## 2 Altered Production of Quinolone Targets  
While target overexpression has been considered a mechanism of resistance to different antibacterial agents because the quantity of the target surpasses the inhibitory capacity of antibiotics, with overexpression of \(\beta\)-lactam inhibitor-sensitive TEM-1 leading to intermediate-to-full resistance to \(\beta\)-lactam plus \(\beta\)-lactam inhibitor combinations being a classic example [83; 84; 85], quinolones show an opposite phenomenon: lower levels of quinolone targets lead to higher levels of resistance. This phenomenon was first described in an in vitro selected quinolone-resistant _Staphylococcus aureus_ in 2003 [86]. Using pre  
\begin{table}
\begin{tabular}{l c c c c c c} \hline  & & & & & & & \\ \cline{2-6}  & **Low Levels of** & **Decreased** & **Transcriptomic/Metabolic** & **Quinolones** & **Resistance** & **Tolerance** \\  & **Targets** & **Antibiotic Access** & **Alterations** & **Inactivation/Degradation** & & & \\ \hline Expression levels & Y & NA & NC & – & Y & – \\ \multicolumn{6}{l}{} \\  
malfloxacin, [PERSON] et al. selected an in vitro mutant exhibiting a 4-fold increase in the MIC levels of premafloxacin (from 0.004-0.008 mg/L to 0.016-0.032 mg/L) and ciprofloxacin (from 0.125-0.25 mg/L to 1 mg/L), with no alterations in either the MICs of ethidium bromide (suggesting no overexpression of efflux pumps) or amino acid substitution in GyrA, GrIA (ParC), GyrB or GrIB (ParE), and with a single nucleotide change G\(\rightarrow\)A in the _grIBA_ operon promoter at position -13, just downstream of a presumptive Shine-Dalgarno sequence [86]. The role of this alteration in the development of quinolone resistance was demonstrated by introducing the mutation in a wild-type _S. aureus_, confirming the increase in ciprofloxacin and premafloxacin MIC levels [86]. When the authors determined the physiological effects of this alteration, they observed a 3-fold reduction in the overall expression levels of _grIA_ and _grIB_[86]. Nevertheless, no apparent effect on bacterial growth was observed, suggesting the presence of compensatory mutations within the chromosome [86].  
The presence of alterations in the _gyrA_ promoter region impacting sensitivity and supercoiling regulation and altering final expression levels has also been described [87], but no data on the effect on final quinolone levels are available in the literature. Of note, quinolones relax DNA by inhibiting supercoiling [88], which, in turn, results in increased expression of _gyrA_[89].  
In relation to the above, potential alterations in the level of expression of genes encoding quinolone targets have been proposed as one of the mechanisms involved in the increased levels of resistance of the so-called \"Small Colony Variants\" (SCVs) [90] (see Section 8: Unconsidered Chromosomal Mutations).  
While to the best of my knowledge no description has been made, it could be hypothesized that mutations at the initial codon of genes encoding quinolone targets leading to modifications from ATG to, for instance, GTG or TTG, may also affect transcription efficiency, as these initial codons are less efficient [91], thereby resulting in lower protein levels and subsequent similar effects on the final quinolone susceptibility levels.