Periprosthetic infection (PPI) continues to be one of the main complications of prosthetic surgery, despite application of measures to prevent it during the perioperative period (Kapadia et al., 2016; Ratto et al., 2016). Biofilm formation favors the persistence of PPI and reduces the effect of antibiotics, thus hampering treatment. The most effective approach to treating PPI is preventing onset (Kapadia et al., 2016; Shoji and Chen, 2020).

Tranexamic acid (TXA) is used routinely in conventional surgery. It is currently indicated for hemostatic control of patients undergoing surgical interventions (Jennings et al., 2016; Waddell et al., 2016; Ockerman et al., 2021). Its mechanism of action is similar to that of lysine, which is capable of binding to plasminogen, preventing fibrinolysis, and, therefore, controlling hemorrhage (Jennings et al., 2016; Draxler et al., 2019).

TXA seems to be associated with a reduction in postoperative complications, including surgical wound infection. Based on the reported decrease in postoperative hematoma, some authors believe this is due to an indirect effect (Klement et al., 2020; Lacko et al., 2020; Sukeik et al., 2020; Yazdi et al., 2020). Other authors reported that TXA exerts a direct antibacterial effect on planktonic cells (Zhang et al., 2021; Benjumea et al., 2022b; Wang et al., 2022) and, moreover, that it produces a synergistic effect with antibiotics (Benjumea et al., 2022a). However, these results need to be validated.

The aim of our study was to assess the synergistic effect of TXA with vancomycin and gentamicin in clinical strains isolated from patients with staphylococcal PPI and to corroborate our findings in an in vivo model.

The study was carried out at a tertiary teaching Institution in Madrid, Spain.

Our in vivo model corroborates our previous findings regarding the antimicrobial effect of TXA in PPI (Benjumea et al., 2022a). However, when we tested in vitro TXA against a large collection of clinical strains, we were able to demonstrate its synergistic effect in only 30% of them.

TXA has been associated with a decrease in the incidence of surgical wound complications, including PPI, in clinical studies (Klement et al., 2020; Lacko et al., 2020; Yazdi et al., 2020). However, the main mechanism of action through which the drug acts has not yet been established. It has been hypothesized that TXA could have a direct or indirect effect on the growth of microorganisms, and while we previously demonstrated the direct antibacterial effect of TXA (Benjumea et al., 2022a,b), some authors support an indirect effect by immunomodulation, which favors the host immune defense mechanisms against the pathogens that cause the infection. In addition, other studies support a direct hemostatic mechanism, which reduces the frequency of postoperative hematoma and thus the possibility of infectious complications (Draxler et al., 2019; Lacko et al., 2020; Sukeik et al., 2020; Yazdi et al., 2020).

In a previous study, we demonstrated the synergistic effect of TXA with vancomycin and gentamicin, which are the most widely used, locally applied antibiotics in orthopedic surgery (Benjumea et al., 2022a). However, in the present study, we decided to analyze the synergistic effect of 10 mg/ml TXA with these antibiotics against a large collection of clinical strains isolated from patients with musculoskeletal infection. We used a 10-mg/ml concentration for a number of reasons: first, because we had previously demonstrated that it had a direct antibacterial effect on planktonic cells and that its effect was not dose-dependent (Benjumea et al., 2022a; Wang et al., 2022); second, because it has been shown that ≥20 mg/ml can cause cellular toxicity (to chondral cells, epithelial cells, and fibroblasts), with the result that wound healing capacity could be compromised at higher doses (Parker et al., 2018; Eikebrokk et al., 2019; Bolam et al., 2021); and, third, because 10 mg/ml is the most widely used concentration in clinical studies (Sun et al., 2017; Montroy et al., 2018; Masaryk et al., 2022; Bi et al., 2023). Our analysis showed an overall reduction in turbidity of 10.9% for S. aureus and 2.2% for S. epidermidis strains when treated with TXA alone. However, in specific strains, this reduction ranged between 10% and 23%. Therefore, we found that the effect of TXA may be strain-dependent.

Furthermore, with the aim of assessing the synergistic effect in an in vivo model, where the environment could affect the previous in vitro results, we created a murine PPI model, which, to our knowledge, is the first to assess the synergistic effect of TXA with antibiotics. Only 2 previous studies have been based on in vivo models testing TXA, although neither of them used other compounds in combination (Zhang et al., 2021; Wang et al., 2022). We combined 10 mg/ml TXA and gentamicin-loaded bone cement (PMMA) in an S. aureus murine PPI model and demonstrated a synergistic effect (86.2% decrease in bacterial growth, compared with the group treated with TXA alone, as observed in the SEM images). In the group of mice in which only gentamicin-loaded PMMA was used, this reduction in bacterial growth reached 53.1%, corresponding to an approximately 35% reduced antibacterial effect. Although we only tested an ATCC S. aureus strain in the in vivo model, which already showed good results in the in vitro experiments, additional factors, such as the host's immune factors, also positively affected the synergistic effect. As hypothesized by Wang et al. (2022), TXA affects the expression of polysaccharides and proteins that favor the formation of bacterial aggregates, making biofilm formation difficult. This probably accounts for the effect of TXA that would make bacteria more susceptible to the action of both phagocytic cells and the antibiotic.

In addition, we found that the incidence of colonized brain samples was higher (66.7%) in the group treated with TXA only than in the group treated with TXA and gentamicin (20.0%), although no differences were observed with respect to the group treated with gentamicin only (20.0%).

Regarding the potential risks of TXA, including thromboembolic disease, it has not been demonstrated that the combination of TXA with locally applied antibiotics (joints) causes adverse effects. In orthopedic surgery, the use of antibiotic-loaded cement is a very widespread practice. Similarly, TXA is widely used topically and systemically, and no increase in the incidence of adverse effects has been described in any of its applications (Franchini et al., 2018; Montroy et al., 2018; Oliva-Moya et al., 2022).

One of the main limitations of our study is that our findings suggests that TXA effect is strain-dependent, so they cannot be applied universally across all staphylococcal strains encountered in clinical practice. In addition, as it was a prospective study, we included a small number of clinical strains. Based on this, future clinical studies assessing the use of TXA directly in the clinical scenario and including other antibiotics commonly used in the treatment of staphylococcal infections are required. There is also needed to better elucidate the molecular mechanisms underlying the observed synergistic effect of TXA and whether its addition to PMMA would affect the adhesion of bone cement.

In summary, our study is the first to analyze the synergistic effect of TXA and locally applied gentamicin on the growth of microorganisms in an in vivo model. We also demonstrated that the in vitro synergistic effect of TXA with gentamicin and vancomycin is strain-dependent. The main limitation of the study is that we only tested a single strain in the murine model.

Our results showed that the ability of TXA to reduce bacterial growth is based on a direct anti-aggregative effect on biofilm formation, thus reducing the extracellular matrix, which enables antimicrobials to better reach bacteria. This may explain the synergism between 10 mg/ml TXA and vancomycin/gentamicin. However, this effect is not always observed, as it is strain-dependent.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The requirement of ethical approval was waived by Gregorio Marañón Local Ethics Committee on research involving medicinal products for the studies involving humans because the strains used are from clinical isolates without using patient data. The studies were conducted in accordance with the local legislation and institutional requirements. The Ethics Committee/institutional review board also waived the requirement of written informed consent for participation from the participants or the participants' legal guardians/next of kin because the strains used are from clinical isolates without using patient data. The animal study was approved by Dirección General de Agricultura, Ganadería y Alimentación. Consejería de Medio Ambiente, Vivienda y Agricultura (PROEX no. 257.6/22). The study was conducted in accordance with the local legislation and institutional requirements.

AB: Conceptualization, Data curation, Investigation, Methodology, Writing—original draft. MD-N: Data curation, Formal analysis, Investigation, Methodology, Writing—original draft. ÁG-C: Investigation, Methodology, Writing—original draft. AV: Investigation, Methodology, Writing—original draft. RH: Investigation, Methodology, Writing—original draft. EC: Conceptualization, Supervision, Writing—review & editing. MS-S: Conceptualization, Investigation, Supervision, Writing—review & editing. PM: Supervision, Validation, Writing—review & editing. JV: Conceptualization, Supervision, Validation, Writing—review & editing. FC: Conceptualization, Supervision, Validation, Writing—review & editing. MG: Funding acquisition, Project administration, Resources, Supervision, Validation, Writing—review & editing.