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1Department of Medicine, Surgery and Dentistry, Clinic of Infectious Diseases, San Paolo Hospital University of Milan, Italy
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Our experiment will show how to perform a sequencing analysis of bacterial species translocating in peripheral blood of HIV positive patients.
Merlini, E., Bellistri, G. M., Tincati, C., d'Arminio Monforte, A., Marchetti, G. Sequencing of Bacterial Microflora in Peripheral Blood: our Experience with HIV-infected Patients. J. Vis. Exp. (52), e2830, doi:10.3791/2830 (2011).
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.
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 or capillary tubes) must be limited to situations in which there is no alternative.
Laboratory surfaces must be decontaminated with an appropriate chemical disinfectant after a spill of blood and when work activities are completed.
Contaminated materials used must be decontaminated before reuse or must be disposed of correctly via the clinical waste route.
Every incident of occupational exposure to potentially infectious blood or fluids (i.e., those requiring universal precautions) should be treated as a medical emergency as interventions must be initiated promptly to be effective.
The protocol requires 5 days for its completion. The timeframe is detailed in Figure 1.
Bacterial species are identified using the methods described in Figure 2.
1. Sample Collection
2. DNA Extraction from Plasma Samples
DNA is extracted using a commercial kit following manufacturer’s instructions (Easy-DNA Kit, Invitrogen, Carlsbad CA, USA).
3. 16S rRNA Gene PCR
PCR amplification is performed as previously described 9.
4. PCR Product Purification
Only PCR positive samples should be purified. Purification is performed using a commercial kit following manufacturer’s instructions (PureLink PCR microkit, Invitrogen, Carlsbad CA, USA).
Lysogeny Broth (LB) Plate Preparation
Competent Cells Transformation
Transformation is performed using a commercial kit following manufacturer’s instructions (Topo TA cloning kit, Invitrogen, Carlsbad CA, USA).
Following incubation, white and blue colonies develop on plates. White colonies are positive for PCR product insertion and blue colonies are negative for PCR product insertion.
6. Sequencing Analysis
7. Representative Results
Figure 1. Timeline of the procedure.
Figure 2. Flow charts of the entire bacterial identification procedure.
Figure 3. 2% agarose gel showing broad range 16S rRNA gene PCR products. Lane 1 contains a 100bp DNA ladder, lane 2 contains the PCR positive control, lane 3 shows the negative PCR control. Lane 4 shows samples from an HIV-positive patient, and lane 5 contains water. Lane 6 shows samples from a healthy individual and a negative PCR reaction; lane 7 contains water. Only HIV positive patients displays a positive PCR amplification. Ultrapure water used as a negative control during the extraction step, indicates that no contamination occurred.
Figure 4. 0.7% agarose gel showing plasmid extracted with miniprep procedure. Lane 1 contains a 1 Kilobase DNA ladder, lane 2 contains the blue control colony, lanes 3 through 12 contain white colonies. The plasmid from the blue colony does not contain the PCR product insert. All 10 white colonies contain plasmid with the correct insert.
Figure 5. Shows an example of bacterial sequencing analysis in plasma from one HIV-positive individual. Our results show that microbial translocation in HIV disease involves a polimicrobic flora, which is not seen in HIV-negative subjects, suggesting substantial failure of gut immunity in controlling bacteria translocation.
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 loss of bacterial DNA should be avoided.
Plasma samples should be used within 12 hours after thawing.
Optimization of a DNA extraction protocol was performed in order to obtain the maximum quantity of DNA from donors’ peripheral blood. More specifically, plasma instead of whole blood is used , because the latter contains low amounts of bacterial DNA which is also under-represented by the great amount of DNA derived from peripheral blood cells.
We deliberately chose to exclude from our study subjects with signs and/or symptoms of acute infection. Nonetheless, given that subjects with HIV-related immunedepression present impairment of the gastro-intestinal barrier and translocation of bacterial products, we could not exclude translocation of entire microorganisms. Thus the rationale behind the use of lysozyme: given that this enzyme damages the bacterial wall, its use allows for the release of bacterial DNA from extremely low numbers of circulating microorganism which do not necessarily account for overt clinical manifestations.
The major issue in the present experiment is the risk of contamination. Given the high risk of PCR contamination from environment- and reagent-borne bacteria, we optimized laboratory procedures to avoid contamination in each step of the experiment, starting from sample collection.
In particular, for best sample preparation, it is essential to avoid contamination of plasma samples and surfaces of plastic consumables and containers. This is achieved by using disinfectants, powder free gloves and by respecting standard laboratory procedures. Within the described protocol the most critical steps in terms of contamination risk are DNA extraction and amplification, due to the possible presence of contaminating bacterial DNA, deriving, for example, from Taq polymerase.
Using this approach, we demonstrated that HIV-infected patients display translocation of polymicrobic bacteria in peripheral blood. No microbes are detected in the peripheral blood of healthy individuals, possibly reflecting the integrity of the gastrointestinal barrier. Future research will focus on the investigation of the role of peripheral blood microbiota in the pathogenesis of HIV/AIDS and response to anti-retroviral therapy.
No conflicts of interest declared.
We are grateful to Gianni Scimone for excellent assistance with video making.
|Easty-DNA Kit||Invitrogen||K 180001|
|Lysozyme||Fluka||62970||10 μg/mL in Distillated Water|
|AmpliTaq Gold||Applied Biosystems||26478701|
|Microcon 100||EMD Millipore||42413|
|Purelink PCR micro kit||Invitrogen||K310050|
|LB Agar, powder||Invitrogen||22700025|
|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|
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