horizontal bars represent mean values © 2013 ISHAM, Medical Mycology, 51, 884–887 Med Mycol Downloaded from informahealthcare.com by University New South Wales on 11/03/13 For personal use only C krusei colonization in gastrointestinal tract Candida species colonizing the human GI tract are considered a component of the resident flora, and C albicans is the most common species recovered from such circumstances [2] However, C albicans was not detected in the present study, possibly because the levels of the yeast in the stomach were too low to reach the detection limit of the C albicans-specific real-time PCR assay Instead, C krusei seemed to be the best adapted to survive and to establish itself as the predominant fungal colonizer in the stomach microbiota of patients with three types of gastric conditions We cannot rule out the possibility that other Candida species were present, but may not have been detected by our assays due to the complexity of the mixed organisms, as well as the limited spectrum of the assays used in this study However, the high (48%) C krusei colonization rates in patients with GI diseases is unexpected and of clinical significance given that C krusei is an emerging fungal nosocomial pathogen producing a spectrum of clinical manifestations and intrinsically resistant to fluconazole [13] H pylori competes with other microbes in the stomach microbiota In this study, C krusei colonization was detected in 52% of ulcer and 100% of gastritis patients, whose H pylori infection rates were only 35% and 30%, respectively In contrast, 82% of gastric bleeding patients had H pylori infections but only 20% these individuals were found to be C krusei positive Moreover, C krusei colonization in the absence of H pylori infection was found in 7/7 (100%) of gastritis and 14/34 (41%) of ulcer patients, but was not seen in samples from normal controls, indicating a possible causative role of C krusei in gastritis and peptic ulcer In fact, the contributing role of C albicans in gastritis development in mice with altered microbiota [14], and the etiologic role of C tropicalis in gastric ulcer in patients without H pylori infection [15], has been reported recently Nevertheless, more detailed and proactive investigations are warranted to further study the predominance of C krusei colonization observed in the present study In summary, C krusei was found to be the predominant fungal colonizer in Vietnamese patients with gastrointestinal diseases The exact role of this opportunistic Candida pathogen in GI tract diseases and its interplay with other GI microbiota components needs further exploration Declaration of interest: The authors report no conflicts of interest The authors alone are responsible for the content and the writing of the paper This work was supported by research project no 50/2010/HD-NDT from the Ministry of Sciences and This paper was first published online on Early Online on July 2013 © 2013 ISHAM, Medical Mycology, 51, 884–887 887 Technology, Vietnam, and research project RO1 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