Evidence for polymicrobic flora translocating in peripheral blood of HIV-infected patients with poor immune response to antiretroviral therapy

Abstract

In advanced HIV infection, the homeostatic balance between gastrointestinal indigenous bacteria and gut immunity fails and microbes are able to overcome the intestinal barrier and gain the systemic circulation. Because microbial translocation is not fully controlled by antiviral therapy and is associated with inefficient CD4+ reconstitution, we investigated the profile of translocating bacteria in peripheral blood of 44 HIV-infected patients starting therapy with advanced CD4+ T-lymphopenia and displaying poor CD4+ recovery on virologically suppressive HAART. According to CD4+ reconstitution at 12-months HAART, patients were considered Partial Immunological Responders, PIRs (CD4+≥250/µl, n = 29) and Immunological non Responders, INRs (CD4+<200/µl, n = 15)). We show that PIRs and INRs present similarly elevated plasma levels of lipopolysaccharide (LPS) and its ligand sCD14 that were not lowered by virologically suppressive therapy. Bacterial 16S rRNA gene amplification and sequencing resulted in a highly polymicrobic peripheral blood microbiota both prior and after 12-month HAART. Several differences in bacterial composition were shown between patients' groups, mainly the lack of probiotic Lactobacillaceae both prior and after therapy in INRs. Failure to control microbial translocation on HAART is associated with a polymicrobic flora circulating in peripheral blood that is not substantially modified by therapy.

Figure 1. Characterization of translocating microflora in HIV+ antiretroviral naive patients.

Patients' plasma samples were examined before and after 12 months of HAART for the presence and identification of DNA bacterial fragments using a broad-range 16S rRNA gene PCR amplification followed by sequencing analysis. At baseline, 14/44 (32%) HIV+ patients yielded a positive PCR amplification, whereas at T12, 7/44 (16%) HIV+ patients were PCR-positive. Sequencing analysis of HIV positive patients as a whole revealed multiple bacterial orders for each patient with no differences in the bacterial composition between baseline and T12. Positive (%) = # patients displaying a specific bacterial order/total # of PCR positive patients. Negative (%) = # patients negative for a specific bacterial order/total # of PCR positive patients.

Figure 2. Characterization of translocating microflora in PIRs and INRs.

Bacterial microflora translocating in peripheral blood was evaluated in 44 HAART-naive patients with severe immune depression (CD4+≤200/µL) at baseline and after 12 months of stable HAART (T12). At T12, 29 patients were partial immunological responders -PIRs- (CD4+≥250/µL; VL<60 cp/mL) and 15 were immunological-non-responders -INRs- (CD4+<200/µL; VL<60 cp/mL). At baseline, we found a significantly higher proportion of PCR-positive INRs (INRs: 8/15, 53%; PIRs: 6/29, 20%; p = .04). At T12 a non significantly higher proportion of INRs yielded a positive 16S gene PCR amplification: INRs: 4/15 (27%); PIRs: 3/29 (10%); p = .21. PIRs and INRs displaying a similar bacterial orders both at baseline and T12, except Pseudomonadales order *(FRs 5/6, 83% vs 1/8, 12% p = .026). Positive (%) = # patients displaying a specific bacterial order/total # of PCR positive PIRs (or INRs). Negative (%) = # patients negative for a specific bacterial order/total # of PCR positive PIRs (or INRs).

Figure 3. Identification of bacterial families in PIRs and INRs.

Positive (%) = # patients displaying a specific bacterial family/total # of PCR positive PIRs (or INRs). Negative (%) = # patients negative for a specific bacterial family/total # of PCR positive PIRs (or INRs). PIRs and INRs presented a different profile in term of bacteria families. (A) PIRs displayed a similar composition of bacterial families between baseline and T12. (B) No major changes in bacterial families were seen in INRs after 12 months-HAART. At baseline, the significant differences between the two groups concerned Lactobacillaceae *(PIRs 3/6, 50% vs INRs 0/8 0%; p = .05) and Pseudomonadaceae *(PIRs 5/6, 83% vs INRs 1/8, 13%; p = .026). No differences between PIRs and INRs at T12.

Figure 4. Bacterial genera identified in plasma sample of PIRs and INRs.

Positive (%) = # patients displaying a specific bacterial genera/total # of PCR positive PIRs (or INRs). Negative (%) = # patients negative for a specific bacterial genera/total # of PCR positive PIRs (or INRs). (A–B) A similar composition of bacterial genera were seen in PIRs both at T0 and T12. (C–D) INRs presented no variation in bacterial genera following 12 months of HAART. At baseline, significant differences between PIRs and INRs were seen for Lactobacillus sp *(PIRs 3/6, 50% vs INRs 0/8 0%; p = .05) and Pseudomonas sp *(PIRs 5/6, 83% vs INRs 1/8, 13%; p = .026). No differences between the two groups at T12.