The findings of previous studies indicated Sub-MICs of antibiotics can influence bacterial virulence. The results of this study showed that ciprofloxacin and azithromycin Sub-MICs can alter the expression of the virF in Shigella flexnneri and EIEC. Some studies have been shown that ciprofloxacin and trimethoprim-sulfamethoxazole Sub-MICs promote the synthesis of Shiga-toxin (Stx) in enterohemorrhagic E .coli by inhibiting DNA gyrase and activating the SOS response which can increase the risk of the hemolytic uremic syndrome (HUS) in patients (19). Similar findings have been observed that Sub-MICs of azithromycin can promote the release of Shiga toxin in E. coli (15), however, another study reported that azithromycin considerably decreases Stx levels (19). Furthermore, it has been shown that ciprofloxacin at Sub-MICs may inhibit Uropathogenic Escherichia coli (UPEC) adhesion to epithelial cells via reducing hydrophobicity (16). Moreover, enhanced expression of antibiotic resistance genes in Enterococcus faecium was observed in response to ciprofloxacin Sub-MICs (20). Similarly, there is a report that shows multidrug resistance in E. coli was induced by Sub-MICs of ciprofloxacin (23). Also, some studies showed azithromycin Sub-MICs can reduce biofilm formation in Pseudomonas aeruginosa (17) and had an increasing effect on E. coli growth (18).
Bacterial sensory systems frequently respond to environmental stimuli by changes in gene expression; as a result of the cell adjusting to new environments (9). In antibiotic stress conditions, alterations in gene expression in genes involved in surface structures, reflux systems, and enzymes linked to antibiotic inactivation, have been observed. The regulation of these genes are usually under control of bacterial sensory systems (10, 24). A substantial increase in the expression of the virF of Shigella and EIEC was seen in response to ciprofloxacin Sub-MICs in our study. The virF protein is a major regulator of the transcription of bacterial genes involved in invasion in both species. The virF protein is encoded on an invasion plasmid (pINV) and is an upstream regulator of other virulence gene regulators like virB. Most virulence genes encoded by pINV are under the direct control of the VirB protein, thus, transcriptional activation of operons implicated in invasion is dependent on virF expression (25). The physicochemical changes in the environment like temperature, pH, and osmolarity usually alter the regulation of virF gene expression, resulting in changes in bacterial virulence (26). CsrA, a carbon storage regulator in E. coli and Shigella, is a protein involved in cellular metabolism, flagella biosynthesis, and biofilm development. Potts et al. reported that a two-component regulatory system BarA-SirA, can promote CsrA expression in response to a decrease in carbon sources like glucose and accumulation of the intermediate metabolites like fumarate and acetate (27). Gore et al. also found that bacterial attachment and invasion in cell culture were decreased in Shigella flexnneri mutant for the csrA gene, compared to the wild-type strain. They concluded that this reduction in bacterial virulence happens because of a lower virF gene expression in csrA mutants and subsequent reduction of expression of pfkA; a gene involved in bacterial glycolysis (phosphofructokinase (28). Sub-MICs of bactericidal antibiotics can enhance bacterial respiration by stress induction in the bacteria, which it can lead to cell death by accumulating toxic compounds such as respiratory oxygen species (ROS) (29). High concentration of ROS increases glycolysis, that leads to reducing the glucose resources and increasing pyruvate and acetyl CoA formation (30). The decreased carbon sources probably can induce expression of CsrA and PfkA proteins that leads to up-regulation of virF gene in bacteria (31, 32). Unlike ciprofloxacin, a down-regulation of virF gene by Sub-MICs of azithromycin have been observed in the present study. Bacteriostatic antibiotics, such as azithromycin have an inhibiting effect in the bacterial protein translation process. Inhibition of protein translation can lead to reducing cellular respiration by repressing glycolysis and tricarboxylic acid cycle (TCA). This may result in the accumulation of ADP and AMP, a significant increase in NADH, and a depletion in cell ATP level (33). In this regard, an hypothesis is that Sub-MICs of antibiotics cause the impaired balance of these metabolites, reduce the expression of CsrA and PfkA proteins, and subsequently decreasing of virF gene expression in these bacteria. However, it needs to be assessed by a study on bacterial global gene expression assay.
It has been proposed that virF gene is also under control of a regulator protein called YhjC. Wanwu et al. reported Shigella flexnneri mutants for the yhjC gene have a decreased ability to adhere and penetrate the host cell. Their results showed the deletion of the yhjC gene down-regulated the virF and subsequently all virF-dependent genes as well. The factors that have effect on the expression of the yhjC gene are unknown, however, it has been observed that expression of this gene increases, when the temperature rises from 30 to 37 degrees. These findings increase the prospect that this gene is under control of a two-component regulation system CpxA/R (34). In the present study, temperature, pH, and osmolarity for all antibiotic-treated and untreated samples were equal, thus, the probability that Sub-MICs of antibiotics alter virF expression via CpxA/R, is diminished.
In conclusion, the antibiotics in different bacteria have different effects. Therefore, it is impossible to generalize the findings to antibiotic accessory effects in diverse microorganisms. Hence, evaluation of accessory mechanisms of antibiotics that are major choice for the treatment of infections with particular bacteria is more practical. The results showed, ciprofloxacin and azithromycin Sub-MICs can have effect on virulence of EIEC and Shigella flexnneri. These antibiotics are the primary choices of treatment of acute infections by Shigella and EIEC. Given the potential of azithromycin for the reducing of pathogenicity of these bacteria, it seems that it is a more suitable choice with a lower risk for treatment of acute infections with EIEC and Shigella flexnneri that need antibiotic therapy.