## 2 Results  
### Distribution of Virulence Factors and Phylogenetic Clustering of Isolates  
First, we decided to evaluate the genetic background of the tested isolates, employing PCR analysis with previously published primer pairs regarding the above described virulence factors (VFs). According to the results of our genetic analysis, a relatively high distribution of VFs among the isolates studied (Figure 1a) is an important phenotypic characteristic, as one or more VFs were expressed in 100% of the strains. All of them encoded the _fimH_ gene, and among the other factors analyzed, the most frequently identified were _sitA_ (92.5%), _ser_ (75%), and sfa (70%). The ability to express HlyA and/or PapC proteins, as well as antigen 43 occurred at a medium level (42.5-56.7%). In contrast, the lowest distribution was obtained for the _cnf_, _ibeA_, and _iss_ genes (24.2%, 19.2%, and 18.3%, respectively). For 117 of the 120 strains, phylogroups were determined by extended quadruplex PCR. Of the 8 phylogroups described [17], 6 were identified in our collection (Figure 1b). A large number of isolates was assigned to group B2 (71.7%), followed by groups B1 (10.8%) and D (7.5%). UPEC isolates from groups F, A, and C were less frequently represented (3.3%, 2.5%, and 1.7%, respectively). Figure 1: Genetic characteristics of the UPEC collection. (**a**) Percentage distribution of virulence factors identified by PCR in UPEC isolates. (**b**) Percentage distribution of phylogroups determined by phylogenetic typing using the quadruplex PCR method [17]. Based on the findings above, a series of analyses were carried out to determine the co-occurrence of virulence factors in relation to each other. Correlations were statistically confirmed with [PERSON]'s exact test (Figure 2). In particular, many correlations were determined for the _sfa_, _papC_, and _agn43_ genes. The least correlated with other VFs were the _ibeA_, _sitA_, and _aer_ genes (Figure 2). For each pair, the direction of correlation was determined to be either positive or negative, using [PERSON]'s test (Figure 2). A strong correlation was observed for the presence of the _cntf_ gene, which was always present with the _hlyA_ gene, while the presence of _hlyA_ was not dependent on the presence of _cntf_ (See supplementary material for details). Moreover, by including Clermont's classification in the analysis, we observed that different phylogenetic groups had different quantitative distributions of VF. The B2 group had the highest mean value (6.01), the A, C, and D groups had intermediate values, while B1 had the lowest value. Thus, the correlation between individual virulence factors and Clermont's phylogroups was analyzed. Some peculiarities should be noted, such as the occurrence of the _cntf_ gene exclusively in the B2 phylogeny. Similarly, the _hlyA_ or _ibeA_ gene sequences are present only in this group, except for two isolates (Table 1). Due to the size of the groups, only the differences between the largest B1 and B2 groups were evaluated. Statistical analysis shows that these groups differ in the distribution of all analyzed genes except for _fimH_, _ibeA_, and _aer_, with the _fimH_ sequence present in all strains, regardless of the group (Table 1). Figure 2: Co-occurrence matrix of virulence factors. Statistically significant results ([PERSON]’s exact test, \(p<0.05\)) are shown in green, while non-significant correlations are shown in gray. The direction of correlations was determined using [PERSON]’s test, where “+” indicates a positive correlation and “-” a negative correlation (\(p<0.05\)). The matrix presents horizontal values vs. vertical values.