## 8 Possible Mechanisms of Resistance of _Borrelia burgdorferi_ s.l. In Vitro
### Evidence from Interactions between Borreliae and Antimicrobial Agents and Eukaryotic Cells  
_Borrelia burgdorferi_ is.l. can be recovered long after the initial infection, even from patients treated with antibiotics. This indicates that it can resist eradication by host defense mechanisms and antibiotics [52]. One study found that borreliae are protected from the lethal action of a 2-day exposure to 1 \(\mathrm{\SIUnitSymbolMicro g}\)/mL of ceftaxone (10-20 times the measured MBC) when in the presence of human foreskin fibroblasts [140]. In the absence of fibroblasts, the study found that the spirochetes did not survive and, similarly, borreliae were not protected from ceftaxone by glutaraldehyde-fixed fibroblasts or fibroblast lysate. These findings suggest that living cells are necessary for protection. The ability of the organism to survive in the presence of fibroblasts was not related to its infectivity. Fibroblasts can protect borreliae exposed to ceftaxone for at least 14 days. Mouse keratinocytes, HEp-2 cells, and Vero cells showed the same protective effect, however, Caco-2 cells did not [140]. This is consistent with the findings of [PERSON] et al. and demonstrates that eucaryotic cells can protect _B. burgdorferi_ is.l. from the action of penicillin and ceftaxone, but not from the action of doxycycline and erythromycincin [141]. The intracellular location of spirochetesobserved in this study suggests that antibiotics that penetrate cells might have greater efficiency under such conditions. Doxycycline and erythromycin were more efficacious than penicillin or ceftaxone in killing borreliae in vitro when the organisms were grown in the presence of eukaryotic cells [141]. Thus, several eukaryotic cell types seem to provide the borreliae with a protective environment and may contribute to its long-term survival, at least in vitro [140].  
Figure 5: Representative two-dimensional gel analysis of _B. burgdorferi_ s.s. isolate LW2 (adapted from [129]). Twenty micrograms of protein derived from a culture in the log-phase of growth were focused on a pH-gradient ranging between pH 3 and 10. After isoelectric focusing (IEF), the sample was separated through a 10% Tris/tricine SDS-PAGE and subsequently silver stained. Location and molecular mass of differentially expressed proteins in samples of borreliae exposed to penicillin G and doxycycline at concentrations around the MIC are indicated by straight lines. Such protein spots were then cored from preparative gels and further analyzed by MALDI TOF MS (see Table 3). The positions of molecular mass standard in kDa are indicated on the right. The linear pH gradient ranging from pH 3 to 10 is indicated at the bottom of the photograph.  
Bacterial persistence in the presence of antimicrobials, with no apparent increase in the MIC of the organism, appears to be a possible mechanism of antimicrobial resistance in borreliae [7; 127]. _B. burgdorferi_ s.l. can form both round body cells and biofilm-like structures in vitro, which appear more resistant to antimicrobial agents [142; 143]. Consequently, the formation of drug-tolerant persister cells, which has been demonstrated in vitro, may be another explanation for the persistence of borreliae and a lack of responsiveness to antimicrobial activity in vitro [72]. These findings offer a possible explanation for why, under certain circumstances, individual LB patients suffer from relapses, or why borreliae can be re-cultured from some patients after having undergone appropriate administration of antibiotic treatment [7; 60]. These observations point to the necessity to explore and develop assays capable of creating experimental in vitro conditions, which mimic more closely the in vivo situation of patients treated for LB.