## 8 Possible Mechanisms of Resistance of _Borrelia burgdorferi_ s.l. In Vitro
### Changes in Protein Expression in Borreliae Exposed to Antimicrobial Agents  
Proteomics, a snapshot-like analysis of a certain protein composition that is regarded as representative of the functional status of a defined biological compartment, is a growing field of research that encompasses environmental factors [52; 129]. Here, proteins, the acting macromolecules in bacterial cells, can serve as crucial targets to better understand infectious diseases [130]. Currently, two-dimensional electrophoresis (2-DE) is the only method available for separating proteins from complex mixtures, such as cells or tissues. The approach with the highest resolution separates up to 10,000 protein species [131]. Therefore, it represents a very promising approach for providing additional insights into the physiological processes of bacterial cell metabolism. Additionally, morphological changes in _B. burgdorferi_ is.l. in response to adverse environmental conditions have been reported to occur when cells are exposed to physiological stress. Typical stress factors known to induce such alterations are changes in pH, depletion of energy metabolism, serum starvation while growing in BSK medium, the presence of antimicrobial agents, and ageing [18; 132; 133; 134; 135]. These data suggest that borreliae can respond rapidly to altered environmental conditions through variable protein expression [135]. Exposure of the pathogens to penicillin and doxycycline under defined test conditions revealed that fifteen protein spots in the samples treated with penicillin G were down-regulated (Figure 5; Table 3). Similarly, four protein spots were identified on the gels of the doxycycline-treated samples that were downregulated, as revealed by a \(\geq 50\%\) decrease in spot intensity in comparison to untreated controls [17; 52]. In three spots, borreliae exposed to both penicillin and doxycycline at the MIC and at \(\frac{1}{2}\) the MIC led to a downregulation of protein synthesis. After 72 h, one additional protein could be identified that was strongly over-expressed after cultivation of borreliae in the presence of doxycycline at the MIC. It is interesting to note that, in addition to other proteins, the p66 protein, a pore-forming protein of _B. burgdorferi_ is.l., is downregulated in the presence of penicillin G [17; 52]. Porins are known to be vital for the influx of antimicrobial agents into the cytoplasm of Gram-negative bacteria [136]. In addition, the absence of porins is associated with resistance to \(\delta\)-lactams, which occurs in porin-deficient mutants of Gram-negative bacteria with increased resistance to antimicrobial agents [137]. Accordingly, one is inclined to speculate that the downregulation of porins by borreliae in the presence of \(\delta\)-lactams may be an adaptive process to hinder the influx of these drugs through the outer membrane into the periplasmic space, thereby lowering their inhibitory activity in peptidoglycan synthesis [17; 52]. Interestingly, a significant upregulation of the triosphosphate isomerase, a key enzyme of glycolytic carbohydrate metabolism, was observed in the presence of doxycycline. This enzyme is also known to be variably expressed in the amastigote stage during the complex development cycle of _Leishman_ spp. [138]. In borreliae, its upregulation in the presence of a bacteriostatic antibiotic agent may, again, be interpreted as a counteracting initiation of increased metabolic activity due to an adaptive or evasive mechanism on the part of the pathogen being exposed to the drug [17; 52]. Similarly, borreliae are known to react to serum starvation by a complex protein expression response involving more than 20 proteins [135]. Equally, _E. coli_ reacts to fatty acid starvation of the culture medium by accumulating spot-dependent guanosine tetraphosphate (ppGpp) and inhibiting rRNA synthesis [139]. These findings suggest that, although _B. burgdorferi_ is.l.