Patients who require a spacer exchange as part of a two-stage procedure for the treatment of periprosthetic hip and knee joint infections (PJI) have high failure rates. Little is known about the clinical impact of microbiological results and changes in the microbiological spectrum and resistance pattern in these patients. Between 01/2011 and 12/2019, 312 patients underwent a total of 327 two-stage revision arthroplasties at our institution. A spacer exchange was required in 52/312 (16.7%) patients (27 knee/25 hip). Microbiological results, antibiotic resistance patterns, patient's host factors as well as re-revision and re-infection rates at a median follow-up of 47.8months (range 12.2-116.7months) were analyzed. A propensity score (PS)-matched analysis of patients who underwent spacer exchange and patients treated with standard two-stage procedure was performed. We found a high number of microbiological spectrum changes in patients with multiple culture positive procedures between explantations and spacer exchanges (10/12 [83.3%]), spacer exchanges and reimplantations (3/4 [75%]) as well as between reimplantations and subsequent re-revision surgeries (5/6 [83.3%]). In 9/52 (17.3%) patients, same microorganisms were detected repeatedly in two different procedures. We observed changes in the antibiotic resistance patterns in 6/9 (66.7%) of these patients. High re-infection rates were found in patients with culture positive reimplantations (10/12 [83.3%]), and low re-infection rates were found in patients with culture negative reimplantations (2/40 [5%]; p < 0.001). Between patients with and without spacer exchange, no differences were found in the re-revision rates (13/52 [25%] with vs. 13/52 [25%] without; p = 1.00) as well as re-infection rates (12/52 [23.1%] with vs. 8/52 [15.4%] without; p = 0.32). Changes in microbiological spectrum and antibiotic resistance patterns between stages are common in patients who require a spacer exchange. If eradication of the microorganism at reimplantation can be accomplished, comparable re-revision rates to standard two-stage procedures can be achieved.