## 3 Results
### Transcriptional Profiling of PmS, PmR and PmHR by RNA Sequencing  
In order to search for genes that played an important role in the formation of fluoroquinolone resistance in _Pm_, we sequenced the transcriptome of three strains under the sub-inhibitory concentration of ENR (Supplementary Table S2). The results showed that more than 97% of the reads can be mapped to the reference genome sequence (GenBank: 8172276). Raw read data were sequenced and filtered for further analysis, yielding approximately 1.76-2.15 billion clean reads. The average length of the reads for _PmS_, _PmR_ and _PmHR_ was 300 bp, and their GC content was 42.77%, 43.17% and 42.35%, respectively, indicating successful sequencing of the _Pm_ with different levels of ENR resistance (Supplementary Table S3).  
In total, 14,055,756 clean reads were detected. Based on the FDR threshold \(\leq\)0.05 and 1 log2 fold-change (FC) \(|>\) 1, a total of 1,499 significant DEGs were identified. Simply put, compared with _PmS_, _PmR_ has 144 up-regulated and 292 down-regulated genes. Compared with _PmS_, 181 genes were up-regulated and 382 genes were down-regulated in _PmHR_. Compared with _PmR_, 104 genes were up-regulated and 172 genes were down-regulated in _PmHR_ (Supplementary Figures S2 and S3). The GO enrichment analysis and KEGG pathways were performed for DEGs. Enrichment analysis revealed significant changes in 19 GO biology processes, among which, seven biology processes were relating biological process, seven biology processes were relating cellular components and five biology processes were related to molecular function. These processes involve the intrinsic components and compositions of cell membranes, intracellular organelles, adhesion proteins and membrane-protein-related pathways. (Supplementary Figure S4). On the other hand, the top 20 of KEGG pathways were annotated. We annotated 650 DEGs and enriched them into 215 metabolic pathways. We further screened the enriched metabolic pathways, and categorized them into metabolic pathways such as antigen presentation and processing, cell adhesion, phagocytosis and cell killing and virulence. Among them, we found that antigen presentation and processing enriched DEGs were the most common. (Supplementary Figure S5 and Table S4).  
Furthermore, in order to find the difference between _PmR_ and _PmHR_, significantly expressed genes were screened with the improvement of drug resistance, and the _satP_ (gene 1687), _foCA_, _napF_, _glgP_ and _napD_ genes were significantly up-regulated (Table S5). As the most significantly expressed gene, the _satP_ gene is mainly involved in encoding intimal proteins. By analyzing the expression of _satP_, we found that the expression of _satP_ in the _PmR_ group was 17 times higher than that in the _PmS_ group, and the expression of _satP_ in the _PmHR_ group was 20 times higher than that in the _PmS_ group (Figure 1A,B).  
\begin{table}
\begin{tabular}{c c c c c c c c c} \hline \hline \multirow{2}{*}{**Strains**} & \multicolumn{6}{c}{**MIC Range (\(\mathrm{\SIUnitSymbolMicro g}\)/mL)**} & \multicolumn{6}{c}{**Substitution in**} \\ \cline{2-10}  & **CIP** & **ENR** & **ENR + CCCP** & **ENR + Verapamil** & **GyrA** & **GyrB** & **ParC** & **ParE** \\ \hline _PmS_ & 0.25 & 0.25 & 0.125 & 0.125 & - & - & - & - \\ _PmR_ & 4 & 4 & 2 & 2 & Arg88 Ile & Ser467 Phe & Glu84 Lys & - \\ _PmHR_ & 64 & 64 & 64 & Arg88 Ile & Ser467 Phe & Glu84 Lys & - \\ \hline \hline \end{tabular}
\end{table}
Table 1: QRDR mutation genotypes and fluoroquinolone MICs for _PmS_, _PmR_ and _PmHR_.  
The qRT-PCR analysis was performed to validate the expression of _satP_, _focA_, _npF_, _glgP_ and _mapD_ in _Pm_ with different levels of drug resistance. As shown in Figure 1C-G, qRT-PCR results of _satP_ agreed with the FPKM data. At the same time, the relative expression of _satP_ in the _PmR_ group was significantly higher than in the _PmS_ group, and in the _PmHR_ group significantly higher than in the _PmR_ group. The expression level of _satP_ gene was in accordance with the trend of drug resistance.