AbstractPurposeTo characterize the microbiome composition in peri‐implant pocket of peri‐implantitis and peri‐implant sulcus controls using 16S rRNA gene sequencing.Materials and MethodsIn this controlled clinical cross‐sectional study, 23 subjects with control implants (n = 14) and diseased implants (peri‐implantitis, n = 21) were included. The peri‐implant pocket/sulcus was sampled and used to extract DNA and amplify the 16S rRNA gene using universal primers targeting the V3‐V4 regions. The resulting 16S PCR amplicons were sequenced on Illumina MiSeq, and the sequences were processed using DADA2 and the Human Oral Microbiome Database (HOMD) as references. Alpha and Beta diversity, as well as core microbiome and differential abundance analyses, were performed using the MicrobiomeAnalyst workflow.ResultsThere were no significant differences in microbial diversity between control implants and implants with peri‐implantitis (Shannon p = 0.82). Overall bacterial community structure assessed through beta diversity analysis was also not significantly different between the two groups (p = 0.18). However, high levels of Gram‐negative bacteria were detected in peri‐implant pockets compared to the control sulcus. Abundant species in peri‐implantitis were Capnocytophaga leadbetteri, Treponema maltophilum, Peptostreptococcus, Neisseria, P. gingivalis, and Porphyromonas endodontali, Lactococcus lactis and Filifactor alocis (p < 0.05). Gram‐positive bacteria such as Streptococcus salivaris, Prevotella melaninogenica, L. wadei, and Actinomyces spp. serve were more abundant in peri‐implant control sulcus.ConclusionsPeri‐implant sulcus in control implants harbors predominantly Gram‐positive bacteria, whereas pockets of implants with peri‐implantitis harbor predominantly Gram‐negative bacteria.