Shilpa Ray[1], Aswathy Narayanan[1], Christian Giske[1, 2], Ujjwal Neogi[1], Anders Sönnerborg[1, 3], Piotr Nowak[1, 3]

Affiliates

Department of Laboratory Medicine, Division of Clinical Microbiology, ANA Futura, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden[1], Department of Clinical microbiology, Karolinska University Hospital, Solna, Stockholm, Sweden[2], Department of Medicine Huddinge, Division of Infectious Diseases, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden[3]

Abstract

Background: Millions of individuals currently living with HIV globally are receiving antiretroviral therapy (ART) that suppresses viral replication and improves host immune responses. The crucial involvement of gut microbiome during HIV infection has been extensively studied; correlating microbiome changes with immune deficiency and inflammation. However understanding the direct effect of ARTs on the diversity of gut commensals of HIV infected individuals has been mostly overlooked in microbiome studies.

Methods: The microbiota composition was determined by 16S rRNA sequencing (Illumina MiSeq) of stool samples from 16 patients before and during ART. We also tested the direct effect of 15 antiretrovirals on predominant gut microbes of high clinical relevance namely, Escherchia coli, Enterococcus faecalis, Bacteroides and Prevotella spp. using agar dilution and broth microdilution assay to assess their individual antibacterial effect. Thus, we screened 35 isolates of E. coli, 20 isolates of E. faecalis, 22 isolates of Bacteroides spp and 10 isolates of Prevotella spp. which were cultured from patient samples received at the Department of Clinical microbiology, Karolinska University Hospital.

Results: Our 16srRNA analysis showed that effective ART does not reestablish the microbiome diversity within HIV infected patients, thereby showing moderate reduction in α-diversity (Shannon index P=0.24, Simpson index P=0.46, Fisher index P=0.28) and ß-diversity (NMDS2 and NMDS1) for clustering of microbial communities before and after ART. Escherichia genus (phylum Proteobacteria) showed enrichment under therapy. On the contrary, the relative richness of Prevotella (phylum Bacteroidetes) was significantly reduced after ART. Our study also found the direct effects of antiretrovirals on gut microbes, where the thymidine analog zidovudine (ZDV) showed in vitro antibacterial activity with an MIC of 0.5-1 mg/l against E. coli, 4-8 mg/l against Bacteroides fragilis and 4 mg/l against Prevotella spp. Efavirenz (EFV) also showed a consistent MIC of 32 mg/l against E. faecalis, 32 mg/l for Prevotella spp. and 32-128 mg/l against Bacteroides spp. In addition, among the nine tested drug combinations, the combination containing EFV (as well as emtricitabine [FTC] and tenofovir disoproxil fumarate [TDF]) showed antibacterial effect against E. faecalis, Bacteroides and Prevotella spp. Even the drug combinations of EFV with lamivudine (3TC) and ZDV affected growth of E. coli, E. faecalis, Bacteroides and Prevotella spp.

Conclusion: HIV infection induces gut microbiome dysbiosis and ART does not reverse these changes after one year follow-up. In this study, we found that the antivirals directly have a broad effect on the gut bacteria, which in HIV infected individuals might aggravate microbiota alterations. Therefore restructuring the microbiota could be a potential therapeutic target in HIV-1 patients since effective ART itself may not be sufficient to reverse the microbiota dysbiosis.