The ability of MRSP to form biofilm may be an important virulence factor and while it is closely related to MRSA, it appears that there are important cross-species differences. While biofilm production was common amongst this collection of MRSP isolates from different major clones and geographic regions, there was no evidence that CLA inhibits biofilm formation, in contrast to previous reports on MRSA
[13, 22, 23]. Accordingly, results do not support the use of CLA for prevention of biofilm formation, and since there was no significant impact on biofilm formation regardless of classification, it is unlikely that CLA would have any impact on biofilm eradication.
CLA resistant strains of MRSP were chosen for this study to ensure that any impact of CLA was from its anti-biofilm effect, not simply from inhibition of bacterial growth. The Clinical and Laboratory Standards Institute interpretive breakpoint for clarithromycin resistance (≥ 8 μg/ml)
 was chosen to represent a breakpoint concentration that is readily achieved through in vitro studies. One cannot exclude the possibility that an impact might have been present with higher concentrations of CLA, but the clinical relevance would be questionable.
The mechanism of biofilm prevention by macrolides seen in MRSA and other bacteria is not completely understood but current studies speculate that they also act through modification of the immune system’s inflammatory response to infection, and/or through a direct effect on bacterial virulence
[24, 26, 31]. For this reason it cannot be excluded that CLA may be effective in vivo, however in vitro studies involving MRSA still support a preventative effect on biofilm formation
. It has also previously been shown that macrolide antibiotics affect quorum sensing—the initial mechanism behind bacterial biofilm formation and cell-cell communication—within the biofilm leading to reduced polysaccharide synthesis and instability of biofilm architecture
[20, 25, 32].
It is possible that CLA does not impose a preventative biofilm forming mechanism on MRSP, as seen in MRSA, due to genetic variances not yet revealed between the two species. Currently, ica is considered to be the major operon responsible for staphylococcal biofilm formation
 but its study in MRSP strains has not been performed. Alternative pathways for quorum sensing could also cause the mitigation of the previously demonstrated effect of macrolides. Dru typing results suggest a varying geographic distribution and representative chosen isolate population across the two current internationally predominant MRSP strains, ST68 and ST71 (Table
. From this we can infer that genetic differences and therefore the effect of CLA on different strains of MRSP are likely minimal.
Time assessed biofilm development, while only one isolate was studied, suggests that biofilm formation occurs rapidly in vitro, since adherent bacteria and exopolysaccaride matrix were evident within 4 h by both the crystal violet MPA and through qualitative SEM evaluation of growth on surgical screws. Objective assessment of the impact of CLA on biofilm formation on screws was not possible since only one isolate was studied in a qualitative manner, yet these subjective data are in support of the crystal violet MPA and provide further evidence of a lack of efficacy of CLA for prevention of biofilm formation. The irregular biofilm patterns on screws, most notably the circular biofilm accumulations on screw heads, is consistent with preferential biofilm adhesion to invisible surface defects or irregularities in the machining process. This suggests that minor surface alterations, either from inherent defects or damage to implants during placement, could facilitate biofilm attachment in vivo.
In vitro evaluations of CLA on MRSP biofilm formation were performed on polystyrene and one orthopedically relevant biomaterial providing potential limitations to this study. Although no significant inhibitory effect of CLA on these materials was found, material properties of biofilm attachment sites could play a minor role in the susceptibility to and persistence of staphylococci infections
. Combinational therapy with CLA and varying antimicrobials has also been shown to have appreciable effects against MRSA biofilm formation
[13, 22, 23]. Because of the potential benefit to biofilm formation prevention and the safety of macrolides shown in long-term randomized macrolide therapy, further study and use of CLA in combinational therapy and on varying biomaterials is recommended
Large variances in both the amount of bacteria and antimicrobial in suspension in the 200 μl sample before addition to the microtiter plate could add to uncertainty in crystal violet microtiter plate assay results as previously described
. Optical determination of 0.5 McFarland and quick doubling-time of S. pseudintermedius could also contribute to the large standard deviations found for each averaged isolate OD570 reading. Microtiter plate washing techniques, as described in Stepanovic et al., also leave room for interpretation and could lead to the removal of transient bacterial biofilms, further adding to the variance in quantitative results for each isolate. Although the two currently most prevalent MRSP strains internationally were represented (ST68 and ST71) other biofilm forming strains of MRSP susceptible to the biofilm prevention mechanism in CLA might exist. Finally, the SEM study only accounts for one of 20 screened isolates across 11 biological replicates. Though the analysis was only for comparison the images are not representative of strains seen in the MPA.