Biz laboratuvarda tekniklerini uygulamak için onların yardım için Lozan Üniversitesi Hastanesi Merkezi bakteriyoloji laboratuarının teknisyenleri teşekkür ederim.

Bu protokol geliştirilmiş ve rutin bir aracı olarak uygulamaya geçirilmeden önce kurumun araştırma ve geliştirme süreçleri ve etik kurallarını takip valide edilmiştir. Amonyum Klorür Eritrosit parçalayan Usulü ile bir Kan Kültürü Bakteriyel Pelet 1. Hazırlık <ol><li> Daha sonra santrifüjleme için pozitif kan kültürünün hazırlanması. <ol><li> Kan kültürü kapağını sterilize edin. Şişenin kapağı üzerinde% 70 etanol ekleyin ve yakmak. NOT: Bir laminer akış kaputun altında bu adımı yapmayın. </li><li> Laminar akış için şişe taşıyın. 20 G şırınga ile kan kültürü 5 ml toplayın. </li><li> Steril su (H2O) 45 ml içeren 50 ml&#39;lik bir santrifüj tüpüne kan kültür 5 ml ilave edilir ve örnek karıştırın. </li></ol></li><li> Liziz santrifüjleme ile kan kültürü bakteryel tanelerin hazırlanması. <ol><li> Oda sıcaklığında 10 dakika boyunca 1000 x g&#39;de santrifüjleyin. </li><li> Remolaboratuar vakum pompası ile (özellikle H2O ve kan hücreleri içeren) süpernatant ettik. </li><li> Solüsyonu (0.15 M NH4CI, 1 mM KHCO 3) yokedici ev yapımı amonyum klorür 1 ml pelletini. Kazıyarak ve tüp vorteks pelet bölmek ve oda sıcaklığında 10 dakika boyunca 140 x g&#39;de santrifüjleyin. </li><li> Ağırlıklı olarak kırmızı kan hücreleri enkaz içeren süpernatant atın. <ol><li> Topak hemorajik gerçekleşmemesi durumunda, başka bir artık kırmızı kan hücreleri lize etmek için ve pelet yıkamak için steril damıtık H 2 0 2 ml pelletini. Oda sıcaklığında 10 dakika boyunca 140 x g&#39;de santrifüjleyin ve süpernatant atılır. </li></ol></li><li> H2O 200 ul pelletini </li></ol></li><li> Çeşitli seçici ve seçici olmayan agar ortamında kan kültürünün alt kültür. <ol><li> 20 G şırınga ile kan kültürü 1 ml toplayın. </li><li> Kan kültürü int 1 damla aşılamakkan agar, çikolata agar, McConkey agar ve Schaedler agar dört kadran o. </li><li> % 5 CO2 atmosferinde 37 ° C&#39;de kan agarı ve çikolata agar plakaları inkübe, normal bir atmosferde Mc Conkey agar ve anaerobik koşullarda Schaedler agar. </li><li> Farklı medya üzerinde bakteri büyümesini izleyin. </li><li> Bakteriyel saflık için kontrol edin. </li></ol></li></ol> MALDI-TOF MS ile 2. Tanımlama <ol><li> Kan pelet doğrudan tanımlama. <ol><li> Transferi 1 MALDI hedef plaka üzerinde kan pelet ul ve 42 ° C ısıtma platformu üzerinde kurumaya bırakın. </li><li> 1 ul% 70 formik asit ile örnek Yerleşimi ve 42 ° C ısıtma platformu üzerinde kurumaya bırakın. </li><li> MALDI matris 1 ul numuneyi, kaplama ve bir 42 ° C ısıtma platform üzerinde kurumasına izin (α-siyano-4-hidroksisinamik asetonitril-% 2.5 trifloroasetik asit,% 50 asit çözeltisi, doymuş). </li><li> MAL geçinÜretici tarafından tarif edildiği gibi, bir MALDI-TOF MS sistemi DI-TOF MS analizi. </li><li> Kimlik puanı (puan &lt;2 ile örneğin) tür düzeyinde geçersiz ise, kan numunesinin pelet bir protein çıkarımı gerçekleştirin. </li></ol></li><li> Kan kültürü topağından proteini ekstraksiyon işleminden sonra tanımlanması. <ol><li> Oda sıcaklığında 2 dakika boyunca 13000 x g&#39;de 70% etanol ve 1 ml santrifüj ile pelet 20 ul karıştırın. </li><li> Süpernatant atılır ve% 70 formik asit ve 25 ul pelet% 100 asetonitril 25 ul ekle. Vortex şiddetle pelet tekrar süspansiyon. Vorteks önce pipet uçları ile onları parçalayarak sert pelet süspanse. </li><li> Oda sıcaklığında 2 dakika için 13,000 x g&#39;de santrifüjleyin. Aktarım 1, bir MALDI hedef plaka üzerinde yüzer ul (ekstre proteinleri ihtiva etmektedir) ve 42 ° C&#39;de ısıtma platformunda kurumaya bırakın. 2.1.2 anlatıldığı gibi devam edin. </li></ol></li></ol> 3. Bakteriyel IDENTIFBir Otomatik Mikrobiyal Sistemi ile ication ve Antibiyotik Duyarlılık Testleri <ol><li> Otomatik bir sistem ile bakteriyel mikrobiyal kimlik ve AST için bakteriyel süspansiyonun hazırlanması. <ol><li> Mikrobiyal otomatik sistem ile uyumlu steril% 0.45 NaCI çözeltisi 3 ml içeren plastik bir tüp hazırlanır. </li><li> 0,6-0,8 bir McFarland bulanıklığa tekabül eden bir bakteri süspansiyonu elde etmek için% 0,45 NaCl çözeltisi içinde kan kültürü pelet yeterli hacim. Bir densitometer makine kullanarak bulanıklığı ölçün. Not: Kan kültürü bakteri topağın hacminin 0,6-0,8 bir McFarland bulanıklık elde etmek için 50 ila 200 ul arasında değişir. </li></ol></li><li> Üretici tarafından tarif edildiği şekilde tanımlanması için otomatik Gram-pozitif (GP) veya Gram-negatif (GN) kartları ve Gram pozitif koklar ve Gram-negatif bakterilerin antibiyotik duyarlılık testi için otomatik AST kartları inoküle. NOT: identificatioMALDI-TOF MS kimlik skor değeri&gt; 2 ise n GP veya GN kartlarla uygulanmaz. Kan agar (GP ve GN) ve MacConkey agarı (GN) plakaları üzerinde bakteri süspansiyonu alt-kültürleme ile bakteri süspansiyonu saflığını kontrol edin. </li></ol> Disk Difüzyon Testi ile 4. Antibiyotik Duyarlılık Testleri Disk difüzyon deneyi antimikrobiyal duyarlılık testi (EUCAST, Sürüm 3.0 Nisan 2013 www.eucast.org) Avrupa komitesi tarafından açıklanmıştır. <ol><li> Bakteriyel süspansiyonun hazırlanması disk difüzyon deneyi ile antimikrobiyal duyarlılık testleri. <ol><li> Steril% 0.9 NaCl çözeltisi, 3 ml ihtiva eden bir cam tüp hazırlanır. </li><li> 0.5 McFarland bulanıklığa tekabül eden bir bakteri süspansiyonu elde etmek için% 0.9 NaCI çözeltisi içinde kan kültürü pelet yeterli hacim. Bir densitometer makine kullanarak bulanıklığı ölçün. NOT: Kan kültürü hacmiBakteriyel küçük topak, 0,6-0,8 arasında bir McFarland bulanıklık elde etmek için 50 ila 200 ul arasında değişir. </li><li> Bakteriyel süspansiyonun vorteksleyin. </li></ol></li><li> Mueller-Hinton agar (MH) veya Mueller-Hinton agar-agar titiz organizmalar (TKF) üzerine bakteri süspansiyonu aşılanması (MH,% 5 at kanı defibrine ve 20 mg / β-nikotinamid adenin dinükleotid L ile takviye edilmiştir). <ol><li> Bakteriyel süspansiyon içinde bir steril pamuklu çubuk daldırma ve hafifçe cam tüpün iç duvarında çubukla çevirerek fazla sıvıyı çıkarın. </li><li> Üç yönde silmeli veya otomatik bir plaka rotator ile MH / TKF agar levhasının tüm yüzeyi üzerine eşit şekilde yayılır bakteri süspansiyonu. </li></ol></li><li> Aşılanmış agar plakaları üzerinde antimikrobiyal disklerin uygulanması. <ol><li> El ile kurutuldu, aşılanmış agar plakalarının yüzeyi üzerine veya bir duyarlılık bir disk dağıtıcısı kullanılarak yakından diskleri uygulanır. </li><li> Uygun tempe inkübe plakasıdüşene ve antimikrobiyal duyarlılık testi EUCAST disk difüzyon yöntemi tarif edildiği gibi atmosfer (Sürüm 3.0 Nisan 2013 www.eucast.org). </li></ol></li></ol> 5. Otomatik MRSA Belirlenmesi için Tanı Testi PCR tabanlı <ol><li> Bir mikropipet kullanarak, 20 sn boyunca PCR tabanlı tanı test kiti ve vorteks otomatik MRSA ile sağlanan örnek reaktif şişenin içine kan kültürü pelet 50 ul aktarın. </li><li> 1 ml&#39;lik bir mikropipet kullanılarak kartuşun örnek portuna numuneyi dağıtmak. PCR sisteminin otomatik olarak kartuşunun takılması ve üretici tarafından tarif edildiği gibi tahlil başlar. </li></ol>

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	<li>Alberti, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+of+sepsis+and+infection+in+ICU+patients+from+an+international+multicentre+cohort+study.">Epidemiology of sepsis and infection in ICU patients from an international multicentre cohort study.</a> Intensive care medicine. 28, 108-121 (2002).</li><li>Angus, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+of+severe+sepsis+in+the+United+States:+analysis+of+incidence,+outcome,+and+associated+costs+of+care.">Epidemiology of severe sepsis in the United States: analysis of incidence, outcome, and associated costs of care.</a> Critical care medicine. 29, 1303-1310 (2001).</li><li>Vincent, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sepsis+in+European+intensive+care+units+results+of+the+SOAP+study.">Sepsis in European intensive care units results of the SOAP study.</a> Critical care medicine. 34, 344-353 (2006).</li><li>Micek, S. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Empiric+combination+antibiotic+therapy+is+associated+with+improved+outcome+against+sepsis+due+to+Gram-negative+bacteria+a+retrospective+analysis.">Empiric combination antibiotic therapy is associated with improved outcome against sepsis due to Gram-negative bacteria a retrospective analysis.</a> Antimicrob Agents Chemother. 54, 1742-1748 (2010).</li><li>Seifert, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+clinical+importance+of+microbiological+findings+in+the+diagnosis+and+management+of+bloodstream+infections.">The clinical importance of microbiological findings in the diagnosis and management of bloodstream infections.</a> Clin Infect Dis. 48, S238-S245 (2009).</li><li>Barenfanger, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decreased+mortality+associated+with+prompt+Gram+staining+of+blood+cultures.">Decreased mortality associated with prompt Gram staining of blood cultures.</a> American journal of clinical pathology. 130, 870-876 (2008).</li><li>Munson, E. L., Diekema, D. J., Beekmann, S. E., Chapin, K. C., Doern, G. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+treatment+of+bloodstream+infection:+laboratory+reporting+and+antimicrobial+management.">Detection and treatment of bloodstream infection: laboratory reporting and antimicrobial management.</a> J Clin Microbiol. 41, 495-497 (2003).</li><li>Clerc, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+matrix-assisted+laser+desorption+ionization+time-of-flight+mass+spectrometry+on+the+clinical+management+of+patients+with+Gram-negative+bacteremia+a+prospective+observational+study.">Impact of matrix-assisted laser desorption ionization time-of-flight mass spectrometry on the clinical management of patients with Gram-negative bacteremia a prospective observational study.</a> Clin Infect Dis. 56, 1101-1107 (2013).</li><li>Meex, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+identification+of+bacteria+from+BacT/ALERT+anaerobic+positive+blood+cultures+by+MALDI-TOF+MS+MALDI+Sepsityper+kit+versus+an+in-house+saponin+method+for+bacterial+extraction.">Direct identification of bacteria from BacT/ALERT anaerobic positive blood cultures by MALDI-TOF MS MALDI Sepsityper kit versus an in-house saponin method for bacterial extraction.</a> Journal of medical microbiology. 61, 1511-1516 (2012).</li><li>Moussaoui, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix-assisted+laser+desorption+ionization+time-of-flight+mass+spectrometry+identifies+90%+of+bacteria+directly+from+blood+culture+vials.">Matrix-assisted laser desorption ionization time-of-flight mass spectrometry identifies 90% of bacteria directly from blood culture vials.</a> Clin Microbiol Infect. 16, 1631-1638 (2010).</li><li>Seng, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ongoing+revolution+in+bacteriology+routine+identification+of+bacteria+by+matrix-assisted+laser+desorption+ionization+time-of-flight+mass+spectrometry.">Ongoing revolution in bacteriology routine identification of bacteria by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.</a> Clin Infect Dis. 49, 543-551 (2009).</li><li>Stevenson, L. G., Drake, S. K., Murray, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+identification+of+bacteria+in+positive+blood+culture+broths+by+matrix-assisted+laser+desorption+ionization-time+of+flight+mass+spectrometry.">Rapid identification of bacteria in positive blood culture broths by matrix-assisted laser desorption ionization-time of flight mass spectrometry.</a> J Clin Microbiol. 48, 444-447 (2010).</li><li>Croxatto, A., Prod&amp;34hom, G., Greub, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+MALDI-TOF+mass+spectrometry+in+clinical+diagnostic+microbiology.">Applications of MALDI-TOF mass spectrometry in clinical diagnostic microbiology.</a> FEMS Microbiol Rev. 36, 380-407 (2012).</li><li>Chen, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+bacterial+identification+in+positive+blood+cultures+by+use+of+two+commercial+matrix-assisted+laser+desorption+ionization-time+of+flight+mass+spectrometry+systems.">Direct bacterial identification in positive blood cultures by use of two commercial matrix-assisted laser desorption ionization-time of flight mass spectrometry systems.</a> J Clin Microbiol. 51, 1733-1739 (2013).</li><li>Prod&amp;34hom, G., Bizzini, A., Durussel, C., Bille, J., Greub, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix-assisted+laser+desorption+ionization-time+of+flight+mass+spectrometry+for+direct+bacterial+identification+from+positive+blood+culture+pellets.">Matrix-assisted laser desorption ionization-time of flight mass spectrometry for direct bacterial identification from positive blood culture pellets.</a> J Clin Microbiol. 48, 1481-1483 (2010).</li><li>Prod&amp;34hom, G., Durussel, C., Greub, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+blood-culture+bacterial+pellet+preparation+for+faster+accurate+direct+bacterial+identification+and+antibiotic+susceptibility+testing+with+the+VITEK+2+system.">A simple blood-culture bacterial pellet preparation for faster accurate direct bacterial identification and antibiotic susceptibility testing with the VITEK 2 system.</a> Journal of medical microbiology. 62, 773-777 (2013).</li><li>Clerc, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix-assisted+laser+desorption+ionization+time-of-flight+mass+spectrometry+and+PCR-based+rapid+diagnosis+of+Staphylococcus+aureus+bacteraemia.">Matrix-assisted laser desorption ionization time-of-flight mass spectrometry and PCR-based rapid diagnosis of Staphylococcus aureus bacteraemia.</a> Clin Microbiol Infect. , (2013).</li><li>Martiny, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+rapid+microbial+identification+directly+from+positive+blood+cultures+using+matrix-assisted+laser+desorption/ionization+time-of-flight+mass+spectrometry+on+patient+management.">Impact of rapid microbial identification directly from positive blood cultures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry on patient management.</a> Clin Microbiol Infect. 19, E568-E581 (2013).</li><li>Stoneking, L. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Would+earlier+microbe+identification+alter+antibiotic+therapy+in+bacteremic+emergency+department+patients.">Would earlier microbe identification alter antibiotic therapy in bacteremic emergency department patients.</a> The Journal of emergency medicine. 44, 1-8 (2013).</li><li>Nordmann, P., Dortet, L., Poirel, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+detection+of+extended-spectrum-beta-lactamase-producing+Enterobacteriaceae.">Rapid detection of extended-spectrum-beta-lactamase-producing Enterobacteriaceae.</a> J Clin Microbiol. 50, 3016-3022 (2012).</li></ol>

Yazarlar hiçbir rakip mali çıkarlarının olmadığını beyan ederim.

Geleneksel pozitif kan kültürü tanı yaklaşımları ile karşılaştırıldığında, amonyum klorür parçalama santrifüj yaklaşımı kullanarak bakteriyel pelet hızlı edinimi 24 saat ve 48 saat 24 ile AST rapor süresi (Şekil 1 ve 16&#39;dan kimlik rapor süresini azaltmak 3). Uygun antibiyotik tedavisi hızlı bir giriş kan dolaşımı enfeksiyonları muzdarip hastaların sonuçlarını iyileştirmek esastır. Böylece, yaklaşık 8 ila 16 saat ...

Hastanede yatan hastalarda kan dolaşımı enfeksiyonları ve sepsis morbidite ve mortalitenin önemli bir nedenidir. Böylece, enfeksiyonlar kan dolaşımına bağlı mortalite hastanede yatan hasta% 37 ila% 14 gözlenir ve yoğun bakım ünitelerinde hasta 1-3% 35 artabilir. Enfeksiyöz ajan hızlı kimlik optimum antimikrobiyal tedaviyi yönlendirmek ve antimikrobiyal tedavi 4,5&#39;den başarılı sonucunu artırmak için çok önemlidir. Pozitif kan kültürü Gram lekelerin hızlı analiz zaten antimikrobiyal tedavinin 6,7 ama enfeksiyöz ajan doğru kimlik uyarlaması üzerinde önemli bir etkiye sahiptir hastalara iyi adapte antibiyotik tedavi sağlamak için gereklidir. Örneğin, farklı antibiyotik tedavi rejimleri enterokok ve Gram boyama ile ayırt etmek zordur streptokoklar ile bakteriyemi takip uygulanması gerekir. Benzer şekilde, türlere tanımlama levEl 8-laktamlara β karşı arttırılmış bir direnç kazandıran bir kromozom AMPc geni kodlayan, Gram negatif enterobakteriler tespit etmek için gereklidir. Pozitif kan kültürü ile, geleneksel tanısal yaklaşım biyokimyasal testler, farklı seçici medya büyüme ve otomatik mikrobik tanımlama sistemleri dahil olmak üzere çeşitli yaklaşımlar ile ilave inkübasyon öncesinde kimlik birkaç saat gerektirir farklı agar plakaları üzerinde bulaşıcı ajan, altkültürü etmektir. Geleneksel bir yaklaşım teşhis sonuçlarına süresi yaklaşık 1-3 gün taşımaktadır. Mikroorganizmaların hızlı tespit edilmesi için matris destekli lazer desorpsiyon / iyonizasyon time-of-flight kütle spektrometresi (MALDI-TOF) teknolojisinin ortaya hızlı bir şekilde pozitif kan şirketinden de agar plakaları üzerinde büyüyen kolonilerin mikroorganizmaların değil tanımlamak için yeni bir aleti kültürler (Şekil 1) 9-12. ThKan kültürlerinden bir enfeksiyon ajanı tanımlamak için MALDI-TOF E kullanımı önemli bir kaç dakika yerine geleneksel yöntemlerle gerekli saat ve gün sonuçlarına süresini azalttı. Croxatto ve ark. 13 tarafından ele alındığı şekilde, MALDI-TOF tanımlama etkinliği mikroorganizmaların saflık ve miktarı da dahil olmak üzere farklı parametrelere dayanır. Bu iki kriter kolaylıkla agar plakaları üzerinde büyüyen koloniler elde edilen ayrı fakat MALDI-TOF kimlik müdahale edebilir bir çok selüler ve protein bileşenlerini içeren kan kültürü gibi kompleks örnekleri, bakteriyel zenginleştirme ve arıtması için bir ön tedavi analitik gerekmektedir. Kan kültüründen çeşitli mikroorganizmaların izolasyon yöntemleri bakteriyel çıkarma 9,14, serum ayırıcı yöntemi 10 saponin ya da diğer hafif deterjanlar yöntemi dahil, bir çok çalışma kullanılmıştır, lizis santrifüj yöntemleri 12 </skadar&gt; ve bu sepsityper kiti gibi ticari çözümleri. Bizim bakteriyoloji tanısal laboratuvar MALDI-TOF ile bakteri ve maya hızlı tanımlama ve otomatik tanımlama sistemleri (Şekil 2) 15 izin amonyum klorür eritrosit-lizis dayalı basit bir kan kültürü, bakteriyel pelet hazırlık geliştirmiştir. Bu kan kültürü pelet hazırlık aynı zamanda Gram boyama gibi diğer doğrudan aşağı uygulamalar için bir örnek sunmak, metisilin dirençli Staphylococcus aureus hızlı tespiti (MRSA) için POCT-PCR&#39;lerine ve antibiyotik duyarlılık testi gibi otomatik PCR tabanlı tanı testleri AST otomatik sistemleri ve / veya agar plakaları üzerinde disk difüzyon deneyleri (Şekil 3) ile incelenmiştir. Prod&#39;hom ve arkadaşları tarafından açıklandığı gibi, bu çalışmada, kan kültürü bakteryel tanelerin hazırlanması için farklı adımları açıklar. 15 (Şekil 4). Biz de de olacakkâtip kan kültürü pelet yapılabilir ana üç uygulama için protokoller: otomatik Enterobacteriaceae için sistemleri 16 ve stafilokok ve otomatik PCR-ile MALDI-TOF 15, kimlik (ID) ve antibiyotik duyarlılık testi (AST) tarafından Identification MRSA 17&#39;nin saptanması için tanısal test göre.

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			<td height="20" style="height:20px;width:268px;">20 needle gauge&#160;</td>
			<td style="width:268px;">Terumo, Leuven, Belgium</td>
			<td style="width:239px;">NN-2038R</td>
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		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">50 ml Falcon tube</td>
			<td>BD, Franklin Lakes, NJ, USA</td>
			<td>352070</td>
			<td>50 ml centrifuge tubes</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Ammonium chlorure</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>101145</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Potassium hydrogen carbonate</td>
			<td>Fluka, St. Louis, MO, USA&#160;</td>
			<td>60340</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Formic acid</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA&#160;</td>
			<td>F0507</td>
			<td>Flammable, corrosive</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">a-Cyano-4-hydroxycinnamic acid</td>
			<td>Fluka, St. Louis, MO, USA&#160;</td>
			<td>70990</td>
			<td>Acute toxicity</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Acetonitrile</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA&#160;</td>
			<td>271004</td>
			<td>Flammable, acute toxicity</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Trifluoroacetic acid</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA&#160;</td>
			<td>T6508</td>
			<td>Corrosive, acute toxicity</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Vitek 2 60 instrument</td>
			<td>Biom&#233;rieux, Marcy-l&#39;Etoile, France</td>
			<td>27202</td>
			<td>automated microbial system instrument</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Vitek 2 Gram-positive (GP) card</td>
			<td>Biom&#233;rieux, Marcy-l&#39;Etoile, France</td>
			<td>21342</td>
			<td>automated GP&#160; identification card</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Vitek 2 AST-P580 card</td>
			<td>Biom&#233;rieux, Marcy-l&#39;Etoile, France</td>
			<td>22233</td>
			<td>automated microbial AST system</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Vitek 2 Gram-negative (GN) card</td>
			<td>Biom&#233;rieux, Marcy-l&#39;Etoile, France</td>
			<td>21341</td>
			<td>automated GN&#160; identification card</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Vitek 2 AST-N242 card</td>
			<td>Biom&#233;rieux, Marcy-l&#39;Etoile, France</td>
			<td>413391</td>
			<td>automated microbial AST system</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Xpert MRSA</td>
			<td>Cepheid, Sunnyvale, Ca, USA</td>
			<td>&#160;GXMRSA-100N-10</td>
			<td>nucleic acid amplification technology MRSA</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">GeneXpert IV instrument</td>
			<td>Cepheid, Sunnyvale, Ca, USA</td>
			<td>GXIV-4-D</td>
			<td>nucleic acid amplification technology instrument</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Microflex LT MALDI-TOF MS instrument</td>
			<td>Bruker Daltonics, Bremen, Germany</td>
			<td>BDAL microflex LT/SH</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">MSP 96 target&#160; steel BC</td>
			<td>Bruker Daltonics, Bremen, Germany</td>
			<td>280799</td>
			<td>MALDI target plate</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Densitometer Densicheck instrument</td>
			<td>Biom&#233;rieux, Marcy-l&#39;Etoile, France</td>
			<td>27208</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">MALDI Sepsityper kit 50</td>
			<td>Bruker Daltonics, Bremen, Germany</td>
			<td>8270170</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Mac Conkey agar</td>
			<td>Biolife, Milano, Italy</td>
			<td>4016702</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Mueller-Hinton agar</td>
			<td>Oxoid, Hampshire, England</td>
			<td>CM0337</td>
			<td>Mueller-Hinton agar (MH)&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">MHF agar</td>
			<td>Biom&#233;rieux, Marcy-l&#39;Etoile, France</td>
			<td>43901</td>
			<td>Mueller-Hinton agar-fastidious organisms agar (MHF)</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">BD columbia III agar</td>
			<td>BD, Franklin Lakes, NJ, USA</td>
			<td>254071</td>
			<td>blood agar</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">BD chocolate agar</td>
			<td>BD, Franklin Lakes, NJ, USA</td>
			<td>254089</td>
			<td>chocolate agar</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">BD schaedler agar</td>
			<td>BD, Franklin Lakes, NJ, USA</td>
			<td>254084</td>
			<td>Schaedler agar</td>
		</tr>
	</tbody>
</table>

preparation of a blood culture pellet for rapid bacterial identification and antibiotic susceptibility testing

Kan dolaşımı enfeksiyonları ve sepsis morbidite ve mortalitenin önemli bir nedenidir. Bakteriyemi muzdarip hastaların başarılı bir sonuç optimum antibiyotik tedavisine rehberlik enfeksiyöz ajan hızlı bir tanımlanmasına bağlıdır. Pozitif kan kültürü Gram leke analizi hızla yapılmış ve zaten önemli ölçüde antibiyotik rejimi etkileyebilir edilebilir. Bununla birlikte, enfeksiyöz ajanın doğru tespit hala en iyi hedef tedaviyi saptamak için gereklidir. Burada, disk difüzyon testi ile, MALDI-TOF MS, antibiyotik duyarlılık testi (AST) tarafından tespit edilmesi veya otomatik AST sistemleri gibi çeşitli gerekli alt uygulama için bir numune olarak kullanılabilecek bir pozitif kan kültürü hızlı ve basit bir Bakteri taneleri bir preparasyonunu sunmak ve otomatik diagnostik testi PCR tabanlı. Kan kültürü, bakteriyel pelet üzerine doğrudan uygulanabilir bu farklı kimlik ve AST sistemlerinin performansı çok si olanperformansına Milar normal agar plakaları üzerinde büyüyen koloniler izole elde. Geleneksel yöntemlere göre, bir bakteri pelet hızlı edinimi önemli ölçüde belirlenmesini ve AST hem rapor süresini azaltır. Böylece, aşağıdaki kan kültürü pozitifliği, MALDI-TOF ile tanımlama 8 içinde sırasıyla 18 saat, otomatikleştirilmiş AST sistemleri veya disk difüzyon deneyleri ile AST sonuçları ise daha az 1 saat içinde rapor edilebilir. Benzer bir şekilde, hızlı, PCR bazlı tahlilin sonuçları klinisyene bakteremi raporu müteakiben en az 2 saat iletilebilir. Birlikte, bu sonuçlar, kan kültürü bakteri pelet hızlı hazırlanması kimlik ve AST geri dönüş süresi ve dolayısıyla kan dolaşımı enfeksiyonları olan hastaların başarılı sonuç üzerinde önemli bir etkiye sahip olduğunu göstermektedir.

Prod&#39;hom ve ark. 15 tarafından yapılan çalışmada, 78 hastadan 122 pozitif kan kültürü amonyum klorür liziz santrifüj ile elde edilen bakteriyel peletler MALDI-TOF MS ile analiz edilmiştir. 122 pozitif kan kültürü üzerinden, 95 (% 77.9) doğru cins düzeyinde tür düzeyinde ve bir (% 0.8) olarak tespit edilmiştir. Geri kalan 26 (% 21.3), kan kültürü topaklar, MALDI-TOF ile güvenilir bir kimlik verdi. Bunlar arasında, 21 13 streptokok ve 5 koagülaz-negatif stafilokok içeren ...

Watch this Scientific Journal Video about Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing at JoVE.com

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

Pozitif kan kültürü hızlı bir Bakteri taneleri bir preparasyon, MALDI-TOF ile tanımlama, Gram boyama antibiyotik duyarlılık testi ve PCR-esaslı testi gibi uygulamalar için bir numune olarak kullanılabilir. Sonuçlar hızla kan dolaşımı enfeksiyonlarından muzdarip hastaların sonuçlarını iyileştirmek için klinisyenler için tebliğ edilebilir.

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 ...

preparation-blood-culture-pellet-for-rapid-bacterial-identification

Research

JoVE Journal

Immunology and Infection

35.4K Görüntüleme.  University Hospital Center and University of Lausanne.  Pozitif kan kültürü hızlı bir Bakteri taneleri bir preparasyon, MALDI-TOF ile tanımlama, Gram boyama antibiyotik duyarlılık testi ve PCR-esaslı testi gibi uygulamalar için bir numune olarak kullanılabilir. Sonuçlar hızla kan dolaşımı enfeksiyonlarından muzdarip hastaların sonuçlarını iyileştirmek için klinisyenler için tebliğ edilebilir. 

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

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third most common infection in US hospitals. Different manifestations of candidiasis are associated with biofilm formation, both on host tissues and/or medical devices (i.e. catheters). Biofilm formation carries negative clinical implications, as cells within the biofilms are protected from host immune responses and from the action of antifungals. We have developed a simple, fast and robust in vitro model for the formation of C. albicans biofilms using 96 well microtiter-plates, which can also be used for biofilm antifungal susceptibility testing. The readout of this assay is colorimetric, based on the reduction of XTT (a tetrazolium salt) by metabolically active fungal biofilm cells. A typical experiment takes approximately 24 h for biofilm formation, with an additional 24 h for antifungal susceptibility testing. Because of its simplicity and the use of commonly available laboratory materials and equipment, this technique democratizes biofilm research and represents an important step towards the standardization of antifungal susceptibility testing of fungal biofilms.

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

We describe a simple, rapid and robust method for the formation of Candida albicans biofilms using 96 well microtiter plates and its utility in antifungal susceptibility testing of cells within biofilms.

İzleme Oluşumu ve Antifungal Duyarlılık Testi için 96 Şey microtiter Levha tabanlı Yöntemi Candida albicans Biyofilmler

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third ...

İmmünoloji ve Enfeksiyon

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 ...

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium. Because resistance to ampicillin and vancomycin in E. faecalis is still uncommon compared to resistance in E. faecium, the development of rapid tests allowing differentiation between enterococcal species is important for appropriate therapy and resistance surveillance. The E. faecalis OE PNA FISH assay (AdvanDx, Woburn, MA) uses species-specific peptide nucleic acid (PNA) probes in a fluorescence in situ hybridization format and offers a time to results of 1.5 hours and the potential of providing important information for species-specific treatment. Multicenter studies were performed to assess the performance of the 1.5 hour E. faecalis/OE PNA FISH procedure compared to the original 2.5 hour assay procedure and to standard bacteriology methods for the identification of enterococci directly from a positive blood culture bottle.

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

A rapid protocol for the direct identification of Enterococcus faecalis and other Enterococcus species from a positive blood culture using a Peptide Nucleic Acid fluorescent in situ hybridization assay (PNA FISH).

Belirlenmesi için 1.5 Saat Usulü Enterococcus</emDoğrudan Kan Kültürleri> Türler

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium.  Because resistance to ampicillin and ...

Antimicrobial Susceptibility Testing of Mycobacterium Tuberculosis Complex for First and Second Line Drugs by Broth Dilution in a Microtiter Plate Format

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). Antimicrobial susceptibility testing (AST) of Mtb has traditionally been performed by the agar method of proportion or by 
macrobroth testing on an instrument such as the BACTEC (Becton Dickinson, Sparks, MD), VersaTREK (TREK Diagnostics, Cleveland, OH) or BacT/ALERT (bioM&eacute;rieux, Hazelwood, MO). The agar proportion method, while considered the &#x201C;gold&#x201D; standard of AST, is labor intensive and requires calculation of resistance by performing colony counts on drug-containing agar as compared to drug-free agar. If there is &ge;1% growth on the drug-containing medium as compared to drug-free medium, the organism is considered resistant to that drug. The macrobroth methods require instrumentation and test break point ("critical") drug concentrations for the first line drugs (isoniazid, ethambutol, rifampin, and pyrazinamide). The method described here is commercially available in a 96 well microtiter plate format [MYCOTB (TREK Diagnostics)] and contains increasing concentrations of 12 antimicrobials used for treatment of tuberculosis including both first (isoniazid, rifampin, ethambutol) and second line drugs (amikacin, cycloserine, ethionamide, kanamycin, moxifloxacin, ofloxacin, para-aminosalicylic acid, rifabutin, and streptomycin). Pyrazinamide, a first line drug, is not included in the microtiter plate due to its need for acidic test conditions. Advantages of the microtiter system include both ease of set up and faster turn around time (14 days) compared with traditional agar proportion (21 days). In addition, the plate can be set up from inoculum prepared using either broth or solid medium. Since the microtiter plate format is new and since Mtb presents unique safety challenges in the laboratory, this protocol will describe how to safely setup, incubate and read the microtiter plate.

Antimicrobial susceptibility testing of Mycobacterium tuberculosis is challenging but critical for patient care. This microtiter plate format offers testing of 12 antimycobacterial drugs and provides minimum inhibitory concentration (MIC) values, which will aid in appropriate treatment.

Microtiter Plate Format Broth Seyreltme Birinci ve İkinci Line İlaçları Mycobacterium tuberculosis kompleksi Antimikrobiyal Duyarlılık Testi

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). ...

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 ...

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

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. Therefore, rapid administration of adequate antibiotic therapy is of foremost importance in the treatment of bloodstream infections. The critical element in this process is timing, heavily dependent on the results of bacterial identification and antibiotic susceptibility testing. Both of these parameters are routinely obtained by culture-based testing, which is time-consuming and takes on average 24-48 hours2, 4. The aim of the study was to develop DNA-based assays for rapid identification of bloodstream infections, as well as rapid antimicrobial susceptibility testing. The first assay is a eubacterial 16S rDNA-based real-time PCR assay complemented with species- or genus-specific probes5. Using these probes, Gram-negative bacteria including Pseudomonas spp., Pseudomonas aeruginosa and Escherichia coli as well as Gram-positive bacteria including Staphylococcus spp., Staphylococcus aureus, Enterococcus spp., Streptococcus spp., and Streptococcus pneumoniae could be distinguished. Using this multiprobe assay, a first identification of the causative micro-organism was given after 2 h.
Secondly, we developed a semi-molecular assay for antibiotic susceptibility testing of S. aureus, Enterococcus spp. and (facultative) aerobe Gram-negative rods6. This assay was based on a study in which PCR was used to measure the growth of bacteria7. Bacteria harvested directly from blood cultures are incubated for 6 h with a selection of antibiotics, and following a Sybr Green-based real-time PCR assay determines inhibition of growth. The combination of these two methods could direct the choice of a suitable antibiotic therapy on the same day (Figure 1). In conclusion, molecular analysis of both identification and antibiotic susceptibility offers a faster alternative for pathogen detection and could improve the diagnosis of bloodstream infections.

The design of a straightforward one-day workflow scheme for bacterial pathogen diagnostics enables the rapid recognition of bloodstream infections. The inclusion of eight clinically relevant bacterial targets and their antibiotic resistance profiles offers the clinician an initial insight on the same day, which can lead to more adequate therapy.

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. ...

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin&#160;contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth expedites the identification of bacteria. The presented method is a rapid and reliable method for identification of Gram negative bacteria directly from blood culture broth.

MALDI-TOF kütle spektrometrisi kullanılarak kan kültür besi yerinden, Gram negatif bakterilerin hızlı belirlenmesi

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, which are expressed as minimum inhibitory concentrations (MICs) are used in research for antimicrobial development and monitoring of resistance development and in the clinical setting for antimicrobial therapy guidance. Dalbavancin is a semi-synthetic lipoglycopeptide antimicrobial agent that was approved in May 2014 by the Food and Drug Administration (FDA) for the treatment of acute bacterial skin and skin structure infections caused by Gram-positive organisms. The advantage of dalbavancin over current anti-staphylococcal therapies is its long half-life, which allows for once-weekly dosing. Dalbavancin has activity against Staphylococcus aureus (including both methicillin-susceptible S. aureus [MSSA] and methicillin-resistant S. aureus [MRSA]), coagulase-negative staphylococci, Streptococcus pneumoniae, Streptococcus anginosus group, &#946;-hemolytic streptococci and vancomycin susceptible enterococci. Similar to other recent lipoglycopeptide agents, optimization of CLSI and ISO broth susceptibility test methods includes the use of dimethyl sulfoxide (DMSO) as a solvent when preparing stock solutions and polysorbate 80 (P80) to alleviate adherence of the agent to plastic. Prior to the clinical studies and during the initial development of dalbavancin, susceptibility studies were not performed with the use of P-80 and MIC results tended to be 2-4 fold higher and similarly higher MIC results were obtained with the agar dilution susceptibility method. Dalbavancin was first included in CLSI broth microdilution methodology tables in 2005 and amended in 2006 to clarify use of DMSO and P-80. The broth microdilution (BMD) procedure shown here is specific to dalbavancin and is in accordance with the CLSI and ISO methods, with step-by-step detail and focus on the critical steps added for clarity.

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Here, we present a protocol for determination of dalbavancin susceptibility of clinically relevant Gram-positive bacteria using a broth microdilution reference methodology.

Dalbavansin için Başvuru sıvı mikrodilüsyon yöntemi<em&gt; İn Vitro</emAerobik Olarak Büyüyen Bakterilerin&gt; Duyarlılık Testi

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, ...

A RAPID Method for Blood Processing to Increase the Yield of Plasma Peptide Levels in Human Blood

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the correct determination of a peptide&#39;s concentration, it should be stable during blood processing. However, this is not the case for several peptides which are quickly degraded by endogenous peptidases. Recently, we developed a blood processing method employing Reduced temperatures, Acidification, Protease inhibition, Isotopic exogenous controls and Dilution (RAPID) for the use in rats. Here, we have established this technique for the use in humans and investigated recovery, molecular form and circulating concentration of food intake regulatory hormones. The RAPID method significantly improved the recovery for 125I-labeled somatostatin-28 (+39%), glucagon-like peptide-1 (+35%), acyl ghrelin and glucagon (+32%), insulin and kisspeptin (+29%), nesfatin-1 (+28%), leptin (+21%) and peptide YY3-36 (+19%) compared to standard processing (EDTA blood on ice, p&#160;&#60;0.001). High performance liquid chromatography showed the elution of endogenous acyl ghrelin at the expected position after RAPID processing, while after standard processing 62% of acyl ghrelin were degraded resulting in an earlier peak likely representing desacyl ghrelin. After RAPID processing the acyl/desacyl ghrelin ratio in blood of normal weight subjects was 1:3 compared to 1:23 following standard processing (p&#160;= 0.03). Also endogenous kisspeptin levels were higher after RAPID compared to standard processing (+99%, p&#160;= 0.02). The RAPID blood processing method can be used in humans, yields higher peptide levels and allows for assessment of the correct molecular form.

The RAPID blood processing method can be used in humans and yields higher peptide levels as well as allows for assessment of the correct molecular form. Therefore, this method will be a valuable tool in peptide research.

Kan işleme için hızlı bir yöntem insan kanı plazma peptid düzeylerinin veriminin arttırılması için

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the ...

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation of experiments, fresh or frozen monocytes are cultured in flasks for 1 week with M-CSF to induce their differentiation into macrophages. Then, the macrophages can be harvested and seeded into culture wells at required cell densities for carrying out experiments. The use of defined numbers of macrophages rather than defined numbers of monocytes to initiate macrophage cultures for experiments yields macrophage cultures in which the desired cell density can be more consistently attained. Use of cryopreserved monocytes reduces dependency on donor availability and produces more homogeneous macrophage cultures.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. The protocol utilizes cryopreservation of monocytes coupled with their bulk differentiation into macrophages. Then harvested macrophages can then be seeded into culture wells at required cell densities for carrying out experiments.

Makrofaj koloni stimüle edici faktör farklılaştırılmış insan monosit türevli makrofajlar Kültür

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation ...