## 2 Results
### Biofilm-Forming UPECs Are More Virulent to Galleria Mellonella Larvae  
Finally, in order to determine if the biofilm-forming UPEC are more virulence than non-forming examples we employed a _G. mellonella_ larvae model. As discussed in our previous work [38] this larvae serves as a surrogate model of infection. The model is cost  
Figure 4: Phenotypic evaluation of the UPEC strains. Non-biofilm forming (n = 24) and biofilm-forming (n = 23) strains were grown aerobically in MOPS minimal medium (0.2% glucose), with shaking. (**a**) The relative accumulation of the (p)ppGpp alarmones was assessed by the \({}^{32}\)P labeling of nucleotides and revealed chromatographically on PEI cellulose plates by TLC in 1.5M KH2 PO4, pH 3.4. The presented data were collected at an exponential phase of growth (A600 = 0.3) (**b**) Doubling time was determined spectrophotometrically with a plate reader. (**c**) Linear regression plot obtained by combining both parameters (generation time and alarmone accumulation), with R2 values indicated. Results are obtained from at least three independent experiments and are expressed as the mean with SEM. Statistical significance was determined by unpaired Student’s t-test.  
\begin{table}
\begin{tabular}{c c c c} \hline \hline \multirow{2}{*}{**Group**} & **Number of** & **\% of the Group** & \multirow{2}{*}{**Fisher’s Exact Test**} \\  & **Resistant Strains** & **(\% of Multiple** & \\  & **(Multiple** & **Resistant within** & \\  & **Resistant)** & **Resistant Strains)** & \\ \hline Biofilm forming & \multirow{2}{*}{16 (12)} & 30.8\% & \multirow{2}{*}{0.0410} \\ n = 39 & & (75\%) & \\ Non-biofilm forming & & 19.7\% & \\ n = 81 & & (43.2\%) & \\ \hline \hline \end{tabular}
\end{table}
Table 3: Biofilm-forming ability on CRA vs. the presence of a multiple resistance phenotype.  
efficient, effective, reliable, and does not raise ethical concerns. Moreover, its use, has been described to study UPEC virulence [39; 40]. The larvae were injected with a bacterial inoculum (\(10^{4}\) to \(10^{7}\) cfu/larvae) to determine the mortality rate of insects exposed to each isolate. Five strains from each of the CRA+/\(-\) groups were tested. The survival of larvae was monitored for several days, and the results were collected in the form of Kaplan-Meier curves (Figure 5a). The _G. mellonella_ larvae showed different survival profiles, depending on the strain used. However, in all cases, only the relatively high infectious dose \(10^{7}\)/larvae resulted in 100% mortality rate within 48 h. In addition, a quantified LD50 value between 24 and 72 h post-inoculation and the LD50 reduction level (LD50 72h/LD50 24 h) were calculated for each isolate (Figure 5b). The virulence of biofilm-forming isolates was higher in this model than that in the non-biofilm-forming samples.  
Figure 5: Evaluation of virulence of CRA+/\(-\) UPEC strains. Evaluation of virulence of biofilm-forming (gray part of panel) and non-biofilm-forming (colorless part of panel) UPEC strains in a surrogate infection model of _G. mellonella_. The 5 biofilm producers chosen were selected based on the greatest ability to form biofilm, and the 5 non-biofilm forming samples were randomly selected. **(a)** Kaplan–Meier survival curves of _G. mellonella_ after injection of \(10^{4}\)-\(10^{7}\) cells/larva. **(b)** LD50 values calculated between 24 and 72 h. Percentage changes in LD50 between the 1 st and 3 rd days are indicated by arrows, with their value above them. The values of growth rate (min) and alarmome relative concentration (%) in particular strains are indicated under the plot.