Although S. epidermidis is associated primarily with its commensal nature and is predominantly present on the skin and mucous membranes41, there has been growing scientific interest in investigating its pathogenic characteristics owing to its typical epidemiological association with implant-related MSIs11,29,37. Indeed, in cases of FRI and delayed- and late-onset PJI (3–12 months and > 12 months, respectively), CoNS, particularly S. epidermidis, is the most commonly identified pathogen23,37. In a recently published French cohort of patients with bone and joint infections assessing nearly 12,000 cases of PJI, CoNS represented 25% of the cases11. Moreover, there is limited knowledge concerning the impact of S. epidermidis, previously known as a critical phenotypic and genomic determinant of invasiveness, on the clinical outcome of patients treated with MSIs. By comparing phenotypic and genomic data from a collection of commensal and MSI-associated S. epidermidis isolates, we assessed the importance of adaptive processes likely involved in the transition from a commensal nature to a state of infection. We attempted to provide insights into the molecular basis of MSI caused solely by invasive S. epidermidis, thus excluding MSIs caused by associations with pathogens. Polymicrobial peri-implant tissue, synovial and sonication fluid cultures yielding other skin and mucous-colonizing pathogens, such as S. aureus and gram-negative bacilli, have been described in up to 30% of MSIs2,3,4,5,6,7, which hampers the inclusion of a greater number of patients. Despite the limited number of MSI patients prospectively followed, biofilm formation and methicillin resistance were the key phenotypes, whereas the presence of IS256 was a genomic independent marker of poor outcomes.
In the present study, S. epidermidis isolates were obtained from 31 patients with different MSIs, including patients with FRI, PJI, bacterial septic arthritis, and chronic osteomyelitis. Importantly, all patients were prospectively followed for one year to evaluate their clinical outcomes accurately. FRI cases were the most commonly evaluated (n = 16) due to the high rates of trauma observed in Brazil. In a recent prospective cohort study conducted by our group, the overall FRI rate was 15.9%42. Among surgically removed orthopaedic implants, knee arthroplasty is the most common intervention. According to the findings of Lourtet-Hascoët et al.43, implant-associated complications occurred mainly in the knees (54.2%), followed by the hips (39.1%) and other areas (6.7%). We did not identify any other associations between MSI type, infection location, and clinical outcome in patients with MSI caused by S. epidermidis.
The mechanisms involved in the evolution from the commensal to the pathogenic (invasive) form of S. epidermidis include horizontal gene transfer (HGT), which is associated with pathogenic characteristics such as adhesion to tissues and host implants, biofilm formation, cellular toxicity, and antimicrobial resistance18,44,45,46. However, few studies have focused on the correlation between clinical outcomes after treatment and the molecular epidemiology (clonality) of S. epidermidis isolates causing MSI. In this study, the molecular epidemiology analysis of the 46 S. epidermidis isolates subjected to NGS revealed an expected predominance of the ST2 clade (21.7%) exclusively associated with MSI20. These ST2 isolates, belonging to CC2, which is the most widespread CC worldwide, appear to be the most pathogenic. In the study by Méric and colleagues, 415 S. epidermidis isolates from colonized and MSI samples were evaluated, where the increasing presence of accessory genes in clinical samples suggested the dissemination of genes by HGT between colonizing and invasive isolates47. As expected, there was significant variability in the frequency of sequence types (STs). However, most were grouped in the clonal complex (CC2). The model published in these studies effectively demonstrated how a commensal isolate of S. epidermidis can become invasive. Another molecular epidemiology study of S. epidermidis in PJIs conducted in Sweden revealed that nearly 300 S. epidermidis isolates from PJI and nasal commensals presented distinct genomic characteristics. PJI isolates were exclusively identified as MRSE ST2 strains (43%), ST215 strains (25%), and ST5 strains (9%)20. Additionally, Trobos et al. reported that in 50 S. epidermidis isolates from PJI, genotypic and phenotypic characteristics were positively associated with treatment failure, especially in ST2 isolates (49%)48. In the study by Sánchez et al., S. epidermidis ST2 isolates were predominant in 44% of PJIs, followed by ST640 and ST523. Despite the variability of STs in our study (25 different varieties), ST2, ST23, ST6, ST885, and ST1199 were identified exclusively in MSI, and 37% of the isolates were grouped in CC2 (ST2, ST23, ST5, ST73, and ST89), which is associated with bacterial resistance and strong biofilm production. Sanchez et al. reported that the combination of pathogenic factors may contribute to the adaptation and survival of invasive clones, such as ST2, to specific physiological conditions during infection, thus overcoming the host’s natural and antibiotic-induced immunological protection23.
Recent studies have demonstrated that invasive S. epidermidis isolates expressing antimicrobial-resistant phenotypes to β-lactams or multidrug-resistant and strong/moderate biofilm formers harbour the mecA gene, mobile genetic elements (IS256, SCCmec) and other genes (ica locus) associated with biofilm formation20,23,38,39,47,48,49,50. Frequently, these pathogenic isolates belong to the ST2 or ST23 lineage of global and hospital spread and multidrug resistance, containing mutations in the rpoB gene that confer resistance to rifampicin and glycopeptides such as teicoplanin21. In the present study, isolates resistant to methicillin were significantly associated with MSI and, interestingly, with infection recurrence (univariate analysis). Indeed, the blaZ and mecA genes were carried by 84.8% and 56.5% of the isolates, respectively. Isolates resistant to quinolones and aminoglycosides were also significantly more common in MSIs. Quinolone resistance was associated with a mutation in the parE gene (D432V) found in 13% of the MSI isolates. Similarly, aminoglycoside resistance genes varied from 6.5% (ant(9)-Ia) to 37% (AAC(6’)-Ie-APH(2’’)-Ia), being found almost exclusively in MSI isolates. Additionally, the rpoB gene, which confers resistance to rifampicin, was present in 21.7% of the isolates and was found in only patients with MSI. Rifampicin resistance develops through a single-step mutation in the rpoB gene encoding the β subunit of bacterial DNA-dependent RNA polymerase. We identified specific mutations D471E and I527M in the rpoB gene, the latter associated with the multidrug-resistant global dissemination clone of hospital-origin S. epidermidis (MRSE), previously referred to as the most common cause of rifampicin resistance, which comprises the ST2 and ST23 lineages21. Indeed, five of the six S. epidermidis isolates presenting the I527M mutation in the rpoB gene presented MICs > 128 µg/mL for rifampicin. Rifampicin resistance is concerned owing to the clinical impact of this drug on MSI treatment. Recently, Lazarines et al.49 demonstrated a significant increase in the risk of treatment failure in PJIs caused by rifampicin-resistant S. epidermidis. The findings of this study are regionally essential and unprecedented in Brazil, as they demonstrate that this MRSE lineage is adapted to the local hospital environment and is often associated with a worse clinical prognosis. According to Lee and colleagues, rifampicin resistance in MSI is currently needed, as these two concurrent mutations in rpoB (D471E and I527M) can lead to concomitant heteroresistance to vancomycin and teicoplanin21,49.
S. epidermidis expresses genes that produce numerous surface proteins capable of adhering to multiple substrates and forming biofilms11,12,37,51. These characteristics have been attributed to the primary virulence mechanisms of this species52. Indeed, the well-adapted mechanisms of adhesion and biofilm formation of S. epidermidis contribute to the high frequency of chronic infections associated with implants.
In our results, the presence of IS256 (regulating biofilm formation and genes encoding resistance to aminoglycosides) in the S. epidermidis isolates was a strong independent predictor of MSI with poor outcomes, with an odds ratio of 8.002. Moreover, weak biofilm production in the S. epidermidis isolates was protective, reducing the chance of MSI recurrence by 93% (1–0.070). These results suggest that the IS256 mobile genetic element and weak biofilm formation are significant genomic markers predictors of MSI recurrence caused by S. epidermidis, which may have future implications to the clinical setting. In practical terms, the application of this model can be envisaged in the stratification of patients into risk groups. Based on these predicted probabilities (IS256), it is possible to identify patients with a higher likelihood of treatment failure. These individuals could be candidates for more aggressive therapeutic interventions or more frequent clinical follow-ups, while low-risk patients would be managed less intensively. This personalised approach, when refined by future studies, can optimise resources and improve clinical outcomes. Considering what is currently known, S. epidermidis expresses a striking genetic flexibility by easily repurposing itself depending upon local challenging environmental conditions through continuous generation of novel phenotypic and genotypic variants to switch from a colonizing to an invasive state9,39,50,51. Nosocomial S. epidermidis strains typically harbour multiple copies of the highly active IS256 in their genomes affecting the expression of virulence and drug resistance genes and represents an obvious driving force for the flexibility of the S. epidermidis genome18. It has been speculated by previous studies and confirmed by our results, that the acquisition of readily IS256 mobile genetic element is correlated with increased biofilm formation, while it has been regarded as a positive molecular marker of invasive hospital-origin isolates and associated with unfavourable prognoses after MSI treatment11,12,20,23. In fact, our results provide evidence that IS256 is not only a genetic marker of invasive isolates but also strongly associated with the clinical outcome of infection recurrence, as we identified that this mobile genetic element was carried by 41.3% of the isolates, exclusively those derived from MSI. Meanwhile, several other genetic traits which have not been tested in the present study is likely important to the multifactorial processes of infection adaptation and would also play a role in the outcome of MSI such as enhanced growth in iron-free and nutrient-poor media, reduced production of hemolysins and mutation in the acetate kinase (ackA) and the β-subunit of the RNA polymerase (rpoB) gene50.
The presence of the complete ica operon (icaA, icaB, icaD, icaC) was more common in MSI isolates (39.1%) and in patients with recurrent MSI (32.2% vs. 25.8%); however, the difference was not statistically significant between these groups, which may be associated with the small number of isolates evaluated in the present study. While previous studies have successfully associated the complete ica operon (associated with biofilm formation) harbouring the insertion sequence IS256, with invasive S. epidermidis isolates53 our findings are in agreement with others study’s conclusions, from which ica operon is not a useful diagnostic marker of the invasive capacity of S. epidermidis in MSI10,24,54,55.
The most significant limitation of the present study is the small number of patients evaluated, and isolates tested, especially those isolated from only 31 patients with musculoskeletal infections. However, the study design favoured the inclusion of patients with MSIs caused solely by S. epidermidis isolates, which could demonstrate the isolation and real pathogenic role of this agent. MSIs identified in S. epidermidis are often accompanied by other gram-positive and gram-negative bacteria. Additionally, the prospective observation of patients with MSI for at least one year to reduce the bias of underdiagnosis of recurrence in late-diagnosed infections was also a concern of the authors. On the other hand, this study advanced the search for independent associations of the phenotypic and genotypic characteristics of infected S. epidermidis isolates with those of colonized isolates and evaluated the clinical outcomes of infection recurrence and cure after one year of follow-up. Logistic regression models optimized with the stepwise method were used to test the accuracy of the independent forces of the genomic variables (virulence and resistance genes and IS256) in predicting the clinical outcome. Additionally, the model’s performance was evaluated via Nagelkerke’s R, which resulted in 47.21% confidence in the model. The objective of the predictive analysis of recurrence in this study was exploratory and investigative, acknowledging the limitations imposed by the current sample size. Although our findings are promising, we emphasise that they should be tested in new populations before a safe clinical application can be developed and implemented. Nevertheless, the robust metrics obtained reinforce the potential of the model as a clinical decision support tool, particularly in the management of patients with treatment for S. epidermidis-MSI. In the final model, the variables IS256 and biofilm production were significant independent predictors of the recurrence outcome. Another limitation of the study is the performance of nasal swabs to identify control isolates in healthy individuals, which is not related to patients who evolve to infection. In contrast the ideal approach would be to analyses isolates from the skin/mucosa of patients undergoing orthopaedic surgeries, such as arthroplasty or fracture correction, that do not result in infection. However, to include only isolates from patients who underwent orthopaedic surgery that evolved to infection, collecting samples from thousands of patients before hospitalization for more than ten years would be necessary. A relevant limitation is the decision to evaluate only a single colony of S. epidermidis from each patient and healthy individual, as the infection could be caused by several clones (polyclonal), while expressing significant spatiotemporal diversity and genomic flexibility dependent on environmental selective pressures within the same individual55. Another technical limitation concerns the clonal relatedness analysis of commensal and invasive isolates by applying MLST, which is based on sequence comparisons of seven core genome loci, allowing classifications into global lineages rather than the gold standard to determine clonal relatedness in staphylococci via pulsed-field gel electrophoresis (PFGE), which is more discriminatory than MLST54. Finally, we acknowledge that certain clinical variables, such as the type of antibiotic therapy administered to MSI patients, were not included in the analytical model evaluating independent factors for treatment failure in MSI patients with S. epidermidis. This omission may have influenced the outcomes of our multivariate analysis. Nonetheless, it is worth mentioning that all patients received antibiotic treatments guided by the standard bacterial sensitivity profiles (EUCAST/ BRCAST) and were administered for the appropriate durations.
In conclusion, genomic analysis revealed evidence of the presence of specific phenotypes and genomic traits associated with the pathogenicity and risk of treatment failure in MRSE isolates causing MSI. Commensal and invasive S. epidermidis isolates are not the same. Differences between invasive isolates and those derived from colonization are associated with pathogenic characteristics such as strong/moderate biofilm formation and bacterial resistance to multiple antibiotics, such as β-lactams, aminoglycosides, and quinolones. These traits should be considered important risk factors to guide the microbiological diagnosis and clinical treatment of MSIs. The detection of a pathogenic MRSE lineage causing MSI reinforces recommendations for the implementation of rigorous measures to control the spread of MDR strains in the hospital environment. Future studies should include a greater number of patients with MSI in a prospective design to reinforce the clinical relevance and impact of findings related to genetic variants and virulence factors associated with treatment failure.
