Bu çalışma Profileringsfonds Azm (PF245) tarafından desteklenmiştir.

BÖLÜM I: Patojen TANIMLANMASI 1. Numune Hazırlama Not: aşağıdaki protokol açıklandığı gibi tüm moleküler iş akışı moleküler tanı kalite güvencesi 3 önerileri doğrultusunda yapılmalıdır. <ol><li> A 1:10 seyreltilmiş örnek olmak için 1.5 ml&#39;lik tepkime tüp içine% 0.9 NaCl 0.9 ml kan bir kültür 0.1 ml bir örnek ekleyin. (Seyreltilir qPCR inhibisyonu önlemek için). </li><li> Pelet 5 dk bakteriyel DNA 13400 xg&#39;de örnek santrifüjleyin. </li><li> 100 ul steril demineralize H 2 O bakteri pelletini </li><li> 4 ° C ye kadar daha fazla kullanımda DNA örneği saklayın. </li></ol> 2. Tanımlama Testi: Real-time PCR 16s rDNA <ol><li> Aşağıdaki gibi reaksiyon karışımı hazırlayın. Assay numune başına dört ayrı reaksiyonlar meydana gelir. Her karışım 12.50 ul ana içerirkarışım, 0.9 uM ileri astar (5-TCCTACGGGAGGCAGCAGT-3) 7, 0.6 uM ters astar (5-GGACTACCAGGGTATCTAATCCTGTT-3) 8 ve probları bir panel. Prob miktarı aşağıda yer alan dört ayrı reaksiyonların her biri için verilmiştir. </li><li> İlk tepki içerir: <ul><li> 0.2 uM evrensel bir sonda (5-FAM-CGTATTACCGCGGCTGCTGGCAC-3-BHQ1) 8 </li><li> 0.2 uM P. aeruginosa prob (5-JOE-CCAAAACTACTGAGCTAGAGTACG-3-BHQ1) </li></ul></li><li> İkinci reaksiyon içerir: <ul><li> 0.2 uM E. coli prob (5-JOE-GGAGTAAAGTTAATACCTTTGCTCATT-3-BHQ1) </li><li> 0.2 uM Pseudomonas spp. Sondaya (5-NED-CCTTCCTCCCAACTTAAAGTGCTT-3-MGBNFQ) </li></ul></li><li> Üçüncü reaksiyonu içerir: <ul><li> 0.2 uM Staphylococcus spp. Sondaya (5-NED-AATCTTCCGCAATGGGCGAAAGC-3-MGBNFQ) </li><li> 0.2 uM S. aureus prob (5-FAM-AGATGTGCACAGTTACTTACACATAT-3-BHQ1) </li><li> 0.2 uM Enterococcus spp. (5-JOE-TCCTTGTTCTTCTCTAACAACAGAG-3-BHQ1) </li></ul></li><li> Dördüncü reaksiyonu içerir: <ul><li> 0.2 uM evrensel bir sonda (5-FAM-CGTATTACCGCGGCTGCTGGCAC-3-BHQ1) </li><li> 0.3 uM Streptococcus spp. Sondaya (5-NED-CCAGAAAGGGACSGCTAACT-3-MGBNFQ) </li><li> 0.2 uM S. pneumoniae prob (5-JOE-CCAAAGCCTACTATGGTTAAGCCA-3-BHQ1) </li></ul></li><li> 20 ul toplam hacimde ulaşmak için steril demineralize H2O ekleyin. Bir 96-kuyulu plakanın PCR kuyucuklara Her reaksiyon karışımı 20 ul ekleyin. </li><li> Her bir kuyucuğa örnek 5 ul ekleyin. </li><li> 96-PCR plakanın damgalanması yapışkan film kullanın. </li><li> Aşağıdaki en iyi termal siklus koşullarını kullanarak ABI PRISM 7900HT Real Time PCR Sistemi plaka çalıştırın: <ul><li> 10 dakika süre ile 50 ° C de ön-ısıtma </li><li> 15 dakika boyunca 95 ° C&#39;de ön denatürasyon </li><li> 42 döngü <ul type="circle"><li> 15 sn 95 ° C &#39;de denatürasyon </li><li> 1 dakika boyunca 60 ° C&#39;da tavlama </li></ul></li></ul></li></ol> 3. Sonuçların Analizi <ol><li> Sekmesi Analiz Ayarlar 0.1 Ct Analiz eşiği ayarlayın. 6 ve End (Cycle): 15 Başlat temel yapılandırmaları (Cycle) daraltın. </li><li> Tüm numuneler için döngüsü eşiği (Ct) değerini kaydedin. Pozitif bir PCR sonucu dikkate cut-off değeri 35 bir Ct-değere ayarlanabilir. Kan kültürlerinde mevcut bakteri miktarı Ct-değerleri 35 altında üreten, 10 7 10 11 CFU / ml arasında değişmekteydi. </li></ol> BÖLÜM II: ANTİBİYOTİK DUYARLILIK TESTİ 4. Pozitif Kan Kültürleri 9 Bakteri İzolasyonu <ol><li> Pozitif kan kültürü şişe suyu 5 ml aspire ve serum ayırıcı tüpüne aktarın. </li><li> 10 dakika boyunca 2000 x g&#39;de santrifüje serum ayrıştırıcı tüp. </li><li> Serum ayırıcı tüpten atılır. </li><li> Transferi bacTeria steril pamuklu ile tüp jel tabakası den% 0.9 &#39;luk tuzlu su içine bir 0,5 McFarland standart bir süspansiyon elde edilinceye kadar. </li></ol> 5.. Mikro Titre Tabaklar Aşılanması <ol><li> 5 x 10 5 CFU / ml &#39;lik bir süspansiyon oluşturmak üzere çift konsantre Mueller Hinton II çorbasındaki 0,5 McFarland süspansiyonu ile seyreltilir. </li><li> Antibiyotikler (Tablo 1) bir seçim içeren bir mikro titre plakanın kuyularına Bu süspansiyon ekleyin. </li><li> 6 saat süreyle 37 ° C &#39;de mikro titre plakası inkübe. </li><li> 4 ° C (negatif büyüme kontrolü gibi) de süspansiyon bir kısım saklayın. </li><li> İnkübasyonun 6 saat sonra, steril bir tüp içinde, her sıra bir içerik transfer gibi olumsuz büyümesini kontrol örneği 4 ° C de muhafaza edildi olduğu </li><li> 5 dakika boyunca 16.000 g&#39;de santrifüje borular. </li><li> Dikkatle bakteriyel pelet bozmadan, süpernatant kaldırın. </li><li> Steril demineralize H pelletini <sub&gt; 2 O. </li><li> Steril demineralize H 2 O 10 kat örnekleri sulandırınız </li></ol> 6. Real-time PCR 16&#39;lar rDNA 10 <ol><li> Aşağıdaki gibi PCR karışımı hazırlandı: <ul><li> 12.50 ul iQ SYBR Green Supermix </li><li> İleri 0,5 uM astar 16S-1 (5-TGGAGAGTTTGATCCTGGCTCAG-3) 11 </li><li> 0.25 uM ters primer 16S-2 (5-TACCGCGGCTGCTGGCAC-3) 11 </li><li> 20 ul toplam hacme steril demineralize H2O </li></ul></li><li> Bir 96-kuyulu plakanın PCR kuyucuklara PCR karışımı 20 ul ekleyin. </li><li> Her bir kuyucuğa örnek 5 ul ekleyin. </li><li> 96-PCR plakanın damgalanması yapışkan film kullanın. </li><li> Aşağıdaki optimum ısı bisiklet şartları kullanarak, MyiQ Tek Renk Real-Time PCR Algılama Sistemi plaka çalıştırın: <ul><li> 4 dakika boyunca 95 ° C&#39;de ön denatürasyon </li><li> İlk ° C&#39;de 30 saniye 65 tavlama </li><li> 35 döngü <ul><li> 9 denatürasyon15 sn için 5 ° C </li><li> 1 dakika boyunca 60 ° C&#39;da tavlama </li></ul></li><li> (° C 0.57 ° C lik artışlarla birlikte 20 dakika içinde 60-95 arası) eğrisi analizi eritin </li></ul></li></ol> 7. Sonuçların Analizi <ol><li> Aşağıdaki formül (antibiyotik türüne bağlı olarak) birini kullanarak kesme Ct değeri hesaplanır. <ul><li> Genel olarak: Cut-off Ct değeri = Ct değeri pozitif büyüme kontrolü + 0.5 x (Ct değeri negatif büyüme kontrolü - Ct değeri pozitif büyüme kontrolü) </li><li> Piperasilin, piperasilin / tazobaktam ve Gram-negatif çubuklar seftazidim; Amoksisilin, oksasilin ve trimetoprim / sulfametoksazol S.aureus, Enterococcus spp Amoksisilin:. Cut-off Ct değeri = Ct değeri pozitif büyüme kontrolü + 0.25 x (Ct değeri negatif büyüme kontrolü - Ct değeri pozitif büyüme kontrolü) </li></ul></li><li> Pozitif büyüme kontrol olarak steril demineralize H 2 O ile inkübe örnek kullanın.</li><li> Mikroorganizma bağlı olarak, uygun negatif büyüme kontrolü kullanın: <ul><li> Gram-negatif çubukları örnek antibiyotik karışımı ile inkübe </li><li> Enterococcus spp. Örnek 4 ° C&#39;de saklandı </li><li> S.aureus Örnek 4 ° C&#39;de saklandı </li></ul></li><li> Duyarlılığı (S) ya da aşağıdaki gibi test antibiyotik için suşunun direnç (R) belirleyin: <ul><li> Cut-off Ct değeri daha yüksek bir Ct değeri duyarlılık gösterir </li><li> Cut-off Ct değeri daha düşük bir Ct değeri direnç gösterir </li></ul></li></ol> 8. Temsilcisi Sonuçlar İki model organizmalar, Gram-negatif E. yani coli ve Gram-pozitif S. aureus, bakteriyel patojenler ve bunların antimikrobiyal profilinin belirlenmesi algılama ve tanımlama için kombine prosedür görselleştirmek için seçilir. Protokolünün birinci bölümü patojen bir tanıtımım içerir. Specific probları sekiz klinik mikroorganizmaların saptanması için tasarlanmıştır. Bakteriyel paneli dahil bir hedef varlığında, amplifikasyon eğrileri olarak oluşturulur ve Ct değerleri (Şekil 2) hesaplanır. Pozitif bir PCR sonucu dikkate cut-off değeri 35 bir Ct değeri ayarlanır. Şekil 2A, bir E.coli-enfekte kan kültür tanımlama profili gösterilmiştir. 16S evrensel prob iki ayrı reaksiyon karışımlarının içerdiği ve dolayısıyla iki amplifikasyon eğrileri (25.20 ve 25.95 Ct) üretir. Üçüncü sinyal E. için belirli bir prob türetilmiştir coli (27.04 Ct). Bir S. tanımlama aureus-enfekte kan kültürü Şekil 2B &#39;de gösterilmiştir. 16S evrensel prob 33.35 ve 33.71 amplifikasyonu sinyalleri vardır. Kalan iki sinyalleri Staphylococcus spp için özel problar türetilmiştir. ve S. aureus (32.48 ve 30.59 Ct). Şekil 3 de gösterilmiştir E.coli strain temsil eden bir antibiyotik duyarlılık testi amplifikasyon plot, örnek olarak Şekil 2A. Her bir hat bakteriyel örnek ile inkübe edildi olduğu bir antibiyotik temsil eder. Düşük bir Ct değeri ile bir örnek büyüme test antibiyotik için bir antibiyotik, direnç gösteren varlığında oluştu olduğu bir örnektir. Tersine, bir yüksek CT değeri, herhangi bir gelişme, çünkü test antibiyotiğe antibiyotik, hassasiyet gösteren etkili bir çalışma ile meydana gelmiş olan bir örneği gösterir. Tablo 1, E. antimikrobiyal profilini gösterir belirlenmesi coli ve S. aureus izolatlarının. Tüm Ct değerlerini bildirdi ve protokol metni (7.1) &#39;de belirtilen formülleri kullanarak, iki kesme Ct değerlerini hesaplanan vardır edilirdirenç ve duyarlılık ayırt yonunun. Bildirilen CT değeri hesaplanmıştır cut-off CT değeri (ya da tam tersi) &#39;den daha düşük olması durumunda deformasyon antibiyotiğe dirençli. <img alt="Şekil 1" src="/files/ftp_upload/3254/3254fig1.jpg" /> RDNA PCR gerçek zamanlı 16S kullanarak patojen tanımlanması ve antibiyotik duyarlılık testi prosedürü Şekil 1. Akış çizelgesi. <img alt="Şekil 2" src="/files/ftp_upload/3254/3254fig2.jpg" /> Şekil 2 Tanımlama testi:. Amplifikasyon araziler ve döngüsü eşik değerleri (Ct değerleri) spesifik problar nedensel patojenin tanımlanması için kullanılan ise pozitif kan kültürü, evrensel 16S rDNA sensörü tarafından algılanır.. Kan kültürü A. amplifikasyonu arsa E. içeren coli, S. içeren kan kültüründe B. amplifikasyonu arsa aureus; Pseu ae, Pseudomonaeruginosa gibi; uni, 16S evrensel probu; ecoli, Escherichia coli probu; Pseu sp, Pseudomonas spp. probu; S. pneumoniae, Streptococcus pneumoniae probu; Strep sp, Streptococcus spp. probu; Entero, Enterococcus spp. probu; S. aureus, Staphylococcus aureus probu; Staph sp, Staphylococcus spp. prob. <img alt="Şekil 3" src="/files/ftp_upload/3254/3254fig3.jpg" /> Bir E. antibiyotik duyarlılık testi Şekil 3. Amplifikasyon arsa coli (örnek 1) izole eder. Her eğrisi şekil değiştirme ile inkübe edildi olduğu bir antibiyotik temsil eder. Erken bir sinyal suşu test antibiyotik varlığında büyümüş ve antibiyotiğe dirençli ve böylece anlamına gelir yüksek bir bakteri yükü, kaynaklanır. Geç sinyalleri suşu antibiyotik varlığında yetiştirilen olmadığını göstermektedir, diğer bir deyişle, bu hassastır. <table border="1"><tbody&gt; <tr><td> Örnek 1: E. coli </td><td></td><td></td><td> Örnek 2: S. aureus </td><td></td><td></td></tr><tr><td> AST </td><td> Ct </td><td> R / S </td><td> AST </td><td> Ct </td><td> R / S </td></tr><tr><td> Amoksisilin 8 mg / L </td><td> 16,83 </td><td> R </td><td> Amoksisilin 0.25 mg / L </td><td> 21,03 </td><td> R </td></tr><tr><td> Amoksisilin-klavulanat 8/4 mg / L </td><td> 17,36 </td><td> R </td><td> Oksasilin 2 mg / L </td><td> 25,80 </td><td> S </td></tr><tr><td> Piperasilin 16 mg / L </td><td> 16,67 </td><td> R </td><td> Vankomisin 2 mg / L </td><td> 25,20 </td><td> S </td></tr><tr><td> Piperasilin-tazobaktama 16 /4 mg / L </td><td> 24,15 </td><td> S </td><td> Gentamisin 4 mg / L </td><td> 25,86 </td><td> S </td></tr><tr><td> Ciprofloxacin 1 mg / L </td><td> 29,72 </td><td> S </td><td> Trimetoprim-sulfametoksazol 2/38 mg / L </td><td> 24,62 </td><td> S </td></tr><tr><td> Seftazidim 1 mg / L </td><td> 24,03 </td><td> S </td><td></td><td></td><td></td></tr><tr><td> Seftazidim 8 mg / L </td><td> 26,58 </td><td> S </td><td></td><td></td><td></td></tr><tr><td> Gentamisin 4 mg / L </td><td> 29,83 </td><td> S </td><td></td><td></td><td></td></tr><tr><td> Trimetoprim-sulfametoksazol 2/38 mg / L </td><td> 27,60 </td><td> S </td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Negatif büyüme kontrolü (antibiyotik karışımı) </td><td> 30,41 </td><td></td><td> Negatif büyüme kontrolü (örnek 4 ° C&#39;de saklanır) </td><td> 27,42 </td><td></td></tr><tr><td> Pozitif büyüme kontrolü </td><td> 16,90 </td><td></td><td> Pozitif büyüme kontrolü </td><td> 20,22 </td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Cut-off Ct-değeri 1 * </td><td> 21,76 </td><td></td><td> Cut-off Ct-değeri 1 *** </td><td> 23,82 </td><td></td></tr><tr><td> Cut-off değeri Ct-2 ** </td><td> 18,75 </td><td></td><td> Cut-off Ct-değeri 2 **** </td><td> 22,02 </td><td></td></tr><tr><td> * Amoksisilin, amoksisilin-klavulanat, siprofloksasin, gentamic içintrimetoprim-sulfametoksazol, içinde ** Piperasilin, piperasilin-tazobaktama, seftazidim için </td><td></td><td></td><td> Vankomisin ve gentamisin için *** **** Amoksisilin, oksasilin ve trimetoprim-sulfametoksazol için </td><td></td><td></td></tr></tbody></table> Tablo 1. İki örnek (E.coli ve S.aureus) antibiyotik duyarlılık testi belirlenmesi. PCR testinin Ct değerleri otomatik olarak protokol metninde gösterilen formüller kullanılarak, pozitif ve negatif büyüme kontrolü gelen iki kesim Ct alues ​​hesaplar hangi olarak, bu excel dosyası kopyalandı. Bir antibiyotik cut-off Ct değerinden daha düşük bir değere Ct gösterirse CT değeri cut-off &#39;den daha yüksek ise, suşu, antibiyotiğe dirençli, strain hassastır.

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Biz ifşa hiçbir şey yok.

Burada açıklanan protokol patojenlerin hızlı bir şekilde tespit sağlar ve böylece kan akımı enfeksiyonu olan hastaların prognozu iyileştirmek yeterli antibiyotik erken verilmesinin yol açabilecek fonksiyonel bir antimikrobiyal profili sunmaktadır. Bir test talep koşullarına bağlı olarak, az yani düşük maliyet, yüksek verim, geri-dönüş süresi, test koşulları ayarlanabilir. Tüm bu işlemleri bir iş günü içinde yapılabilir. Dahası, protokol, iki parça önemli ölçüde dönüş süresine azaltır, eş zamanlı olarak gerçekleştirilebilmektedir. Burada sunulan gibi, tanımlama paneli hastanemizde klinik olarak en uygun bakteri bir seçimdir. Temel ilkesi 16S geninin hedef bölgede olduğundan, diğer mikroorganizmalar için özel olarak tasarlanmış probları ile tahlil eklenebilir. Tüm deney başlangıçta kan kültürlerinin hızlı analiz için amaçlanmıştır, fakat, aynı zamanda o işlenmesi için de kullanılabilirtermal örnek malzemeleri. Bu aynı zamanda antibiyotik duyarlılık testi için kullanılan antibiyotikler için böyledir: Daha fazla veya diğer antibiyotikleri yerel direniş kalıpları ve yönergelere göre eklenebilir.

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> Sodyum Klorür (NaCI) </td><td> Merck Kimyasallar </td><td> 106404 </td><td> Su içinde% 0.9 </td></tr><tr><td> Vacutainer SST Serum ayrıştırıcı tüp 5 ml </td><td> BD Diagnostic Systems </td><td> 367986 </td><td></td></tr><tr><td> Mueller Hinton II broth </td><td> BD Diagnostic Dystems </td><td> 212322 </td><td> 44 g / L su içinde </td></tr><tr><td> Rotixa 50 rs santrifüjleyin </td><td> Andreas Hettich GmbH &amp; Co KG </td><td> 4910 </td><td></td></tr><tr><td> 5415 D Santrifüj </td><td> Eppendorf </td><td> Durduruldu </td><td></td></tr><tr><td> Astarlar </td> &lt;td&gt; Sigma-Aldrich </td><td> na </td><td></td></tr><tr><td> Probları </td><td> Sigma-Aldrich/Applied Biyosistem </td><td> na </td><td></td></tr><tr><td> TaqManEnvironmental master miks 2.0 </td><td> Applied Biosystems </td><td> 4396838 </td><td></td></tr><tr><td> iQ SYBRGreen Supermix </td><td> Bio-Rad Laboratories BV </td><td> 170-8880 </td><td></td></tr><tr><td> Mikroamper Optik 96-Well Reaksiyon Plaka </td><td> Applied Biosystems </td><td> N8010560 </td><td></td></tr><tr><td> Mikroamper Optik Yapıştırıcı Film </td><td> Applied Biosystems </td><td> 4311971 </td><td></td></tr><tr><td> iQ 96-Well PCR Tabaklar </td><td> Bio-Rad Laboratories BV </td><td> 223-9441 </td><td></td></tr><tr><td> Microseal B Yapıştırıcı Contalar </td><td> Bio-Rad İşçiatories BV </td><td> MSB-1001 </td><td></td></tr><tr><td> Real-time PCR Algılama Sistemi </td><td> Applied Biosystems </td><td> ABI PRISM 7900HT </td><td></td></tr><tr><td> Real-Time PCR Algılama Sistemi </td><td> Bio-Rad Laboratories BV </td><td> MyiQ Tek Renk </td><td></td></tr></tbody></table>

one-day workflow scheme for bacterial pathogen detection and antimicrobial resistance testing from blood cultures

Kan dolaşımı enfeksiyonları nedeniyle sepsis muhtemel tezahürü, ağır sepsis ve septik şok 1 yüksek mortalite oranları ile ilişkilidir. Bu nedenle, yeterli bir antibiyotik hızlı bir idare kan dolaşımı enfeksiyonlarının tedavisinde önemli bir öneme sahiptir. Bu süreçte kritik unsur bakteri tanımlama ve antibiyotik duyarlılık testi sonuçlarının büyük ölçüde bağımlı zamanlaması vardır. Bu parametreler her iki rutin zaman alıcı ve ortalama 24-48 saat 2, 4 alır kültürü bazlı testi ile elde edilir. Bu çalışmanın amacı, DNA bazlı enfeksiyonu kan hızlı bir şekilde tespit için testler, hem de hızlı bir antimikrobiyal duyarlılık test geliştirmektir. İlk testi tür veya cins-spesifik problar 5 ile tamamlanmaktadır rDNA tabanlı gerçek zamanlı PCR testi bir eubacterial 16S olduğunu. Bu sondalar, Pseudomonas dahil Gram-negatif bakteriler kullanma., Pseudomonas aeruginosave Escherichia coli gibi Staphylococcus spp., Staphylococcus aureus, Enterococcus spp., Streptococcus spp. ve Streptococcus pneumoniae dahil olmak üzere Gram-pozitif bakterilerin ayırt edilebilir. Bu multiprobe deneyi kullanılarak, etken mikro-organizma bir ilk kimlik 2 saat sonra verildi. İkincisi, S. antibiyotik duyarlılık testi için yarı-moleküler testi geliştirdi aureus, Enterococcus spp. ve (fakültatif) aerob Gram-negatif çubuklar 6. Bu test PCR bakteri 7 büyüme ölçmek için kullanılan hangi bir çalışmaya dayanıyordu. Kan kültürlerinde doğrudan hasat bakterilerin antibiyotiklere bir seçim ile 6 saat süreyle inkübasyona ve SYBR Green-tabanlı gerçek zamanlı PCR testi sonrasında büyümesini engellediği tespit edilmiştir. Bu iki yöntem birlikte aynı gün (Şekil 1) uygun bir antibiyotik tedavisinin seçimi yönlendirmek olabilir. Sonuç olarakTanımlama ve antibiyotik duyarlılık hem de, moleküler analizi patojen tespiti için daha hızlı bir alternatif sunar ve kan dolaşımı enfeksiyonu tanısı iyileştirebilir.

Watch this Scientific Journal Video about One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures at JoVE.com

One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures

Bakteriyel patojen teşhis için basit bir günlük akışı düzeninin tasarım enfeksiyonu kan hızlı tanıma sağlar. Sekiz klinik bakteriyel hedefleri ve antibiyotik direnç profilleri dahil olması daha uygun tedaviye yol açabilir aynı gün klinisyen bir ilk fikir sunuyor.

Kan örneklerinden Bakteriyel Patojen Algılama ve Antimikrobiyal Direnç Testi için bir günlük iş akışı şeması

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. ...

one-day-workflow-scheme-for-bacterial-pathogen-detection

Research

JoVE Journal

Immunology and Infection

25.1K Görüntüleme.  Maastricht University Medical Center. Bakteriyel patojen teşhis için basit bir günlük akışı düzeninin tasarım enfeksiyonu kan hızlı tanıma sağlar. Sekiz klinik bakteriyel hedefleri ve antibiyotik direnç profilleri dahil olması daha uygun tedaviye yol açabilir aynı gün klinisyen bir ilk fikir sunuyor.

Video: One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures

Protocol for Recombinant RBD-based SARS Vaccines: Protein Preparation, Animal Vaccination and Neutralization Detection

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide variety of pathogens 1-3. Since the mammalian cell system is capable of post-translational modification, thus forming properly folded and glycosylated proteins, recombinant proteins expressed in mammalian cells have shown the greatest potential to maintain high antigenicity and immunogenicity 4-6.
Although no new cases of SARS have been reported since 2004, future outbreaks are a constant threat; therefore, the development of vaccines against SARS-CoV is a prudent preventive step and should be carried out. The RBD of SARS-CoV S protein plays important roles in receptor binding and induction of specific neutralizing antibodies against virus infection 7-9. Therefore, in this protocol, we describe novel methods for developing a RBD-based subunit vaccine against SARS. Briefly, the recombinant RBD protein (rRBD) was expressed in culture supernatant of mammalian 293T cells to obtain a correctly folded protein with proper conformation and high immunogenicity 6. The transfection of the recombinant plasmid encoding RBD to the cells was then performed using a calcium phosphate transfection method 6,10 with some modifications. Compared with the lipid transfection method 11,12, this modified calcium phosphate transfection method is cheaper, easier to handle, and has the potential to reach high efficacy once a transfection complex with suitable size and shape is formed 13,14. Finally, a SARS pseudovirus neutralization assay was introduced in the protocol and used to detect the neutralizing activity of sera of mice vaccinated with rRBD protein. This assay is relatively safe, does not involve an infectious SARS-CoV, and can be performed without the requirement of a biosafety-3 laboratory 15.
The protocol described here can also be used to design and study recombinant subunit vaccines against other viruses with class I fusion proteins, for example, HIV, respiratory syncytial virus (RSV), Ebola virus, influenza virus, as well as Nipah and Handra viruses. In addition, the methods for generating a pseudovirus and subsequently establishing a pseudovirus neutralization assay can be applied to all these viruses.

This protocol describes a general procedure for studying recombinant receptor-binding domain (RBD)-based subunit vaccines against SARS. It includes methods for transfection and expression of RBD protein in 293T cells, immunization of mice with RBD and detection of neutralization activity of mouse sera using an established SARS pseudovirus neutralization assay.

Rekombinant RBD tabanlı SARS Aşılar için Protokol: Protein hazırlanması, Hayvan Aşılama ve Nötralizasyon Algılama

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide ...

İmmünoloji ve Enfeksiyon

Invasion of Human Cells by a Bacterial Pathogen

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be applied to the study of cell invasion by virtually any culturable bacterium. The stages at which specific aspects of invasion can be studied, such as the role of actin rearrangement or caveolae, will be highlighted. Host cells are grown in flasks and when ready for use are seeded into 24-well plates containing Thermanox coverslips. Using coverslips allows subsequent removal of the cells from the wells to reduce interference from serum proteins deposited onto the sides of the wells (to which S. aureus would attach). Bacteria are grown to the required density and washed to remove any secreted proteins (e.g. toxins). Coverslips with confluent layers of endothelial cells are transferred to new 24-well plates containing fresh culture medium before the addition of bacteria. Bacteria and cells are then incubated together for the required amount of time in 5% CO2 at 37&deg;C. For S. aureus this is typically between 15-90 minutes. Thermanox coverslips are removed from each well and dip-washed in PBS to remove unattached bacteria. If total associated bacteria (adherent and internalised) are to be quantified, coverslips are then placed in a fresh well containing 0.5% Triton X-100 in PBS. Gentle pipetting leads to complete cell lysis and bacteria are enumerated by serial dilution and plating onto agar. If the number of bacteria that have invaded the cells is needed, coverslips are added to wells containing 500 &mu;l tissue culture medium supplemented with gentamicin and incubation continued for 1 h, which will kill all external bacteria. Coverslips can then be washed, cells lysed and bacteria enumerated by plating onto agar as described above. If the experiment requires direct visualisation, coverslips can be fixed and stained for light, fluorescence or confocal microscopy or prepared for electron microscopy.

A general protocol for the study of invasion of host cells by a bacterial pathogen, focusing on Staphylococcus aureus and human endothelial cells.

Bakteriyel Patojen İnsan Hücreleri Invasion

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

Bakteriyel Yapışma Çalışma Kesme Stres Tanıtımı

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

Gribi için yüksek verimlilik Tespit Yöntemi

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

Ixodes scapularis Keneler gelen tükürük, Tükürük Bezi ve hemolimf Koleksiyonu

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Nanomechanics of Drug-target Interactions and Antibacterial Resistance Detection

The cantilever sensor, which acts as a transducer of reactions between model bacterial cell wall matrix immobilized on its surface and antibiotic drugs in solution, has shown considerable potential in biochemical sensing applications with unprecedented sensitivity and specificity1-5. The drug-target interactions generate surface stress, causing the cantilever to bend, and the signal can be analyzed optically when it is illuminated by a laser. The change in surface stress measured with nano-scale precision allows disruptions of the biomechanics of model bacterial cell wall targets to be tracked in real time. Despite offering considerable advantages, multiple cantilever sensor arrays have never been applied in quantifying drug-target binding interactions.
Here, we report on the use of silicon multiple cantilever arrays coated with alkanethiol self-assembled monolayers mimicking bacterial cell wall matrix to quantitatively study antibiotic binding interactions. To understand the impact of vancomycin on the mechanics of bacterial cell wall structures1,6,7.&#160;We developed a new model1 which proposes that cantilever bending can be described by two independent factors; i) namely a chemical factor, which is given by a classical Langmuir adsorption isotherm, from which we calculate the thermodynamic equilibrium dissociation constant (Kd) and ii) a geometrical factor, essentially a measure of how bacterial peptide receptors are distributed on the cantilever surface. The surface distribution of peptide receptors (p) is used to investigate the dependence of geometry and ligand loading. It is shown that a threshold value of p ~10% is critical to sensing applications. Below which there is no detectable bending signal while above this value, the bending signal increases almost linearly,&#160;revealing that stress is a product of a local chemical binding factor and a geometrical factor combined by the mechanical connectivity of reacted regions and provides a new paradigm for design of powerful agents to combat superbug infections.

Acquired resistance to antibiotics is a major public healthcare problem and is presently ranked by the WHO as one of the greatest threats to human life. Here we describe the use of cantilever technology to quantify antibacterial resistance, critical to the discovery of novel and powerful agents against multidrug resistant bacteria.

İlaç-hedef Etkileşimleri ve Antibakteriyel Direnç Algılama Nanomekaniğe

The cantilever sensor, which acts as a transducer of reactions between model bacterial cell wall matrix immobilized on its surface and antibiotic drugs in ...

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

Staphylococcal Cassette Chromosome mec (SCCmec) typing is a very important molecular tool for understanding the epidemiology and clonal strain relatedness of methicillin-resistant Staphylococcus aureus (MRSA), particularly with the emerging outbreaks of community-associated MRSA (CA-MRSA) occurring on a worldwide basis. Traditional PCR typing schemes classify SCCmec by targeting and identifying the individual mec and ccr gene complex types, but require the use of many primer sets and multiple individual PCR experiments. We designed and published a simple multiplex PCR assay for quick-screening of major SCCmec types and subtypes I to V, and later updated it as new sequence information became available. This simple assay targets individual SCCmec types in a single reaction, is easy to interpret and has been extensively used worldwide. However, due to the sophisticated nature of the assay and the large number of primers present in the reaction, there is the potential for difficulties while adapting this assay to individual laboratories. To facilitate the process of establishing a MRSA SCCmec assay, here we demonstrate how to set up our multiplex PCR assay, and discuss some of the vital steps and procedural nuances that make it successful.

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

We demonstrate a simple multiplex PCR assay for quick-screening and typing of Staphylococcal Cassette Chromosome mec (SCCmec) types I-V for methicillin-resistant Staphylococcus aureus, and provide some of the vital steps and procedural nuances that make it successful for adapting this assay to individual laboratories.

Metisiline diren

Staphylococcal Cassette Chromosome mec (SCCmec) typing  is a very important molecular tool for understanding the epidemiology and  clonal strain ...

Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a rapid identification of the infectious agent to guide optimal antibiotic treatment. The analysis of Gram stains from positive blood culture can be rapidly conducted and already significantly impact the antibiotic regimen. However, the accurate identification of the infectious agent is still required to establish the optimal targeted treatment. We present here a simple and fast bacterial pellet preparation from a positive blood culture that can be used as a sample for several essential downstream applications such as identification by MALDI-TOF MS, antibiotic susceptibility testing (AST) by disc diffusion assay or automated AST systems and by automated PCR-based diagnostic testing. The performance of these different identification and AST systems applied directly on the blood culture bacterial pellets is very similar to the performance normally obtained from isolated colonies grown on agar plates. Compared to conventional approaches, the rapid acquisition of a bacterial pellet significantly reduces the time to report both identification and AST. Thus, following blood culture positivity, identification by MALDI-TOF can be reported within less than 1 hr whereas results of AST by automated AST systems or disc diffusion assays within 8 to 18 hr, respectively. Similarly, the results of a rapid PCR-based assay can be communicated to the clinicians less than 2 hr following the report of a bacteremia. Together, these results demonstrate that the rapid preparation of a blood culture bacterial pellet has a significant impact on the identification and AST turnaround time and thus on the successful outcome of patients suffering from bloodstream infections.

A rapid bacterial pellet preparation from a positive blood culture can be used as a sample for applications such as identification by MALDI-TOF, Gram staining, antibiotic susceptibility testing and PCR-based test. The results can be rapidly communicated to clinicians to improve the outcome of patients suffering from bloodstream infections.

Hızlı Bakteriyel Teşhis ve Antibiyotik Duyarlılık Testi için bir Kan Kültürü Peleti hazırlanması

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a ...

Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of Eimeria that Infect Chickens

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the absence of effective control clinical coccidiosis can be devastating. Resistance to the chemoprophylactics frequently used to control Eimeria is common and sub-clinical infection is widespread, influencing feed conversion ratios and susceptibility to other pathogens such as Clostridium perfringens. Despite the availability of polymerase chain reaction (PCR)-based tools, diagnosis of Eimeria infection still relies almost entirely on traditional approaches such as lesion scoring and oocyst morphology, but neither is straightforward. Limitations of the existing molecular tools include the requirement for specialist equipment and difficulties accessing DNA as template. In response a simple field DNA preparation protocol and a panel of species-specific loop-mediated isothermal amplification (LAMP) assays have been developed for the seven Eimeria recognised to infect the chicken. We now provide a detailed protocol describing the preparation of genomic DNA from intestinal tissue collected post-mortem, followed by setup and readout of the LAMP assays. Eimeria species-specific LAMP can be used to monitor parasite occurrence, assessing the efficacy of a farm&#8217;s anticoccidial strategy, and to diagnose sub-clinical infection or clinical disease with particular value when expert surveillance is unavailable.

Loop-mediated Isothermal Amplification LAMP Assays for the Species-specific Detection of Eimeria that Infect Chickens

Diagnosis of Eimeria infection in chickens remains demanding. Parasite morphology- and host pathology-led approaches are commonly inconclusive, while molecular approaches based on PCR have proven demanding in cost and expertise. The aim of this protocol is to establish loop-mediated isothermal amplification (LAMP) as a straightforward molecular diagnostic for eimerian infection.

Türler özgü Algılama Loop-aracılı İzotermal Amplifikasyon (LAMP) Tahliller<em&gt; Eimeria</em&gt; Tavuklar bulaştırmak ki

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the ...

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.