In vitro antibacterial activity of bioactive glass S53P4 on multiresistant pathogens causing osteomyelitis and prosthetic joint infection

Osteomyelitis (OM) is a heterogeneous disease with several pathophysiological mechanisms, making diagnosis and therapy difficult. Infection can occur through the haematogenous route, direct exposure of the tissue to infective agents or from areas of contiguous infection [1‐3]. The microorganisms most commonly involved are the Gram-positive bacteria Staphylococcus aureus, coagulase-negative Staphylococci, Streptococcus sp.; followed by Gram-negative bacteria such as Escherichia coli, Pseudomonas aeruginosa and Acinetobacter spp.. An increasing number of cases of OM caused by multiresistant microorganisms, both in hospital and community-acquired infections, have recently been reported [4, 5]^,. This microbiological profile is most characteristic in OM following surgeries for the treatment of fractures and articular degenerative diseases (osteosynthesis and arthroplasties), or secondary to pressure ulcers. In contiguous OM associated with complex exposed fractures, the presence of polymicrobial infection is also common [6]. The expression of antimicrobial resistance by these agents, coupled with biofilm formation, reduces cure rates and increases disease morbidity, presenting a challenge to the current treatment of OM.

To maintain bone tissue free of microorganisms after debridement, a combination of systemic and local antimicrobial therapy - by filling the bone cavity with antibiotic carriers such as polymethyl-methacrylate (PMMA) - is currently used. Although it is the only material currently approved by the Food and Drug Administration (FDA) in the treatment of osteomyelitis, antibiotic-loaded PMMA has some limitations. Because it is not biodegradable, its use requires several surgical procedures, which may result in loss of bone mass [7]. In addition, the surface of the polymer can act as a substrate for the formation of biofilms, leading to a reduction in the effectiveness of the antimicrobials [8]. Previous studies have also shown that the use of PMMA as a vehicle for local antibiotic therapy can produce inconsistent release of the drug, with a consequent reduction in the efficacy of therapy and induction of bacterial resistance [9, 10]^,. Thus, it is becoming increasingly important to develop alternative treatment strategies and materials for those infections that are difficult to control.

Bioactive Glass (BAG) S53P4 is one of the latest materials to be studied, with its properties presenting solutions to some of the weaknesses of the treatment currently advocated with PMMA or cement. It consists of several ionic compounds (SiO₂, Na₂O, CaO, P₂O₅), and when implanted in the cavity after debridement, releases alkali ions, causing an abrupt increase in pH and, in the process, becoming hydroxyapatite [11, 12]^,. This allows both inhibition of bacterial growth through the osmotic and acid-base imbalance generated – not being dependent on antibiotics to occur [13, 14]^, −, and the formation of new bone matrix, not requiring further implant removal procedures. It is important to notice that, although BAG remains present considerable periods of time after implantation, theoretically being able to act as foreign material, there are no reported foreign body reactions or infections involving S53P4 to date [15]. We aimed to analyze the in vitro antibacterial activity of BAG compared to antibiotic-loaded PMMA in multidrug-resistant bacteria strains producing osteomyelitis and prosthetic joint infections.

The success of the BAG S53P4 intervention in inhibiting bacterial multiplication in this study is in line with the current trend in the literature, which supports the antimicrobial effect of this biomaterial [13‐15, 17‐26]. In addition, although the literature on the subject is still scarce, our results reinforce the hypothesis of some authors [20] who have concluded that the antibacterial activity of antibiotic-loaded PMMA and BAG S53P4 were equivalent, making this material a viable local alternative treatment for medullary osteomyelitis, with no extra need of antibiotic-based therapy. Moreover, in the clinical setting, BAG has consistently proven to be an effective treatment to patients with OM. In a multicentric study, Lindfors et al. [19] analyzed the use of BAG in the treatment of 116 patients with OM and obtained a 90% success rate, very similar to the 88.9% success rate found by Drago et al. [22] and 91.7% found by Fernando et al. (2017). Furthermore, in the same study, Lindfors et al. found that the use of BAG as a one-stage procedure without local antibiotic therapy as an efficient treatment option when compared to a two-stage treatment using antibiotic-loaded PMMA beads prior to the implantation of BAG.

Although our results have shown efficient microbial inhibitory activity against an inoculum of 1,5 × 10⁵ CFU/mL, comparable to patients with OM that are debrided (models range from 10⁴ to 10⁶ CFU/mL) [27‐29], bacterial inhibition did not occur with the same speed as in most studies previously published [13, 14, 17, 20, 25, 26]. In our experiment, PMMA and BAG granules started reducing the bacterial growth only after 24 h, while killing a large number of bacteria took at least 48–72 h after inoculation. In contrast, different studies reported a significant earlier drop (24 to 48 h) in the number of colony forming units when compared the activity of BAG S53P4 with PMMA or calcium sulphate antibiotic beads [17, 18, 20]. This may be explained by the use of test tubes containing the BAG mixtures in this experiment, which reduces the contact surface area between the agent and the culture medium, a factor that may have influenced the release of alkaline ions into the mixture, retarding its effect on bacterial inhibition [30]. In addition to the containers used to hold the mixtures, few other adaptations were necessary to carry out this study. Interestingly, in the present study the bacterial activity of BAG and PMMA demonstrated slightly differences, with a slower bacterial inhibitory activity against Gram-negative bacteria. This phenomenon may be attributed to Gram-negative organisms’ several genetic mechanisms of adaptation to changes in environmental osmolarity and pH [31‐33], Furthermore, the pH threshold value of 10 used to indicate BAG suitability is fairly close to P. aeruginosa natural habitats’ pH values, that range from 4,5 to 9,5. Nonetheless, after 7 days, both BAG and PMMA were able to eliminate all bacteria.

It is also important to point that, since the colony counts were manually performed, we were unable to determine bacterial CFU values above 3.8 × 10⁶ CFU. This matter is especially relevant to the interpretation of control groups’ time-kill curves in Figs. 1a, b, c and 2, because since the first count (seeded immediately after bacterial inoculation), the plates were almost fully covered in colonies, usually more than 100/cm². In this kind of colony distribution pattern, the maximum CFU count possible is defined by the expression [Petri dish area]/[inoculum concentration]. In our case, 58cm²/(1.5 x 10⁵CFU/mL), resulting in about 3.8 × 10⁶ CFU. As a consequence, in Figs. 1, 2, 3 and 4, control groups’ time-kill curve shows a fairly constant bacterial count instead of gradually increasing values. This work, despite its limitations represents one of the first analyses of the antimicrobial activity of BAG S53P4 against clinical species that cause osteomyelitis and prosthetic joint infection from a middle-income country, as well as being the second largest in vitro study in the literature, in which 18 MDR Gram-positive and Gram-negative bacteria were studied. In addition to this, this is the largest in vitro experimental comparison between BAG and the conventional cement-based treatment (PMMA associated with vancomycin or gentamicin) and is of particular importance given its focus on the effect of the intervention on multidrug-resistant species, which have played an increasing role in the etiopathogenesis of osteomyelitis, as can be seen in Table 1.

In conclusion, the results of this study are in line with the trend in the current literature which points to BAG S53P4 being an effective alternative to the current treatment of choice for medullary osteomyelitis. Further prospective studies and clinical trials should be carried out on patients.