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

Summary

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

Abstract

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 system^1,2.

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 activation^4-7. 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.

Protocol

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’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 done in a class II biohazard cabinet hood.
Mechanical pipetting aids should be used.
Use of needles or other sharps (including glass e.g. pipettes o....

Discussion

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°C for approximately 5 days and then at -80°C. Multiple freeze and thaw cycles which may cause.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Easty-DNA Kit	Invitrogen	K 180001
Chloroform	Sigma-Aldrich	C2432
Ethanol	Sigma-Aldrich	E7203
UltraPure Waer	Invitrogen	10977049
Lysozyme	Fluka	62970	10 μg/mL in Distillated Water
AmpliTaq Gold	Applied Biosystems	26478701
Microcon 100	EMD Millipore	42413
PCR primers	Invitrogen
Agarose	Eppendorf	C1343
DNA ladder	Invitrogen	15628019
Purelink PCR micro kit	Invitrogen	K310050
LB Agar, powder	Invitrogen	22700025
Bactoagar	Invitrogen
Ampicillin	Invitrogen	11593027	10 mg/mL in Distillated Water
X-gal	Invitrogen	15520034	40mg/mL in DMF
IPTG	Invitrogen	15529019	100mM in Distillated water
Topo TA cloning kit	Invitrogen	K450002
Purelink Quick Plasmid Miniprep kit	Invitrogen	K210010
Big dye	Applied Biosystems	4337455
DyeEx 2.0 spin kit	Qiagen	63204