The stability of Staphylococcal bacteriophages with commonly used prosthetic joint infection lavage solutions

Abstract

The aim of this study was to assess the viability of four Staphylococcal bacteriophages when exposed to different concentrations of commonly used lavage solutions in the surgical treatment of prosthetic joint infections (PJI). Four tailed Staphylococcal bacteriophages and six different lavage solutions (chlorhexidine 4%, hydrogen peroxide 3%, acetic acid 3%, povidone iodine 10%, sodium hypochlorite 0.5%, and Vashe solution) at 100%, 1%, and 0.01% concentrations were used in this experiment. In addition, the temporal impact of exposing bacteriophages to these lavage solutions was also evaluated at 5-minute exposures and 24-hour exposures. The results show that the titers of the four bacteriophages were statistically significantly decreased for all lavage solutions (100% and 1%) at 5-minute exposures and 24-hour exposures. However, with 0.01% concentrations of the lavage solutions, only acetic acid caused a statistically significant decrease in bacteriophage titers compared to normal saline control. Our findings suggest that tailed Staphylococcal bacteriophages do not remain stable in high concentrations of the most commonly used lavage solutions. However, at very dilute concentrations the bacteriophages do remain viable. This has important clinical ramifications in that it shows when using bacteriophage therapy for PJI it is critical to thoroughly wash out any lavage solutions prior to the introduction of therapeutic bacteriophages especially when acetic acid is used.

Introduction:

Bacteriophage therapy is a promising adjuvant therapy in the treatment of chronic prosthetic joint infections (PJI) because bacteriophages possess innate abilities to eradicate biofilms with the use of depolymerases and endonucleases [1]. At this nascent stage of development, the use of bacteriophage therapy has been shown to be most beneficial when used as an adjuvant with surgical interventions [2]. Consequently, bacteriophage therapy has the potential to treat chronic PJI with either debridement and implant retention surgery (DAIR) or revision surgery to reduce the morbidity and mortality associated with these infections [3].

However, there is a paucity of research proving the effectiveness of phage therapy beyond case studies, and only a few translational studies which have shown that there are readily available bacteriophage therapeutics for Staphylococcal aureus PJI clinical isolates [4–7]. Many aspects of bacteriophage therapy are poorly known, making it challenging for researchers to devise protocols that are effective and reproducible. This is in part because bacteriophages are live viruses and thus are affected by chemicals and pH of environments [8,9]. Therefore, since bacteriophages are mainly used with PJI surgical interventions, it is imperative to know the compatibility of bacteriophages with commonly used lavage solutions that are administrated during the surgical treatments of PJI. This is especially important because many of the lavage solutions commonly used are virucidal [10]. Unfortunately, there is a dearth of translational studies evaluating this and as a result, the aim of this study was to evaluate the compatibility of four Staphylococcal bacteriophages with commonly used lavage solutions to ensure these can be administered concomitantly.

Materials and methods:

Bacteriophages, host bacteria and lavage solutions

The bacteriophages used in this experiment were all tailed bacteriophages. Two myoviridae phages were used: phage Remus (Université Laval) and Phage K (ATCC). As well, two podoviridae were used: Phage P68 (Université Laval) and Phage 44AHJD (Université Laval). The Staphylococcus aureus bacterial strains that were used were the host strains recommended by Université Laval and ATCC. Lavage solutions used in this experiment were Chlorhexidine 4%, hydrogen peroxide 3%, acetic acid 3%, Povidone iodine 10%, Sodium hypochlorite 0.5%, Vashe solution (Hypochlorous acid). Various concentrations (100%, 1% and 0.01%) of the different lavage solutions were made by diluting these lavage solutions in normal saline to achieve those concentrations.

Impact of different lavage solution concentrations on bacteriophage titers

Individual bacteriophages were grown on their host strains by introducing 100 μL of stock bacteriophage solution with 100 μL of host bacteria, that had grown overnight at 37° C, in 10 mL of Luria Bertani broth. These were then incubated at 37 °C for 5 hours and then centrifuged Sorvall ST 40R centrifuge (Thermo scientific). The supernatant was then passed through sterile 0.22 μm filters. Initial titers, as seen with plaque forming units (PFU)/mL of the four bacteriophages were obtained by taking the fluid passed through filters and conducting double agar overlay method with serial dilutions of the fluid. This was conducted in triplicates and repeated to ensure reproducibility of results.

Once titers of the four bacteriophages were known, 100 μL of the different bacteriophages were added to the different lavage solutions and concentrations (100%, 1%, 0.01%). The bacteriophages were left in the different lavage solutions for 5 minutes and 24 hours. The titers of the four bacteriophages, after exposure to the lavage solutions, were then determined as discussed above with double agar overlay method. The titers of the four bacteriophages in normal saline at 5 minutes and 24 hours was used as a control. Differences in titers of bacteriophages in the different lavage solutions were compared to the normal saline control. Statistical comparisons were conducted by comparing mean titers of triplicate samples that were repeated twice with the use of https://medcalc.org. A p value of less than 0.05 was considered statistically significant.

Results:

All four bacteriophages had titers in normal saline that were stable over 24 hours. When all four bacteriophages were placed in lavage solutions that had not been diluted, all lavage solutions except for 3% hydrogen peroxide and 4% chlorohexidine did not allow for any PFU to be detected after 5 minutes or 24 hours. After 5 minutes exposure to 4% chlorohexidine and 3% hydrogen peroxide, all four phages had detectable PFU, albeit at statistically significantly reduced titers (p=0.001). When the four bacteriophages were left in these two solutions for 24 hours, no PFU/mL could be detected for any of the bacteriophages.

When the lavage solutions were diluted to 1% of their initial concentrations all four bacteriophages had no detectable PFU after 24 hours of being exposed to these solutions (Figure 2). However, after 5 minutes of exposure there was detectable PFU with chlorohexidine (0.04%), Vashe (1%) and hydrogen peroxide (0.03%) solutions, but these titers were statistically significantly (P 0.05) reduced when compared to PFU in normal saline for the four phages.

Figure 2:

Plaque forming units (PFU) with different lavage solutions at 100-fold dilution. All lavage solutions caused statistically significant (P 0.05) reduced PFU at 5 minutes and 24 hours.

After the lavage solutions were diluted 1000-fold, there were detectable PFU of all four bacteriophages for all lavage solutions at 5 minutes and 24 hours (Figure 3). There was not a statistical difference in the detectable PFU except for all four phages with acetic acid (0.0003%) at 5 minutes and 24 hours when compared to normal saline control.

Figure 3:

Plaque forming units (PFU) with different lavage solutions at 10,000-fold dilution. Only acetic acid caused a statistically significant (P 0.05) difference in PFU compared to normal saline control.

Discussion:

Lavage solutions are habitually used in prosthetic joint infection surgical interventions to reduce bacterial burdens, thus aiding in eradication of infectious processes [10]. Most of these agents have been used in small studies with documented benefits and limited toxicities, but no randomized clinical trial has been conducted comparing the effectiveness of one over another [11–16]. Moreover, the agents used in this study all have bactericidal and fungicidal properties, and some also have potent known virucidal properties, including povidone iodine, hypochlorous acid, chlorhexidine and sodium hypochlorite [10]. Consequently, given the theoretical benefit of using bacteriophage therapy with surgical interventions, it was paramount to conduct this experiment to assess the stability bacteriophages in the presence of commonly used lavage solutions.

With respect to bacteriophage therapy therapeutics, not all bacteriophages are the same. Tailed bacteriophages are considered the most advantageous therapeutics and tend to be the most stable under extreme conditions [8]. There are 3 classes of tailed phages: myoviridae, podoviridae and sphinovirodae; however, only myoviridae and podoviridae have significant promise in clinical medicine because sphinoviridae often are associated with integrase genes, thus making them prone to be temperate phages. This makes sphinoviridae difficult to use clinically because of the risk for transduction of deleterious genes to bacteria, thus potentially making infections more virulent [17,18]. As a result, here we assessed the use of two myoviridiae and two podoviridae bacteriophages and did not evaluate sphinoviridae phages.

The results show that at high concentrations of commonly used lavage solutions, no detectable concentrations of viable bacteriophages could be detected at 24 hours (Figure 1). Importantly, at five minutes only chlorohexidine (4%) and hydrogen peroxide (3%) had viable bacteriophages that could be detected, albeit at drastically less levels then when compared with the normal saline control. This has important clinical implications in that it shows that phage therapy should not be administered concomitantly with lavage solutions because the level of bacteriophages will be drastically reduced, thereby hindering the potential effectiveness of these therapeutics. As well, this suggests that bacteriophages should not be administered before lavage solutions are infused for the same reasons.

Figure 1:

Plaque forming units (PFU) with different lavage solutions. All lavage solutions caused statistically significant (P 0.05) reduced PFU at 5 minutes and 24 hours.

While the above findings are interesting, typically bacteriophage therapy is administered after lavage solutions have been instilled and washed with normal saline. Consequently, we evaluated the titers of bacteriophages with diluted concentrations of lavage solutions (Figure 2 & 3). It is impossible to know the residual concentrations of lavage solutions left in vivo after normal saline washing, and thus we used 100- and 10,000-fold dilutions to assess reductions in PFU/mL of the four bacteriophages. When the lavage solutions were diluted 100-fold, all lavage solutions still completely prevented detection of viable bacteriophages at 24 hours, and only chlorohexidine (0.04%), hydrogen peroxide (0.03%) and Vashe (1%) solutions had detectable titers at 5 minutes. Yet when the lavage solutions were diluted 10,000-fold all solutions had detectable PFU and only acetic acid (0.0003%) had statistically significant reductions in PFU for all four bacteriophages assessed (Figure 3).

These findings suggest that when using bacteriophage therapy in PJI it is vital to thoroughly wash the joint with normal saline after instilling lavage solutions in order to preserve the concentrations of bacteriophages that would then be added. Furthermore, the use of chlorohexidine, hydrogen peroxide or Vashe solutions offers the most potential leeway in not completely reducing bacteriophage concentrations if significant concentrations of lavage solutions are left in vivo. On the other hand, the use of acetic acid should perhaps not be used with bacteriophage therapy given the potential to reduce bacteriophage concentrations even when significantly diluted.

There are some limitations of our study. For one, we only evaluated four bacteriophages. It would be impossible to evaluate all Staphylococcal bacteriophages given there are an enormous reserve of potential Staphylococcal phages; but the use of four showed reproducibility of results, thus allowing for extrapolation of these findings to other potential phages in the same classes. However, before clinical trials are to be devised with specific Staphylococcal PJI bacteriophage therapeutics, it will be vital to evaluate those other bacteriophages with lavage solutions to then create specific clinical trial protocols. Lastly, we only evaluated Staphylococcus aureus bacteriophages given this is the most virulent and common PJI pathogen [19]. Yet other common pathogens such as coagulase negative Staphylococcus will also need to have similar studies conducted with their respective bacteriophages.

In conclusion, bacteriophages hold promise in aiding in the treatment of PJI, especially when used with surgical interventions. However, bacteriophages are not compatible with all solutions in that titers of bacteriophages can be significantly reduced in presence of certain solutions. Here we show that commonly used PJI lavage solutions drastically reduced the concentrations of four Staphylococcal bacteriophages even when diluted 100-fold but not when diluted 10,000-fold. This reinforces that bacteriophage therapeutics should be administered after ample washing of joints with normal saline and potentially only with certain lavage solutions. However, given the paucity of preclinical bacteriophage research, more studies like this one are needed to clarify many aspects of using bacteriophage therapeutics with commonly used PJI practices to thereby create effective and reproducible bacteriophage therapeutics.