## 7 Drawbacks and Challenges for Susceptibility Testing of Borreliae In Vitro
Consequently, for most antimicrobial agents, a possible loss of activity during incubation is mostly attributable to their poor chemical stability or to side-effects resulting from the enzymatic activity of BSK components [55; 84]. The opposite is true; however, for chloramphenicol as the serum esterase present in BSK medium can reconvert inactive diacetyl-chloramphenicol to the active form of the antibiotic. Thus, the in vitro findings measured in BSK medium do not accurately reflect the level of chloramphenicol activity against borreliae in vivo [84]. Remarkably, on a \(\upmu\)g/mL basis, macrolides are also more efficacious against borreliae than tetracyclines when tested in BSK medium [51; 55]. In contrast, the clinical administration of macrolides, such as erythromycincin and roavithromycin, is frequently unsuccessful in treating LB or is followed by a clinical relapse after the conclusion of the treatment [7; 15; 16; 61]. Similarly, high rates of treatment failure have been reported in patients with primary and secondary syphilis that have been treated with erythromycincin [61; 112]. The reasons for this known variability in the clinical efficacy of macrolides against spirochetes remain, in part, controversial and are a focus of ongoing scientific research. This is why it is so important to perform control experiments. For example, while testing macrolides and ketolides against borreliae, control experiments with a reference strain of a fastidious organism (e.g., _S. pneumoniae_ ATCC 49619) revealed possible interactions of the BSK medium with these antimicrobials. It became obvious that the activity of most of the macrolide derivatives tested increased in BSK medium, as revealed by MIC values below the range published by the Clinical Laboratory Standards Institute (CLSI) for these agents against _S. pneumoniae_[59]. Possibly,the lower protein binding of macrolides in BSK-containing rabbit serum may contribute to the increased activity of the macrolides against borreliae in vitro [61]. Some authors, therefore, speculate that the known high variability of species-dependent protein binding of roxithromycin and erythromycincin (ranging from 7% in rabbits to 86% in humans) may be the reason for the excellent in vitro activity of both drugs against borreliae. This contrasts sharply with the, sometimes, poor clinical performance in cases of human LB [7, 61]. This is important as far as the \"true MIC values\" of the macrolide agents in vivo most likely need to be estimated as being at least one or two log\({}_{2}\) unit dilutions higher than those measured in BSK medium [52]. Furthermore, the analysis of genes that encode ribosomal proteins in eight eubacterial species, including _B. burgdorferi_ s.l., demonstrated that, although several 16s-RNA encoding genes considered to be of structural importance are conserved throughout the bacterial species, the degrees of sequence conservation can differ from one ribosomal protein gene to another [113]. Therefore, impact of this variation on aspects of pharmacokinetic variability and drug interactions must be taken into account. Typical factors that come into play are protein binding, plasma half-life, bioavailability, variable concentrations in various body compartments, and differences in the binding affinities of the traditional macrolides to the corresponding targets of the ribosome. These factors, alongside others, may be responsible for the striking difference between excellent in vitro activity and poor in vivo efficacy of these substances against _Borrelia_ and _Treponema_[52]. Further experiments involving serum-depleted BSK medium or BSK medium that have been supplemented with human serum instead of rabbit serum are needed in order to gain insight into interactions of the test medium with the antimicrobial agents in general, and the quinolones, macrolides and ketolides in particular. Clearly, more studies are needed to resolve the currently known discrepancies between the in vitro and in vivo activity of antimicrobial agents against the Lyme disease spirochete [52].