## 8 Possible Mechanisms of Resistance of _Borrelia burgdorferi_ s.l. In Vitro
### Tolerance of Borrelia burgdorferi s.l. When Exposed to Antimicrobial Agents  
Tolerance to metabolic, chemical and physical challenges, including antimicrobials, is a crucial--if not obligatory--phenotype of _B. burgdorferi_ s.l. It is necessary in order to complete its enzootic cycle in mammalian and possibly avian reservoirs and in ticks. Several pathways and genes have been identified that could be involved in the generation of antimicrobial tolerance in _B. burgdorferi_ s.l. These include the stringent response mediated by Rel and DksA, synthesis of the quorum sensing factor AI-2 mediated by LuxS, and the modulation of the levels of ATP and protein aggregation indirectly mediated by the GTPase CgtA (ObgE) [120]. Another factor that could produce antimicrobial tolerance in _B. burgdorferi_ s.l. is a decrease in growth rate triggered by a scarcity of nutrients [120]. _Borrelia burgdorferi_ s.l. can form antibiotic-tolerant persisters in the presence of microbiota drugs such as doxycycline [121]. It is currently unknown how this occurs. Some authors speculate that the multiple regulatory pathways involved in mediating this tolerance to antimicrobials and environmental stressors by persistence might include genes of the stringent (_rel_ and _dksA_) and host adaptation (_rpoS_) responses, sugar metabolism (_glpD_), and polypeptide transporters (_opp_) [120]. When next-generation RNA sequencing was used on doxycycline-treated spirochetes and spirochetes treated following regrowth, and compared to untreated borreliae, genes upregulated in the treated _B. burgdorferi_ s.l. included a number of Erp encoding genes and _rpUL_,a gene coding for a 50S ribosomal protein. Genes associated with post-treatment regrowth included _bba74_ (Oms28), _bba03_, several peptide ABC transporters, _ospA_, _ospB_, _ospC_, _dbpA_ and _bba62_[121]. One of the reasons proposed by this study for the increased activity of tigecycline against borreliae compared to traditional tetracyclines may be the fact that tigecycline is obviously more resistant to efflux, as mediated by RND-type efflux systems, than are the traditional tetracyclines [57; 122; 123]. This parallels findings where tigecycline was shown to be more efficacious than traditional tetracyclines in many Gram-positive and Gram-negative bacteria, including multi-drug resistant pathogens with RND-efflux pumps [122]. Such RND-type efflux pumps are prevalent in a wide variety of rapid growers, where they appear to decrease the susceptibility of these organisms to antibiotic agents, such as tetracyclines [124]. _Borrelia burgdorferi_ s.l. is pleomorphic and can generate viable but non-culturable bacteria which could also be a component in antimicrobial tolerance. The term \" antimicrobial tolerance/persistence\" is commonly used to describe the small fraction of single-cell heterogeneous antimicrobial-tolerant persister cells. The antimicrobial-tolerant persister phenotype is an epigenetic rather than a genotypic property; a culture of isolated single persister bacteria in fresh medium without an antimicrobial generates a newly heterogeneous bacterial population that mainly contains susceptible cells and a small fraction of antimicrobial-tolerant cells with a new biphasic killing curve on re-exposure [120]. Several studies have confirmed the presence of antimicrobial-tolerant cells in _B. burgdorferi_ cultures. Common to antimicrobial-tolerant persistent bacteria is an increase in the minimum time needed to kill 99.99% of the population (MDK99.99) as well as heterogeneity in cellular susceptibility in culture [120]. This was first suggested by the increased tolerance of stationary-phase borreliae to doxycycline, amoxicillin and nitrofurantoin, and by alterations in spirochete morphology, including the formation of round bodies [125; 126]. Cultures exposed to doxycycline, amoxicillin or ceftaxone displayed biphasic killing curves typical of cultures containing tolerant cells whose numbers increased during the stationary phase. The tolerance to these antimicrobials was not heritable [72; 121].