The increasing ability of ciprofloxacin-resistance or tolerance may lead to high clinical failure rate of bacterial infections and increasing prevalence of infections by antibiotic-resistant bacteria [4, 5]. The canonical mechanisms of ciprofloxacin-resistance include genetic mutations of gyrA and parC or horizontal dissemination of antibiotic resistance genes which protects or modifies the targets the fluoroquinolone [6]. Many studies have shown that transcriptional response in bacterial cells induced by subinhibitory concentration of antibiotics contributes the formation of bacterial resistance [7, 8]. However, less is known about the influence of a transient, high level exposure of ciprofloxacin in bacterial transcriptome. High dose therapy of antibacterial agent has been the commonly used regime when treating infections. The concentration of 2 µg/mL is approximate to the maximum concentration in tissue [9]. For E. coli MG1655 used in this study, this concentration is far exceeding the MIC of ciprofloxacin to the strain.
High concentration of antibiotics caused cellular damage by DNA damage and decrease of motive force.
Ciprofloxacin induces DNA damage such as breaking double-stranded DNA and forking stalled replication, which trigger genetic exchanges [10]. In the present study, we identified some genes related to DNA damage, including gyrA, gyrB, stpA, ygbT, parE, and dnaN, which were elevated in ciprofloxacin treated cells. Among these genes, stpA encodes a H-NS-like protein, which constrains DNA supercoils and influences DNA topology [11]. The gyrA gene contributes to DNA relaxation during DNA replication and mediates breakage and reunion of DNA strand [12]. The elevated expression of these genes suggested that high dose of ciprofloxacin had induced DNA damage in E. coli cells.
In addition, high concentration of antibiotics can inhibit the motive force of bacteria. Among those DEGs in ciprofloxacin treated E. coli, gene clusters that were associated with curli or flagellum biogenesis and metabolisms were lower expressed (Table S2). Decreased expression of motility genes has been viewed as a generalized means of self-protection through energy conservation under particularly harmful conditions [13, 14]. The thrABC operon or threonine operon consists of 4 threonine biosynthesis genes and is mediated by threonine and isoleucine levels in cytoplasm, which are essential for cell growth [15]. The fliL operon is consisted of 7 adjacent genes encode fliL, fliM, fliN, fliO, fliP, fliQ, and fliR, which is required for flagellar biogenesis and normal cell division [16]. The down-regulation of thrABC and fliL operons identified in ciprofloxacin treated E. coli suggests that ciprofloxacin treatment caused cellular damage and destroyed normal development of E. coli cells.
SOS system, toxin/antitoxin system and formaldehyde detoxification system activation, and their significance in drug resistance.
Many studies have demonstrated that subinhibitory concentrations of quinolones activate SOS pathway by DNA damage. The SOS-response in bacteria to antibiotic agents induces ciprofloxacin-resistance and promotes horizontal dissemination of antibiotic resistance genes, which are considered to induce the bacterial adaptive ability and evolution of bacterial resistance to antibiotics [17, 18]. SOS pathway includes the transcription of stress response genes, such as SOS regulon (recA and lexA), and the genes encode DNA repair proteins and polymerases [19]. Our current analysis identified the up-regulation of SOS-response genes including recA gene and its regulator recX, lexA.
Dörr et al demonstrated that SOS response-induced tisB “toxin”, DNA damage inducible toxin, controlled the production of multidrug tolerant cells [20]. Besides, tisB toxin induction from SOS regulon could decrease bacterial growth rate and cause multidrug resistance [21]. In this study, lexA regulated genes tisB (toxic membrane persister formation peptide), ybfE (CopB family), and ydjM were higher expressed after ciprofloxacin treatment. Toxin/antitoxin systems were also higher expressed upon ciprofloxacin exposure. High dose ciprofloxacin induced rapid increase of the tisB toxin within 20 min during exponential phase, indicating that bacterial cells are capable to response to the fluoroquinolone stress promptly that they tend to form persister cells before completing one cycle of binary fission.
The frmRAB operon and rfb clusters were higher expressed after ciprofloxacin treatment by RNA-seq analysis. The frmRAB operon encodes a formaldehyde detoxification system in E. coli and is responsible for glutathione (GSH) -mediated formaldehyde detoxification [22, 23]. GSH-mediated detoxification system prevents the deleterious effects of formaldehyde accumulation in E. coli [24]. We speculate that the cellular GSH induced by ciprofloxacin treatment triggered the formaldehyde degradation pathway and consequently increased the expression of the frmRAB operon in E. coli. RfbABC operon is essential for synthesis of deoxysugar and O-antigen, and multicellular development [25]. These demonstrated that ciprofloxacin treated E. coli cells produced a cellular protection system including enhanced formaldehyde detoxification and auto-repair mechanisms. These were in accordance with the activated SOS response and the ciprofloxacin tolerance or resistance in E. coli cells. In our study, the DEGs in rfbABC operon was also identified to be enriched into “streptomycin biosynthesis” pathway (Table S5).
Significance of increased LPS synthesis induced by high concentration of antibiotics.
Many studies have reported that subinhibitory concentration of quinolones can increase the LPS released by the bactericidal lysis of bacteria [26, 27]. RNA-seq analysis in this study revealed that genes related the biosynthesis of LPS were higher expressed, such as rfb gene cluster, waa operon and kdsA. Further qRT-PCR and LPS assay confirmed that both the LPS release of bacteria and the expression of LPS synthesis related genes increased under the action of ciprofloxacin at bactericidal concentration. These results suggest that high-dose fluroquinolone not only induces bacterial canonical stress response enabling bacterial populations to survive high concentration of antibiotic but also increases LPS production at the genetic level by increasing gene expression. LPS is a major constituent of the outer membrane. Over production of LPS is responsible for sepsis, because it stimulates monocytes and macrophages to produce large amounts of proinflammatory mediators, such as tumor necrosis factor alpha and interleukins [28]. It is known that LPS can trigger inflammatory responses against pathogens in human body, even at very low doses (1 ng/kg body mass) [29]. Therefore, it is very important to reduce the toxicity of bacterial endotoxin while using ciprofloxacin for antimicrobial treatment. In addition to the application of endotoxin scavengers such as polymyxin B to neutralize the endotoxin produced, agents designed to inhibit the expression of endotoxin genes are potential to promote the therapeutic efficiency and reduce the risk of endotoxemia, which is of great clinical importance.