Using pooled human urine and serum samples in experimental designs has certain limitations that may impact the generalizability of the findings. One limitation is the variability in the composition of urine and serum samples from different individuals, which may introduce confounding factors and make it challenging to capture strain-specific or host-specific responses. To address this limitation, future studies could consider using samples from a larger cohort of donors to account for inter-individual variability. Additionally, incorporating host-specific factors such as immune components or specific host cell types in the experimental setup could provide a more accurate representation of the host-pathogen interaction. Furthermore, utilizing strain-specific mutants or isogenic strains in the study design can help elucidate the role of specific genes in different host environments and their impact on virulence.