Amplicon-Based Next-Generation Sequencing as a Diagnostic Tool for the Detection of Phylotypes of Cutibacterium acnes in Orthopedic Implant-Associated Infections

Abstract

The diagnosis of orthopedic implant-associated infections (OIAIs) caused by the slow-growing anaerobic bacterium Cutibacterium acnes is challenging. The mild clinical presentations of this low-virulent bacterium along with its ubiquitous presence on human skin and human-dominated environments often make it difficult to differentiate true infection from contamination. Previous studies have applied C. acnes phylotyping as a potential avenue to distinguish contamination from infection; several studies reported a prevalence of phylotypes IB [corresponding to type H in the single-locus sequence typing (SLST) scheme] and II (SLST type K) in OIAIs, while a few others found phylotype IA1 (more specifically SLST type A) to be abundant. However, phylotype determination has mainly been done in a culture-dependent manner on randomly selected C. acnes isolates. Here, we used a culture-independent amplicon-based next-generation sequencing (aNGS) approach to determine the presence and relative abundances of C. acnes phylotypes in clinical OIAI specimens. As amplicon, the SLST target was used, a genomic fragment that is present in all C. acnes strains known to date. The aNGS approach was applied to 30 sonication fluid (SF) samples obtained from implants removed during revision surgeries, including 17 C. acnes culture-positive and 13 culture-negative SF specimens. In 53% of the culture-positive samples, SLST types were identified: relative abundances were highest for K-type C. acnes, followed by H- and D-type C. acnes. Other types, including A- and C-type C. acnes that are more prevalent on human skin, had low relative abundances. The aNGS results were compared with, and confirmed by a culture-dependent approach, which included the isolation, whole genome sequencing (WGS) and phylotyping of 36 strains of C. acnes obtained from these SF samples. Besides serving as a powerful adjunct to identify C. acnes phylotypes, the aNGS approach could also distinguish mono- from heterotypic infections, i.e., infections caused by more than one phylotype of C. acnes: in eight out of nine culture-positive SF samples multiple C. acnes types were detected. We propose that the aNGS approach, along with the patient’s clinical information, tissue and SF cultures and WGS, could help differentiate C. acnes contamination from true infection.

Shotgun sequencing of sonication fluid for the diagnosis of orthopaedic implant-associated infections with Cutibacterium acnes as suspected causative agent

“…A potential possibility to reduce wound contamination could be to wash the removed implants before sonication; this was not performed in this study due to logistic reasons. Here, the implants were placed in sterile, airtight, single-use plastic containers in the operating room and covered with sterile saline before being transported to the lab ( Ponraj et al., 2022 ).…”

“…In a previous study, 100 implants removed during revision surgery were collected between August 2019 and September 2020 and processed by sonication ( Ponraj et al., 2022 ). SF specimens from 24 of the 100 implants were included in this pilot study.…”

“…The samples for culture-independent analyses were stored at −20°C until processing. DNA extraction of the SF and the negative control was described previously ( Ponraj et al., 2022 ). In brief, SF (40 ml) stored at −20°C was thawed overnight at 4°C, concentrated by centrifugation (15,000 × g for 1 h at 16°C using rotor F 13–14 × 50 Cy, Thermo Scientific™ Sorvall™ RC 6 Plus Centrifuge), and DNA extraction was performed using the DNeasy PowerSoil Kit (QIAGEN, Hilden, Germany) as per manufacturer’s instructions.…”

“…To test the sensitivity of the aNGS approach (in detail described previously in Ponraj et al., 2022 ), a PCR was performed with the single-locus sequence typing (SLST) primers (5′-TTGCTCGCAACTGCAAGCA-3′ and 5′-CCGGCTGGCAAATGAGGCAT-3′) and different amounts (1,000 to 0.001 pg) of C. acnes genomic DNA (strain 266). The PCR contained 5 μl of genomic DNA template, 2.5 μl of AccuPrime PCR Buffer II (Invitrogen, Waltham, MA, USA), 1.5 μl of each primer (10 μM; DNA Technology, Risskov, Denmark), 0.15 μl of AccuPrime Taq DNA Polymerase High Fidelity (Invitrogen, Waltham, MA, USA), and 14.35 μl of PCR-grade water.…”

“…In our previous study, we reported on the sonication of 100 orthopaedic implants that were removed for both presumed aseptic reasons and suspected infection ( Ponraj et al., 2022 ). SF samples were further subjected to culture-dependent and culture-independent analyses.…”