## 7 Drawbacks and Challenges for Susceptibility Testing of Borreliae In Vitro  
Taking into account the aforementioned technical limitations and drawbacks, as well as the lack of internationally accepted standardized methodology [17; 36; 40], current in vitro susceptibility data on MIC and MBC of antimicrobial agents for borreliae are far from consistent [17]. To date, no less than 14 MIC definitions and 11 MBC definitions (Table 1) have been proposed in the scientific literature with regard to susceptibility testing of borreliae [17; 36; 37; 39; 40; 43; 49; 64; 67; 68]. In addition, testing of _B. burgdorferi_ s.l. is significantly influenced by considerable differences in testing conditions, i.e., incubation periods (48 h-10 days), the variable density of the inoculum (\(10^{4}\) to \(\sim 10^{7}\)/mL), the composition of the test medium [17; 36; 84; 107; 108], obvious interactions between the antimicrobial substance and the test medium, the reading mode, the criteria to correctly determineantibiotic-induced killing, and growth inhibition in vitro [36; 40; 64; 107]. Such inconsistencies account for the wide variability of published MIC and MBC values. For example, when reviewing studies in the literature, the reported MIC of penicillin G varies from 0.003 to 8 \(\upmu\)g/mL and the MBC varies from 0.05 to \(>\)50 \(\upmu\)g/mL [17; 38; 51; 109]. Similarly, for doxycycline, the MIC varies from 0.06 to 2 \(\upmu\)g/mL and the MBC varies from 0.25 to 6.4 \(\upmu\)g/mL [17; 39; 51; 64; 109]. The results obtained by these studies are also limited as only small numbers of isolates (two to 30 strains) were examined. In contrast, investigations into common rapid growers typically involve large numbers of clinical isolates [17; 44; 110]. This is additionally a constraint as susceptibility testing is mostly conducted on easily accessible, high-passage isolates from stock cultures known from the literature and is rarely performed on newly isolated low-passage strains, or on isolates directly obtained from patients showing resistance to treatment [51; 53; 81]. _Discrepancies in In Vitro and In Vivo Activities: Interactions of Antimicrobials with BSK Medium_  
The in vitro efficacy of many traditional antimicrobial agents against borreliae has not always correlated with clinical experience [17; 18; 19; 20; 21; 42; 61]. This observation is partly explained by experiments that point to possible interactions between antimicrobial agents and BSK medium, indicating that the chemical instability of some substances, and the side-effects resulting from enzymatic activity of BSK components, are capable of influencing the in vitro testing of borreliae during the lengthy incubation periods (several days to weeks) [17; 36; 40; 52; 84; 108]. Some \(\upbeta\)-lactams, such as penicillin G and amoxicillin, display only moderate to good in vitro activity against _B. burgdorferi_ s.l. but are clinically very efficacious in most patients. This can be explained, on the one hand, by the fact that the activity of some \(\upbeta\)-lactams, both in vitro and in vivo, is temperature-dependent. For example, the in vitro activity of penicillin against borreliae increases up to 16-fold after temperatures are raised from 36 \({}^{\circ}\)C to 38 \({}^{\circ}\)C [110]. On the other hand, for many \(\upbeta\)-lactams, a possible loss of activity during incubation is due, in part, to their poor chemical stability [36; 51; 107; 108]. For instance, a significant decrease in antimicrobial efficacy, ranging from 66.3% for mezlocillin to 85.9% for penicillin, was found in _Bacillus subtilis_ bioassays after 72 h of incubation in BSK medium [36; 40]. Similarly, the activity of trimethoprim is diminished in the presence of BSK medium because this medium contains thymidine and p-aminobenzoic acid. When the medium was modified by reducing the components causing trimethoprim inhibition, the borreliae were shown to be somewhat susceptible to this drugs in vitro [111].