Name: sequencing of bacterial microflora in peripheral blood: our experience with hiv-infected patients
Uploaded: 2011-06-11T15:00:00
Description: Watch this Scientific Journal Video about Sequencing of Bacterial Microflora in Peripheral Blood: our Experience with HIV-infected Patients at JoVE.com

We are grateful to Gianni Scimone for excellent assistance with video making.

Handling of HIV infected blood samples requires some important recommendations.
 
 All specimens of blood must be transported in robust leak-proof containers. Care must be taken when collecting the specimen to avoid contamination of the container&#x2019;s exterior and of 
 any paperwork accompanying the specimen.
 
 All persons processing infected blood must wear gloves. Gloves must be changed and hands washed after completion of specimen processing.
 
 Processing of HIV-infected blood samples must be d...

<ol>
	<li>Hooper, L. V., Macpherson, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+adaptations+that+maintain+homeostasis+with+the+intestinal+microbiota.">Immune adaptations that maintain homeostasis with the intestinal microbiota.</a> Nat Rev Immunol. 10, 159-169 (2010).</li><li>Macpherson, A. J., Harris, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+commensal+intestinal+bacteria+and+the+immune+system.">Interactions between commensal intestinal bacteria and the immune system.</a> Nat Rev Immunol. 4, 478-485 (2004).</li><li>Kanauchi, O., Mitsuyama, K., Araki, Y., Andoh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modification+of+intestinal+flora+in+the+treatment+of+inflammatory+bowel+disease.">Modification of intestinal flora in the treatment of inflammatory bowel disease.</a> Curr Pharm Des. 9, 333-346 (2003).</li><li>Brenchley, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+translocation+is+a+cause+of+systemic+immune+activation+in+chronic+HIV+infection.">Microbial translocation is a cause of systemic immune activation in chronic HIV infection.</a> Nat Med. 12, 1365-1371 (2006).</li><li>Brenchley, J. M., Price, D. A., Douek, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV+disease:+fallout+from+a+mucosal+catastrophe.">HIV disease: fallout from a mucosal catastrophe.</a> Nat Immunol. 7, 235-239 (2006).</li><li>Jiang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+levels+of+bacterial+DNA+correlate+with+immune+activation+and+the+magnitude+of+immune+restoration+in+persons+with+antiretroviral-treated+HIV+infection.">Plasma levels of bacterial DNA correlate with immune activation and the magnitude of immune restoration in persons with antiretroviral-treated HIV infection.</a> J Infect Dis. 199, 1177-1185 (2009).</li><li>Marchetti, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+translocation+is+associated+with+sustained+failure+in+CD4++T-cell+reconstitution+in+HIV-infected+patients+on+long-term+highly+active+antiretroviral+therapy.">Microbial translocation is associated with sustained failure in CD4+ T-cell reconstitution in HIV-infected patients on long-term highly active antiretroviral therapy.</a> AIDS. 22, 2035-2038 (2008).</li><li>Marchetti, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Microbial+Translocation+and+Immune+Hyperactivation+in+Disease+Progression+of+HIV++Patients+with+Preserved+CD4+Count+in+the+Absence+of+ART.">Role of Microbial Translocation and Immune Hyperactivation in Disease Progression of HIV+ Patients with Preserved CD4 Count in the Absence of ART.</a> , (2010).</li><li>Greisen, K., Loeffelholz, M., Purohit, A., Leong, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PCR+primers+and+probes+for+the+16S+rRNA+gene+of+most+species+of+pathogenic+bacteria,+including+bacteria+found+in+cerebrospinal+fluid.">PCR primers and probes for the 16S rRNA gene of most species of pathogenic bacteria, including bacteria found in cerebrospinal fluid.</a> J Clin Microbiol. 32, 335-351 (1994).</li></ol>

We hereby show a PCR/sequencing protocol to characterise the translocation of bacteria in peripheral blood of HIV-infected individuals.
Most reliable results are obtained when using plasma collected in EDTA-containing tubes. After blood sampling, plasma should be separated by 
	centrifugation within 2/3 hours, to avoid haemolysis and DNA fragment degradation. Samples are stored first at -20&deg;C for 
	approximately 5 days and then at -80&deg;C. Multiple freeze and thaw cycles which may cause...

<table border="1">
	<tbody>
		<tr>
			<th>Name of Reagent</th>
			<th>Company</th>
			<th>Catalogue Number</th>
			<th>Comments (optional)</th>
		</tr>
		<tr>
			<td>Easty-DNA Kit</td>
			<td>Invitrogen</td>
			<td>K 180001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Sigma-Aldrich</td>
			<td>C2432</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>E7203</td>
			<td>&nbsp;</td>
		<tr/>
		<tr>
			<td>UltraPure Waer</td>
			<td>Invitrogen</td>
			<td>10977049</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lysozyme</td>
			<td>Fluka</td>
			<td>62970</td>
			<td>10 &mu;g/mL in Distillated Water</td>
		</tr>
		<tr>
			<td>AmpliTaq Gold</td>
			<td>Applied Biosystem</td>
			<td>26478701</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microcon 100</td>
			<td>Millipore</td>
			<td>42413</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PCR primers</td>
			<td>Invitrogen</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Eppendorf</td>
			<td>C1343</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DNA ladder</td>
			<td>Invitrogen</td>
			<td>15628019</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Purelink PCR micro kit</td>
			<td>Invitrogen</td>
			<td>K310050</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>LB Agar, powder</td>
			<td>Invitrogen</td>
			<td>22700025</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bactoagar</td>
			<td>Invitrogen</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Invitrogen</td>
			<td>11593027</td>
			<td>10 mg/mL in Distillated Water</td>
		</tr>
		<tr>
			<td>X-gal</td>
			<td>Invitrogen</td>
			<td>15520034</td>
			<td>40mg/mL in DMF</td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Invitrogen</td>
			<td>15529019</td>
			<td>100mM in Distillated water</td>
		</tr>
		<tr>
			<td>Topo TA cloning kit</td>
			<td>Invitrogen</td>
			<td>K450002</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Purelink Quick Plasmid Miniprep kit</td>
			<td>Invitrogen</td>
			<td>K210010</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Big dye</td>
			<td>Applied Biosystem</td>
			<td>4337455</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DyeEx 2.0 spin kit</td>
			<td>Qiagen</td>
			<td>63204</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

sequencing of bacterial microflora in peripheral blood: our experience with hiv-infected patients

The healthy gastrointestinal tract is physiologically colonized by a large variety of commensal microbes that influence the development 
	of the humoral and cellular mucosal immune system1,2. 
 Microbiota is shielded from the immune system via a strong mucosal barrier. Infections and antibiotics are known to alter both the normal 
	gastrointestinal tract barrier and the composition of resident bacteria, which may result in possible immune abnormalities3.
HIV causes a breach in the gastrointestinal barrier with progressive failure of mucosal immunity and leakage into the systemic circulation of bacterial bioproducts, such as lipopolysaccharide and 
	bacterial DNA fragments, which contribute to systemic immune activation4-7. Microbial translocation is implicated in HIV/AIDS immunopathogenesis and response to therapy 4,8.
We aimed to characterise the composition of bacteria translocating in peripheral blood of HIV-infected patients. To pursue our aim we set up a PCR reaction for the panbacteric 16S ribosomial gene 
	followed by a sequencing analysis.
Briefly, whole blood from both HIV-infected and healthy subjects is used. Given that healthy individuals present normal intestinal homeostasis no translocation of microflora is expected in 
	these patients. Following whole blood collection by venipuncture and plasma separation, DNA is extracted from plasma and used to perform a broad range PCR reaction for the panbacteric 
	16S ribosomial gene9. Following PCR product purification, cloning and sequencing analyses are performed.

Watch this Scientific Journal Video about Sequencing of Bacterial Microflora in Peripheral Blood: our Experience with HIV-infected Patients at JoVE.com

Sequencing of Bacterial Microflora in Peripheral Blood: our Experience with HIV-infected Patients

Our experiment will show how to perform a sequencing analysis of bacterial species translocating in peripheral blood of HIV positive patients.

The healthy gastrointestinal tract is physiologically colonized by a large variety of commensal microbes that influence the development 
	of the humoral ...

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