## 3 Discussion
To test whether reports of increased virulence of such strains would also be confirmed in our study, we screened the infectivity of UPEC differing in (p)ppGpp levels in a _G. mellonella_ model. The results confirmed the initial assumption of increased virulence of CRA+ strains exhibiting higher intrinsic levels of (p)ppGpp (Figure 5). In laboratory _E. coli_ strains, the cellular level of (p)ppGpp is closely related to their growth rate. The increase in the (p)ppGpp level is observed in response to stress factors, such as starvation, and results in a rapid accumulation of high alarmones concentrations (600-1000 pmol OD\({}^{-1}\)) [81]. This leads to the inhibition of rRNA, DNA, and protein synthesis and in turn, yields slower growth rates. However, to date, there are still gaps in the knowledge regarding the role of the basal level of (p)ppGpp, which occurs in the cell under optimal growth conditions, and how it can affect cell physiology. The high heterogeneity of the tested strains, demonstrated by genetic-phenotypic analysis, may arise from the differences in (p)ppGpp levels between clinical isolates. Thesedifferences could be due to either the polymorphism of molecular targets of (p)ppGpp or proteins interacting with ReIA and SpoT, including regulatory proteins or the polymorphism of ReIA and SpoT proteins themselves. Stringent response exhibits pleiotropic effects, triggering vast metabolic adjustments in bacteria to adapt to a challenging environment. However, alarmones are also involved in VFs expression; therefore, we hypothesize that observe variations in (p)ppGpp levels among particular strains served as an adaptation mechanism, increasing competitiveness; however, this theory still awaits verification. Moreover, the observed common assumptions do not always correlate with the expected phenotype. As an example, enterohemorrhagic _E. coli_ (EHEC) strains have a positive correlation between (p)ppGpp and the transcription of virulence factors encoded on the LEE pathogenicity island, while an inverse correlation is observed between high levels of (p)ppGpp and the Shiga toxin production [82]. Similarly, it is well known that high levels of (p)ppGpp result in low growth rates [30]. However, in this study, we observed an inverse correlation, as the biofilm-forming group of UPECs exhibited a significantly higher basal level of (p)ppGpp and, most remarkably, higher growth rates than the non biofilm-forming strains cultivated under the same non-limiting conditions, which is in stark contrast to the knowledge obtained to date. In a way, our observations are supported by a study on ECOR isolates (an evaluation of the regulatory relationship of (p)ppGpp and RpoS), which showed that regulatory pathways are not uniform within one species [83, 84]. Experiments conducted on the K-12 strain suggest that (p)ppGpp should stimulate RpoS synthesis, but results described by [PERSON] et al. indicate that RpoS levels are not equally stimulated by high (p)ppGpp in all ECOR isolates [84]. Furthermore, targets that are upregulated during induction of the stringent response may not be sensitive to basal ppGpp levels, which for some reason, are maintained at higher levels in the cell [85, 86]. [PERSON] et al. described that modest increases in (p)ppGpp (<100 pmol OD\({}^{-1}\)) do not appear to immediately inhibit biomass synthesis (except for stable RNA). High concentrations of pseudomonic acid rapidly increased ppGpp accumulation and canonically abruptly stopped growth. On the other hand, low concentrations of pseudomonic acid also triggered ppGpp synthesis (up to 60-100 pmol OD \({}^{-1}\)) and an immediate, but smaller, decrease in rRNA synthesis. However, the exponential growth rate was not affected in the short term by a moderate increase in ppGpp concentrations [87]. The authors also determined that (p)ppGpp directly inhibits protein synthesis, controlling the rate of ribosome synthesis, albeit only at high concentrations of ppGpp.