Introduction
Prosthetic joint replacement is one of the most important medical innovations of the twentieth century, and it has significantly improved the quality of life for millions of individuals worldwide by providing pain relief and restoring joint function, mobility, and independence [
1,
2]. However, prosthetic joint infection (PJI) following joint replacement is a devastating complication associated with high medical costs and increased in-hospital mortality [
3]. Both PJI diagnosis and treatment are challenging [
4]. Previous studies have shown that
Staphylococcus aureus and coagulase-negative staphylococci (CoNS), and in particular
Staphylococcus epidermidis, are the pathogenic agents in most PJIs [
5].
Currently, the international clinical guidelines for defining PJI diagnosis require two positive periprosthetic cultures with phenotypically ‘identical’ or ‘indistinguishable’ organisms, with the phenotype determined using common laboratory tests for genus and species identification and antibiograms [
6‐
8]. Since phenotypic morphological variations [
9], including the presence of small colony variants and different antibiograms, have been reported in monoclonal CoNS infections [
10‐
12], the term ‘phenotypically identical organisms’ is ambiguous. Furthermore, the assessment of CoNS in clinical cultures is demanding as CoNS are a ubiquitous part of the human skin microbiota and often for
S. epidermidis display high sub-species heterogeneity [
13,
14], which makes distinction between contamination and true infection challenging. Here, we investigated the extent of diversity among CoNS in PJI and characterised in detail the
S. epidermidis in these infections which revealed substantial within-patient diversity further highlighting the complexity and ambiguity in the current phenotypical assessment as the diagnostic criteria.
Discussion
Here, we investigated the diversity among CoNS in PJI and characterised in detail the S. epidermidis isolates from these infections. We found considerable within-patient diversity in S. epidermidis isolates, with variations in phenotypic and genotypic resistance observed in the majority (13/16; 81%) of cases, and also between isolates with the same ST. Additionally, while we considered the inherent difficulty of ruling out the possibility that a single S. epidermidis isolate represents contamination, S. epidermidis isolates belonging to different STs were detected in several PJIs (7/16; 44%). These findings further add to the complexity in assessing whether S. epidermidis identified in multiple cultures in potential PJI cases represent phenotypically identical organisms in two positive periprosthetic cultures. Hence, with the present guidelines, there is a risk that PJI pathogens are incorrectly dismissed as contaminants, which hinders the appropriate microbial diagnosis and treatment.
To date, limited data has been published on the within-patient genetic diversity of CoNS isolates in PJI. Here, we showed that while only a single ST was detected in the majority (9/16, 56%) of the PJIs, polyclonality was detected in 44% (7/16) of all PJIs with between two to five different STs. Importantly, when at least three
S. epidermidis isolates from different tissue samples were characterised in each PJI, the extent of the identified within-patient diversity among
S. epidermidis isolates increased. Within-patient variation in antibiograms was observed comparing
S. epidermidis isolates in almost all patients (10/11, 91%) and different STs were identified in 5/11 (45%). Obviously, among PJIs in which only two
S. epidermidis isolates were available for characterisation, polyclonal infection was detected less frequently (2/5 patients; Table
3). These results are consistent with those of a recent German study in which paired isolates from 55 cases of orthopaedic device-related infection were analysed, and 6/55 (11%) cases assessed as polyclonal [
24]. Therefore, increasing the number of
S. epidermidis isolates for characterisation, and preferably obtaining isolates from different tissue specimens, is important for determining isolate diversity and reduces the risk of incorrect dismissal of isolates as contaminants, and improves the basis for decisions on antibiotic therapy and accurate identification of a relapse or reinfection.
In agreement with previous data, isolates from HA-MDRSE lineages were the cause of most
S. epidermidis PJIs over a period of more than 2 years in the two hospitals in Northern Sweden [
23,
25]. The low pairwise diversity in the ST215 lineage observed in two PJI cases for which isolates were collected more than 1 year apart in the same hospital (2 SNPs), indicates that the ST215 lineage is persistent in the hospital setting in Sweden. In contrast, limited hospital-adapted transmission of genetic lineages has been reported in
S. aureus PJIs [
26]. The findings of this study are in alignment with a previously described scenario of the global dissemination of multidrug-resistant lineages of
S. epidermidis [
27,
28]. The likelihood of hospital-adapted transmission was further corroborated by a recent large study on
S. epidermidis PJI in Sweden [
23]. The adaptation of ST2 and ST215 lineages to the hospital environment includes common genomic traits (
IS256) and high prevalence of antimicrobial resistance genes even though some lineage-dependent differences are evident, i.e., the ST215 lineage lacks the intercellular adhesion gene A (
icaA) gene [
23,
27]. The primary source of HA-MDRSE lineages and the routes of transmission are uncertain. Recent data suggest that current perioperative PJI prevention regimens select MDRSE either from the patient’s normal flora or by facilitating acquisition from the hospital environment [
23].
Polymicrobial infections, including those caused by
S. epidermidis, were common among the PJIs investigated in this study, and consistent with previous data,
Enterococcus faecalis was the most frequent companion microbe [
29]. In most cases, both
S. epidermidis and
E. faecalis were detected in most tissue specimens from each patient, which reduced the chances of contamination; however, the possibility cannot be completely excluded.
The results presented here have practical and clinical implications. The within-patient diversity of
S. epidermidis infections suggests that the clinical microbiology assessment of a PJI requires re-evaluation [
30]. We believe characterising more than two isolates phenotypically and genotypically will improve assessment regardless of whether the PJI microbiological diagnostic criteria are met. In-depth analysis of more than two isolates will also provide additional information for selecting the appropriate targeted antibiotic therapy and help distinguish between a relapse and reinfection. The present clinical microbiology method for genetic heterogeneity assessment is laborious and expensive; however, advances reported in recent studies may change that in the near future. For example, new culture-independent methods that can be applied in clinical laboratories can facilitate the rapid assessment of clonality and population structure of
S. epidermidis communities in PJI [
31]. Another suitable approach is the culturing of several specimens followed by sequencing of multiple microbial isolates during routine PJI microbial diagnostics. Given the high cost of PJI treatment, it is practical to implement routine PJI diagnostics using small-scale rapid sequencing technologies with a turn-around time, including bioinformatics, of 1–2 days [
32].
There were a few limitations to this study. First, the study had a retrospective cohort design. However, this may not affect the investigation of
S. epidermidis populations causing PJIs, as it was shown that the
S. epidermidis population structure in central Sweden has remained fairly stable over the last 10 years [
23]. Second, the collection of more isolates per patient would strengthen our findings. Multiple
S. epidermidis isolates for WGS analysis could only be collected from a few patients with PJI. That said, the microbiological findings of heterogeneity indicate that the present-day criteria for PJI diagnosis is sub-optimal. Third, and most importantly, some of the isolates detected and characterised were potential contaminants and were not truly invasive; however, all consecutive patients with PJI who met the IDSA criteria were included. Further, we used fresh sets of skin incision and subcutaneous incision instruments and a new set of sterile instruments for each tissue specimen to reduce the risk of contamination.
In conclusion, the within-patient genetic diversity in S. epidermidis isolates was substantial, with variation in both antibiotic susceptibility and antibiotic resistance genes. The findings highlight the complexity and ambiguity in the phenotypical assessment of CoNS isolates from periprosthetic tissue cultures as diagnostic criteria for PJI. Larger systematic studies are needed to determine the implications of these findings for microbiological diagnosis and the clinical significance of these results for therapeutic outcomes.
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