Bu eser ulusal anahtar araştırma ve geliştirme programı Çin [2017YFD0500102]; gelen hibe tarafından desteklenmiştir Ulusal Doğa Bilimleri Vakfı Çin [31572544]; Devlet anahtar laboratuvar veteriner etiyolojik biyoloji [SKLVEB2016KFKT005]; Shanghai tarım teknoloji geliştirme programı, Çin [G2016060201] uygulanan.

Fare enfeksiyon deneyler laboratuvar hayvan İzleme Komitesi Jiangsu Province, Çin tarafından onaylanmış ve laboratuvar hayvan Merkezi, Nanjing Tarım Üniversitesi'nde gerçekleştirilen (izin numarası: SYXK (Su) 2017-0007).
1. bakteri hazırlanması
Not: S. suis serotip 2 öldürücü soy P1 7/menenjit19hastalıklı bir domuzla yalıtılmış. Zorlanma P1/7 Todd-Hewitt suyu yetiştirilen (THB, formül en düşük THB litre başına: kalp infüzyon, 3.1 g neopeptone, 20.0 g dekstroz, 2.0 g; sodyum klorür, 2.0 g; disodyum fosfat, 0.4 g; sodyum karbonat, 2.5 g) ve Todd-Hewitt agar (THA, litre başına formül üzerinde kaplama THA: Kalp infüzyon, 3.1 g; neopeptone, 20.0 g; dekstroz, 2.0 g; Sodyum klorür, 2.0 g; disodyum fosfat, 0.4 g; sodyum karbonat, 2.5 g; ağar, 15,0 g) 37 ° C ve % 5 CO2.
<ol><li>Topluluğu P1/7 planktonik hücreleri zorlanma<ol>
<li>Planktonik hücre 5 mL orta log fazı kültüründen toplamak (OD600= 0,6), santrifüj kapasitesi 8000 × g de 3 dakika ve sonra 3 kez PBS ile yıkayın.</li>
		<li>5 mL % 25 gliserol içinde en düşük THB, 5 tüpler içine aliquot de bulunduğu hücreleri resuspend ve-80 ° C'de depolayın</li>
	</ol>
</li>
	<li>Topluluğu P1/7 biyofilm devlet hücreleri zorlanma<ol>
<li>Bir gecede kültür 20 mL al ve taze en düşük THB için 180 mL ekleyin; seyreltilmiş kültür 10 yuvarlak kültür kalıplara eşit, bölmek ve sonra % 5 CO2 37 ° C'de bir kuluçka 24 h için plakaları koymak.</li>
		<li>Kuluçka sonra yavaşça plakasına kalmadığımızı bakteri resuspend için plaka sallamak ve o zaman süpernatant aspirasyon tarafından atın. Not: plaka yavaşça sallayın ve tortu resuspending kaçının.</li>
		<li>Biyofilm devlet hücre hasat, tortu tamamen resuspend ve o zaman örnek için yeni bir tüp transferi için PBS 5 mL ekleyin.</li>
		<li>Aşağıdaki parametreler ile son adım biyofilm devlet hücrelerden solüsyon içeren temizleyicide: 60 W, 4 döngüleri, 5 s off üzerinde ve 10 sn.</li>
		<li>8000 × g de 3 dk santrifüj kapasitesi ve süpernatant-80 ° C'de depolayın Not: Süpernatant biyofilm bileşenleri içerebilir ve biyofilm bakteri daha önce hayvan deneyleri (adım 3) açıklandığı gibi enfeksiyon resuspend için kullanılabilir.</li>
		<li>En düşük THB içinde tortu 10 mL % 25 gliserol ile resuspend ve biyofilm devlet hücreleri-80 ° C'de depolayın</li>
	</ol>
</li>
</ol>2. Tarama elektron mikroskobu (SEM) analiz
<ol><li>Toplanan biyofilm devlet hücreleri ve 12-24 h 4 ° C'de % 4 paraformaldehyde kullanarak planktonik hücreleri tamir</li>
	<li>Örnekleri hızlı distile su ile durulama ve etanol (% 25, % 50, % 70, % 90 ve % 100) 30 dk içinde her çözümü için artan bir konsantrasyon sırayla örnekleriyle kurutmak.</li>
	<li>Kurutulmuş örneklerini sahipleri çift taraflı bant ile metal ve sonunda onları bir buharlaştırıcı altın ve Paladyum içinde kat için uygun.</li>
	<li>Taramalı elektron mikroskobu aşağıdaki parametreleri kullanarak tarafından örnekleri gözlemlemek: EHT (ekstra yüksek gerilim) = 10 kV; WD (mesafe çalışma) = 7.0 veya 8.0 mm; Büyütme = 5000 ×; Sinyal A SE1 =.</li>
</ol>3. hayvan deneyleri
<ol><li>SPF 6-hafta-yaşlı kullanın Bu çalışmada kadın BALB/c fareler. Kafa içi subarachnoidal yolu enfeksiyonu olan enjeksiyon izi bulunduğu 3, 5 mm bregma rostral olduğunu tüm fareler bulaştırmak. Enjeksiyon yeri stereotaksik koordinatlarını açıkça Chiavolini ve ark17tarafından tarif edildi.<ol>
<li>Histopatolojik analiz için iki grup (grup başına 5 fareler) farelerin bulaştırmak planktonik hücreleri veya biyofilm kullanarak devlet bir doz 3 × 107 birim (CFU) koloni oluşturan hücreleri ve başka bir 5 fare denetimi olarak PBS ile enjekte edildi.</li>
		<li>TLR2 ve sitokinler beyin, mRNA ifade tespiti bulaştırmak için farelerin (grup başına 6 fareler) iki ek grupları planktonik hücreleri veya biyofilm kullanarak devlet 3 × 107 CFU bir doz hücreleri.</li>
		<li>Üstünde-e doğru bu seri dilutions kaplama tarafından canlı bakteri sayısını belirleyin.</li>
		<li>12 h sonrası enfeksiyon daha fazla araştırma için tüm fareler ötenazi. Not: Stok orta gliserol enfeksiyonu önce kaldırılması gerekir. Planktonik hücreleri ve biyofilm devlet hücreleri-80 ° C'den kurtarılan 3 kez PBS ile yıkanır. Sonra planktonik hücreleri PBS ile resuspended ve enfeksiyon için uygun doza seyreltilmiş; biyofilm devlet hücreleri saklı süpernatant (Kimden adım 1.2.5) ile resuspended ve enfeksiyon için uygun doza seyreltilmiş. Enfeksiyon için birim fazla 50 µL olmamalıdır.</li>
	</ol>
</li>
	<li>TLR2 ve sitokinler mRNA ifadesi beyin dokusunda algılama<ol>
<li>Toplam RNA, RNA ekstraksiyon kiti kullanılarak beyin dokusundan ayıklayın.<ol>
<li>Her fare için tüm beyin dokusu RNA ayıklamak için kullanın. Bütün beyin dokusu 5 tüpler içine bölün. 100 mg beyin dokusu ile alıp kit tarafından sağlanan lysing matris içeren her tüp için 1 mL lizis çözeltisi ekleyin.</li>
			<li>Bir homogenizer 40 tüpte işlemek bir ayar 6.0 ve daha sonra santrifüj tüpü 12000 × g ve 4 ° C 5 min için s.</li>
			<li>Santrifüjü sonra üst aşaması için yeni bir microcentrifuge tüp aktarın.</li>
			<li>Oda sıcaklığında 5 min için transfer edilen örnek kuluçkaya; kloroform, 10 için girdap 300 µL eklemek s ve oda sıcaklığında 5 min için kuluçkaya.</li>
			<li>Tüp 12000 ×, santrifüj kapasitesi g ve 4 ° C'de 5 dakika süreyle aktarmak üst aşaması için yeni bir tüp.</li>
			<li>5 kez ters çevirme önce Tüp, soğuk mutlak etanol 500 µL ekleyin ve örnek için en az 1 h-20 ° C'de depolayın.</li>
			<li>Tüp 12000 ×, santrifüj kapasitesi g ve 4 ° C'de 15 dakika ve süpernatant kaldır. Yıkama 500 µL RNase free H2o soğuk % 75 etanol ile Pelet</li>
			<li>Kaldır etanol, hava kuru oda sıcaklığında ve resuspend RNA içinde 100 µL RNase free H2o 5 min için Pelet</li>
			<li>Oda sıcaklığında 5 min için RNA kuluçkaya ve RNA bioanalyzer kullanarak RNA konsantrasyonu belirlemek.</li>
		</ol>
</li>
		<li>CDNA sentezi, genomik DNA (gDNA), bir thermocycler bir ters Transkripsiyon (RT) reaktif kiti ile kullanarak kaldırılması da dahil olmak üzere gerçekleştirin.<ol>
<li>Birim ulaşır 10 µL. kuluçkaya kadar 42 ° C'de 2 min için bir tüp içeren 2 µL 5 x gDNA eleme arabelleği, gDNA eleme enzim ve RNase free H2O 1 µL RNA'ın 1 µg ekleyin</li>
			<li>10 µL ana karışımı ekleyin (RT enzim 1 µL Mix I, RT astar Mix 1 µL, 4 µL 5 tampon 2 x ve 4 µL RNase free H2O) tepki çözüm son adım ve sonra hafifçe karıştırın. Hemen RT tepki ile devam: 37 ° C 15 dk ve sonra 85 ° C 5 s.</li>
			<li>SYBR RT-qPCR kiti ile gerçek zamanlı PCR makinasıyla nicel gerçek zamanlı PCR (RT-qPCR) çözümlemesi gerçekleştirin. 2 enzim x 10 µL, ileri astar, 0.8 µL ters astar, 0.4 µL 50 x ROX başvuru boya II, cDNA şablonu ve RNase free H2O 2 µL 0.8 µL toplam hacmi 20 µL kadar ekleyin.</li>
			<li>Her örnek nüsha aşağıdaki termal parametrelerini kullanarak çalıştırın: 30 s 94 ° C'de 5 40 döngüsü tarafından takip s 95 ° c ve 34 s son aşaması 15 ile 60 ° C'de s 95 ° c, 1 dk. 60 ° c ve 15 s 95 ° C'de Astar RT-qPCR için Tablo 1' de listelenmiştir. Temizlik genler B2m ve 18s rRNA iç kontrol kullanılmıştır.</li>
		</ol>
</li>
		<li>2-ΔΔCt yöntemine göre göreli kat değişir hesaplayın.</li>
	</ol>
</li>
	<li>
Histolojik kesitler gözlenmesi
	<ol>
<li>% 10 formalin ile 24 h fiksasyon sonra sabit beyin dokusu içine 2-3 mm doku parçaları uygun ölçüde yeniden.</li>
		<li>Gömme bir kasete doku adet koyun ve dehidratasyon ile otomatik bir vakum kurutucu için yeni bir sabitleştirici çözüm bırakın.</li>
		<li>Sabit doku almak ve etanol (% 70, %80, % 95, % 95 ve % 100) 30 dk içinde her çözüm için konsantrasyonları artan sırayla kurutmak.</li>
		<li>Ksilen 15 dakika ile örnek transparentize, o Sıvı parafin 90 dk 60 ° C'de eğim ve gömme bir makine kullanarak embed.</li>
		<li>Parafin blok gömülü doku 3 µm dilimler halinde kesip, dilim su 45 ° c üzerinde döşeme ve sonra hava kuru.</li>
		<li>60 ° C'de 3 h için bir bölüm kuluçkaya, haematoxylin ve eozin (o) kullanarak yöntemi leke ve tarafsız sakız ile kapatın.</li>
		<li>Histopatolojik değişiklikleri beynin optik mikroskopla gözlemlemek. Bir dört maddelik derecelendirme sistemi histolojik değişiklikler değerlendirmek için kullanıldı. Bir unpaired t testi istatistiksel analizi için kullanın. Tıkanıklık/kanaması: yok, o 0; odak tıkanıklık/kanaması, küçük alanda 1 puan edinildi; geniş alan fokal tıkanıklık/kanaması veya fokal tıkanıklık/kanaması, küçük alanının birden çok site 2 puan edinildi; üç büyük odak tıkanıklık/kanaması siteleri kadar 3 puan edinildi; tıkanıklık/kanaması diffüz, 4 puan. Nekroz: yok, o 0; odak nekroz, 1; puan edinildi 1-10 nekrotik hücre oluşumunu diffüz, o 2 puan; konfluent nekroz, Foci puan edinildi 3; geniş konfluent nekroz, 4 puan edinildi. İltihabi hücre infiltrasyonu: yok, o 0; kaç ve odak infiltrasyonlar, 1 puan edinildi; çok görüşlü infiltrasyon veya toplamları, oluşumu infiltrations 2 puan edinildi; Üç toplamları kadar 3 puan edinildi; ve dörtten fazla toplamları, puan 4.</li>
	</ol>
</li>
</ol>

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Burada açıklanan enfeksiyon intrakranial subarachnoidal rotanın diğer yolları enfeksiyonu üzerinde belirgin avantajları vardır. Ana bilgisayar-bakteri etkileşim ve bakteriyel bileşenleri etkisi ana bilgisayar bağışıklık yanıtı doğrudan beyindeki hangi bakteri giriş merkezi sinir sistemi taklit çalışmaya müfettişler sağlar. Böylece, bu yolu enfeksiyonu diğer bakterilerin neden olduğu menenjit mekanizmalarının araştırılması için genişletilebilir. Üstelik, o da bakteriyel menenjit karş?...

Streptokok suis (S. suis) büyük bir bakteriyel patojen domuz dünya çapında, menenjit, pnömoni, septisemi, endokardit ve artrit1de dahil olmak üzere ağır hastalığa neden oluyor. Ortaya çıkan olabilmekte ajanı da sağlar. Şimdiye kadar bu dokuz serotipleri enfeksiyon serotipleri 2, 4, 5, 9, 14, 16, 21, 24 ve 312,3,4de dahil olmak üzere insanlarda neden olabilir bildirilmiştir. İnsanlar ve domuzlar, menenjit S. suis enfeksiyonlar önemli klinik belirtileri biridir. Vietnam ve Tayland, S. suis menenjit yetişkin5önemli nedenidir. Mikrobiyal biyofilmler birbirine bağlı ve bir arabirim konsantre olan mikroorganizmalar vardır; Onlar bakteriyel virülans, çeşitli ortamlarda ve antibiyotik direnci5hayatta kalmak için gereklidir. Biyofilmler genellikle genellikle polisakkaritler, proteinler ve DNA6içeren bir hücre dışı matriks tarafından çevrili. İkinci ana bilgisayar inflamatuar yanıt ve sitokin üretim7temin edebilmektedir. Biyofilm oluşumu önceki çalışmalarda streptokok menenjit dahil olmak için rapor edildi. Streptococcus agalactiae menenjit tilapia balık modelindeki biyofilmler katkıda ve biyofilm oluşumu ortaya beyin doku içinde ve çevresinde meninjeal vivo içinde ile intra abdominal aşılama8yüzeyler. Menenjit sırasında bir biyofilm benzeri durumda Streptococcus pneumoniae ve böyle bir biyofilm devlet bakterilerde bir fare enfeksiyon modeli9menenjit inducing daha etkili. Buna ek olarak, bizim önceki çalışma, S. suis fare beyin ile ilişkili biyofilm durumu bakteriyel virülans için hayatta kalma analiz10tarafından katkıda bulunur. Ancak, doğrudan kanıt S. suis menenjit biyofilm katılımı için daha fazla araştırma gerektirir.
Mayi (IP)11, burunici (i.n.)12, şarapla (IV)13ve enfeksiyon14, intracisternal (araç ten) yollarını kullanarak farelerde hayvan modelleri S. suis enfeksiyon geliştirilmiştir 15 , 16. ancak, enfeksiyon ı.p., i.n. ve damar içi enjeksiyona yolları menenjit doğrudan beyindeki S. suis yüzey bileşenlerinde rolleri eğitim için uygun değildir. Bu hücre dışı matriks biyofilmler üzerinden içerir. Her ne kadar araç ten aşılama S. suis enfeksiyonu için kullanılan, hassas enjeksiyon yeri bu gazetelerde tanımlanmıştır değil. Buna ek olarak, intrakranial subarachnoidal aşı enjeksiyon izi stereotaksik koordinatlarını açıkça tarif edilmiş bir önceki çalışma17'. Bu aşı noktası ve daha fazla basit Deneysel protokol kolay tanınmasını sağladı. Buna ek olarak, intrakranial subarachnoidal yolu enfeksiyonu taklit eden bakteri giriş merkezi sinir sistemi sinüsler veya orta kulak17ve orta kulak ve S. suis tarafından neden menenjit ilişkisi içine Madsen ve ark18tarafından kanıtlanmıştır. Ayrıca, intrakranial subarachnoidal yolu enfeksiyonu farelerde uygulayarak, biz S. suis küçük RNA rss04 menenjit bizim önceki çalışma10katkıda göstermiştir.
Mevcut çalışma, intrakranial subarachnoidal yolu enfeksiyonu farelerde S. suis menenjit biyofilmler rollerini araştırmak için kullanıldı. Fareler planktonik hücreleri veya S. suis biyofilm devlet hücreleri ile enfeksiyon bu yoldan bulaşmış. Histopatolojik Analizi ve TLR2 ve sitokinler biyofilm devlet hücreleri enjekte farelerin beyin dokusundan artan mRNA ifadesi açıkça S. suis biyofilm menenjit için katkıda belirtti.

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			<td height="46">Todd Hewitt Broth(THB)</td>
			<td style="width:225px;">Becton, Dickinson and Company</td>
			<td style="width:115px;">DF0492078</td>
			<td style="width:304px;">Dissolve 30 g of the powder in 1 L of purified water. Autoclave at 121&#176; for 15 min.</td>
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			<td>Agar</td>
			<td style="width:225px;">DSBIO</td>
			<td style="width:115px;">16C0050</td>
			<td style="width:304px;">Dissolve 15 g of the powder in 1 L of THB. Autoclave at 121&#176; for 15 min.</td>
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			<td height="43" style="height:43px;width:184px;">Milli-Q Reference Water Purification System</td>
			<td style="width:225px;">Merck KGaA</td>
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			<td style="width:304px;">Without Dnase/ Rnase</td>
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			<td>NaCl</td>
			<td style="width:225px;">Tianjin Kemiou Chemical Reagent Co., Ltd</td>
			<td style="width:115px;">10019318</td>
			<td style="width:304px;">Dissolve 8 g NaCl, 0.2 g KCl, 1.42 g Na2HPO3 , 0.27 g KH2PO4 in 1 L of purified water. Autoclave at 121&#176; for 15 min. Use KOH to adjust pH to 7.4.</td>
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			<td>Na2HPO3</td>
			<td style="width:225px;">Xilong Scientific Co., Ltd</td>
			<td style="width:115px;">9009012-01-09</td>
			<td style="width:304px;">Dissolve 8 g NaCl, 0.2 g KCl, 1.42 g Na2HPO3 , 0.27 g KH2PO4 in 1 L of purified water. Autoclave at 121&#176; for 15 min. Use KOH to adjust pH to 7.4.</td>
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			<td>KCl</td>
			<td style="width:225px;">Xilong Scientific Co., Ltd</td>
			<td style="width:115px;">9009017-01-09</td>
			<td style="width:304px;">Dissolve 8 g NaCl, 0.2 g KCl, 1.42 g Na2HPO3 , 0.27 g KH2PO4 in 1 L of purified water. Autoclave at 121&#176; for 15 min. Use KOH to adjust pH to 7.4.</td>
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			<td>KH2PO4</td>
			<td style="width:225px;">Xilong Scientific Co., Ltd</td>
			<td style="width:115px;">9009019-01-09</td>
			<td style="width:304px;">Dissolve 8 g NaCl, 0.2 g KCl, 1.42 g Na2HPO3 , 0.27 g KH2PO4 in 1 L of purified water. Autoclave at 121&#176; for 15 min. Use KOH to adjust pH to 7.4.</td>
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			<td>KOH</td>
			<td style="width:225px;">Xilong Scientific Co., Ltd</td>
			<td style="width:115px;">9009014-01-09</td>
			<td style="width:304px;">Dissolve 8 g NaCl, 0.2 g KCl, 1.42 g Na2HPO3 , 0.27 g KH2PO4 in 1 L of purified water. Autoclave at 121&#176; for 15 min. Use KOH to adjust pH to 7.4.</td>
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			<td>Glycerol</td>
			<td style="width:225px;">Sionpharm Chemical Reagent Co., Ltd</td>
			<td style="width:115px;">10010618</td>
			<td style="width:304px;">Diluted with equal volumu of purified water, autoclave at 121&#176; for 15 min</td>
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			<td height="42" style="height:42px;width:184px;">4% paraformaldehyde</td>
			<td style="width:225px;">Sionpharm Chemical Reagent Co., Ltd</td>
			<td style="width:115px;">80096675</td>
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			<td height="42" style="height:42px;width:184px;">25% Glutaraldehyde</td>
			<td style="width:225px;">Sionpharm Chemical Reagent Co., Ltd</td>
			<td style="width:115px;">30092436</td>
			<td style="width:304px;">10-fold diluted with purified water for fixation.</td>
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			<td height="42" style="height:42px;width:184px;">Ethanol</td>
			<td style="width:225px;">Sionpharm Chemical Reagent Co., Ltd</td>
			<td style="width:115px;">10009218</td>
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			<td height="20" style="height:20px;">Chloroform</td>
			<td style="width:225px;">Sionpharm Chemical Reagent Co., Ltd</td>
			<td style="width:115px;">10006818</td>
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			<td height="20" style="height:20px;">Spctrophotometre</td>
			<td style="width:225px;">DeNovix Inc.</td>
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			<td height="42" style="height:42px;width:184px;">Ultrasound cell crusher</td>
			<td style="width:225px;">NingBo Scientz Biotechnology Co.,Ltd</td>
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			<td height="42" style="height:42px;width:184px;">Scanning electron microscope</td>
			<td>Zeiss</td>
			<td style="width:115px;">EVO-LS10</td>
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			<td height="21" style="height:21px;">FastRNA Pro Green Kit</td>
			<td>MP Biomedicals</td>
			<td style="width:115px;">#6045-050</td>
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			<td height="21" style="height:21px;">FastPrep-24 Instrument</td>
			<td>MP Biomedicals</td>
			<td style="width:115px;">116005500</td>
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			<td height="21" style="height:21px;width:184px;">Instrument for PCR</td>
			<td style="width:225px;">SensoQuest GmbH</td>
			<td style="width:115px;">1124310110</td>
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			<td height="21" style="height:21px;">QuantStudio 6 Flex</td>
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			<td height="42" style="height:42px;width:184px;">SYBR Premix Ex Taq II</td>
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			<td style="width:225px;">Takara Biomedical Technology (Beijing) Co., Ltd</td>
			<td style="width:115px;">RR047A</td>
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			<td height="42" style="height:42px;width:184px;">Fully Enclosed Tissue Processor</td>
			<td style="width:225px;">Leica Biosystems Nussloch GmbH</td>
			<td style="width:115px;">ASP200S</td>
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			<td height="21" style="height:21px;width:184px;">Heated Paraffin Embedding Module</td>
			<td style="width:225px;">Leica Biosystems Nussloch GmbH</td>
			<td style="width:115px;">EG1150H</td>
			<td style="width:304px;"></td>
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			<td height="42" style="height:42px;width:184px;">Semi-Automated Rotary Microtome</td>
			<td style="width:225px;">Leica Biosystems Nussloch GmbH</td>
			<td style="width:115px;">RM2245</td>
			<td style="width:304px;"></td>
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			<td height="42" style="height:42px;width:184px;">Water bath for paraffin sections</td>
			<td style="width:225px;">Leica Biosystems Nussloch GmbH</td>
			<td style="width:115px;">HI1210</td>
			<td style="width:304px;"></td>
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			<td height="42" style="height:42px;width:184px;">Autostainer XL</td>
			<td style="width:225px;">Leica Biosystems Nussloch GmbH</td>
			<td style="width:115px;">ST5010</td>
			<td style="width:304px;"></td>
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			<td height="21" style="height:21px;width:184px;">Agilent 2100</td>
			<td style="width:225px;">Agilent Technologies</td>
			<td style="width:115px;">G2939A</td>
			<td style="width:304px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:184px;">Optical microscope</td>
			<td style="width:225px;">Olympus</td>
			<td style="width:115px;">BX51</td>
			<td style="width:304px;"></td>
		</tr>
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intracranial subarachnoidal route of infection for investigating roles of streptococcus suis biofilms in meningitis in a mouse infection model

Streptokok suis sadece domuz dünya çapında büyük bir bakteriyel patojen aynı zamanda gelişmekte olan Hayvansal ajanı var. İnsanlar ve domuzlar, menenjit S. suis enfeksiyonların büyük bir tezahürüdür. Uygun enfeksiyon manken patojenlerin yol açtığı hastalıklar mekanizmaları anlamak için önemli bir araçtır. Enfeksiyon farelerde birçok yolların S. suis enfeksiyon patogenezi okumaya geliştirilmiştir. Ancak, enfeksiyon mayi, etkilerinin ve intravenöz yolları menenjit gibi hücre dışı matriks biyofilmler üzerinden doğrudan beyindeki S. suis yüzey bileşenlerinde rolleri eğitim için uygun değildir. Her ne kadar intracisternal aşılama S. suis enfeksiyonu için kullanılan, hassas enjeksiyon yeri tarif değil. Burada, intrakranial subarachnoidal yolu enfeksiyonu S. suis menenjit biyofilmler rollerini araştırmak için bir fare modeli tanımlanmıştır. S. suis planktonik hücreleri veya biyofilm devlet hücreleri doğrudan 3, 5 mm bregma rostral bulunan enjeksiyon sitesi aracılığıyla farelerin Subaraknoid boşluk içine enjekte edildi. Histopatolojik Analizi ve TLR2 artan mRNA ifade ve sitokinler biyofilm devlet hücreleri enjekte fareler gelen beyin dokusunun S. suis biyofilm S. suis menenjit kesin rol oynar açıkça belirtti. Bu yolu enfeksiyonu diğer yolları enfeksiyonu, ana bilgisayar-bakteri etkileşim çalışma izin üzerinde belirgin avantajları vardır. Ayrıca, bakteriyel bileşenleri etkisi ana bilgisayar bağışıklık yanıtı doğrudan beyindeki değerlendirilmesi için izin verir ve merkezi sinir sistemi bakteriyel giriş taklit eder. Bu yolu enfeksiyonu diğer bakterilerin neden olduğu menenjit mekanizmalarının araştırılması için genişletilebilir. Üstelik, o da bakteriyel menenjit karşı ilaçların etkinliğini test etmek için kullanılabilir.

SEM analiz biyofilm oluşumu deneysel koşullar altında incelemek için gerçekleştirildi. Şekil 1' de gösterildiği gibi biyofilm oluşumu planktonik hücreleri (Şekil 1A) ve biyofilm devlet hücreleri (Şekil 1B) arasında önemli bir fark vardır. SEM analiz biyofilm bakteri kümeleri ve planktonik bakteriler tek tek daha az yoğun ve esas olarak dağınık i...

Watch this Scientific Journal Video about Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model at JoVE.com

Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model

Burada, enfeksiyon biyofilmler streptokok suis menenjit içinde rolleri çalışma farelerde intrakranial subarachnoidal rotanın tarif. Bu enfeksiyon model diğer bakteriyel menenjit patogenezi ve bakteriyel menenjit karşı yeni ilaçların etkinliğini çalışmak için uygundur.

Kafa içi Subarachnoidal rota için soruşturma rolleri streptokok suis biyofilmler menenjit bir fare enfeksiyon modeli içinde enfeksiyon

Streptococcus suis is not only a major bacterial pathogen of pigs worldwide but also an emerging zoonotic agent. In humans and pigs, meningitis is a major ...

intracranial-subarachnoidal-route-infection-for-investigating-roles

Research

JoVE Journal

Immunology and Infection

Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model

7.2K Görüntüleme.  Nanjing Agricultural University. Burada, enfeksiyon biyofilmler streptokok suis menenjit içinde rolleri çalışma farelerde intrakranial subarachnoidal rotanın tarif. Bu enfeksiyon model diğer bakteriyel menenjit patogenezi ve bakteriyel menenjit karşı yeni ilaçların etkinliğini çalışmak için uygundur.

Video: Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

HIV-1 tropizm genotipik Çıkarım kullanarak Nüfus tabanlı Sıralama V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

İmmünoloji ve Enfeksiyon

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 ...

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly constituted by proteins and exopolysaccharides, among other factors 7-10. The microbial community encased within the biofilm often shows the differentiation of distinct subpopulation of specialized cells 11-17. These subpopulations coexist and often show spatial and temporal organization within the biofilm 18-21.
Biofilm formation in the model organism Bacillus subtilis requires the differentiation of distinct subpopulations of specialized cells. Among them, the subpopulation of matrix producers, responsible to produce and secrete the extracellular matrix of the biofilm is essential for biofilm formation 11,19. Hence, differentiation of matrix producers is a hallmark of biofilm formation in B. subtilis.
We have used fluorescent reporters to visualize and quantify the subpopulation of matrix producers in biofilms of B. subtilis 15,19,22-24. Concretely, we have observed that the subpopulation of matrix producers differentiates in response to the presence of self-produced extracellular signal surfactin 25. Interestingly, surfactin is produced by a subpopulation of specialized cells different from the subpopulation of matrix producers 15.
We have detailed in this report the technical approach necessary to visualize and quantify the subpopulation of matrix producers and surfactin producers within the biofilms of B. subtilis. To do this, fluorescent reporters of genes required for matrix production and surfactin production are inserted into the chromosome of B. subtilis. Reporters are expressed only in a subpopulation of specialized cells. Then, the subpopulations can be monitored using fluorescence microscopy and flow cytometry (See Fig 1).
The fact that different subpopulations of specialized cells coexist within multicellular communities of bacteria gives us a different perspective about the regulation of gene expression in prokaryotes. This protocol addresses this phenomenon experimentally and it can be easily adapted to any other working model, to elucidate the molecular mechanisms underlying phenotypic heterogeneity within a microbial community.

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Microbial biofilms are generally constituted by distinct subpopulations of specialized cells. Single-cell analysis of these subpopulations requires the use of fluorescent reporters. Here we describe a protocol to visualize and monitor several subpopulationswithin B. subtilis biofilms using fluorescence microscopy and flow cytometry.

Tek hücreli Analizi Bacillus subtilis</emFloresan Mikroskobu ve Akım Sitometri Kullanma> Biyofilmler

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly ...

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Members of the Burkholderia genus are pathogens of clinical importance. We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.

Toplam Protein Ekstraksiyon ve 2-D Jel Elektroforez Yöntemleri<em&gt; Burkholderia</em&gt; Türler

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca2+, K+, and H+, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Unlike that seen for eukaryotes, there is a paucity of studies that detail membrane depolarization and ion concentration changes in bacteria, primarily as their small size makes conventional methods of measurement difficult. Here, we detail protocols for monitoring such events in the significant Gram-positive pathogen Streptococcus pneumoniae utilizing fluorescence techniques.

Membran Kutup, Membran Dürüstlük ve Hücre içi İyon Konsantrasyonlarında İzleme değişiklikler<em&gt; Streptococcus pneumoniae</em&gt; Floresan boyalar kullanarak

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major cause of morbidity and mortality world-wide. Current vaccines target the pneumococcal capsule, and there are over 90 capsular serotypes. Serotyping pneumococcal isolates is therefore important for assessing the impact of vaccination programs and for epidemiological purposes. The World Health Organization has recommended latex agglutination as an alternative method to the &#8216;gold standard&#8217; Quellung test for serotyping pneumococci. Latex agglutination is a relatively simple, quick and inexpensive method; and is therefore suitable for resource-poor settings as well as laboratories with high-volume workloads. Latex agglutination reagents can be prepared in-house utilizing commercially-sourced antibodies that are passively attached to latex particles. This manuscript describes a method of production and quality control of latex agglutination reagents, and details a sequential testing approach which is time- and cost-effective. 
This method of production and quality control may also be suitable for other testing purposes.

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination testing is a simple, rapid and inexpensive method for serotyping Streptococcus pneumoniae, and has also been widely applied in diagnostic microbiology. This manuscript describes the in-house production of latex agglutination reagents, quality control procedures and the application of this technique to pneumococcal serotyping.

Kapsüler Serotipleme<em&gt; Streptococcus pneumoniae</em&gt; Lateks Aglutinasyon tarafından

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

Bir Zebra balığı Larva Modeli Doğuştan Bağışıklık Tepki Non-invaziv Görüntüleme<em&gt; Streptococcus iniae</em&gt; Enfeksiyon

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

B Grubu bir murin modelinde<em&gt; Streptococcus</em&gt; Vajinal Kolonizasyon

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

Influenza A Virus Studies in a Mouse Model of Infection

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone considering direct medical and hospitalization expenses and work absenteeism2. Animal models are crucial in Influenza A virus (IAV) studies to evaluate viral pathogenesis, host-pathogen interactions, immune responses, and the efficacy of current and/or novel vaccine approaches as well as antivirals. Mice are an advantageous small animal model because their immune system is evolutionarily similar to that found in humans, they are available from commercial vendors as genetically identical subjects, there are multiple strains that can be exploited to evaluate the genetic basis of infections, and they are relatively inexpensive and easy to manipulate. To recapitulate IAV infection in humans via the airways, mice are first anesthetized prior to intranasal inoculation with infectious IAVs under proper biosafety containment. After infection, the pathogenesis of IAVs is determined by monitoring daily the morbidity (body weight loss) and mortality (survival) rate. In addition, viral pathogenesis can also be evaluated by assessing virus replication in the upper (nasal mucosa) or lower (lungs) respiratory tract of infected mice. Humoral responses upon IAV infection can be rapidly evaluated by non-invasive bleeding and secondary antibody detection assays aimed at detecting the presence of total or neutralizing antibodies. Here, we describe the common methods used to infect mice intranasally (i.n) with IAV and evaluate pathogenesis, humoral immune responses and protection efficacy.

Influenza A viruses (IAVs) are important human respiratory pathogens. To understand the pathogenicity of IAVs and to perform preclinical testing of novel vaccine approaches, animal models mimicking human physiology are required. Here, we describe techniques to evaluate IAV pathogenesis, humoral responses and vaccine efficacy using a mouse model of infection.

Grip A virüs enfeksiyonu bir fare modeli çalışmalarda

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone ...

Constructing Mutants in Serotype 1 Streptococcus pneumoniae strain 519/43

Streptococcus pneumoniae serotype 1 remains a huge problem in low-and-middle income countries, particularly in sub-Saharan Africa. Despite its importance, studies in this serotype have been hindered by the lack of genetic tools to modify it. In this study, we describe a method to genetically modify a serotype 1 clinical isolate (strain 519/43). Interestingly, this was achieved by exploiting the Pneumococcus&#8217; ability to naturally acquire DNA. However, unlike most pneumococci, the use of linear DNA was not successful; to mutate this important strain, a suicide plasmid had to be used. This methodology has provided the means for a deeper understanding of this elusive serotype, both in terms of its biology and pathogenicity. To validate the method, the major known pneumococcal toxin, pneumolysin, was mutated because it has a well-known and easy to follow phenotype. We showed that the mutant, as expected, lost its ability to lyse red blood cells. By being able to mutate an important gene in the serotype of interest, we were able to observe different phenotypes for loss of function mutants upon intraperitoneal and intranasal infections from the ones observed for other serotypes. In summary, this study proves that strain 519/43 (serotype 1) can be genetically modified.

Constructing Mutants in Serotype 1 Streptococcus pneumoniae strain 519/43

Here, we describe a S. pneumoniae serotype 1 strain 519/43 that can be genetically modified by using its ability to naturally acquire DNA and a suicide-plasmid. As proof of principle, an isogenic mutant in the pneumolysin (ply) gene was made.

Serotip 1 Streptococcus pneumoniae suşunda Mutantların İnşası 519/43

Streptococcus pneumoniae serotype 1 remains a huge problem in low-and-middle income countries, particularly in sub-Saharan Africa. Despite its importance, ...