In the initial research stage, we conducted amplification (using F5 and R2 primers) of the mutS-rpoS chromosomal region to examine the presence and type of the o454-nlpD region in our E. coli collection. While performing polymorphism analyses on the o454-nlpD region, we discovered an additional pattern with a different length of 1660 bp, differing from the previously described pattern [2]. To identify complementary regions in the tested genomes, we utilized the F5 and R2 primer sequences and the online software available at http://insilico.ehu.es/PCR/. Subsequently, sequences with a length of 1660 bp (representing the atypical o454-nlpD pattern) were explicitly chosen to confirm the amplified region’s accuracy.

HaeIII restriction analysis of the 1660 bp PCR products yielded restriction fragments consistent with the predictions from in silico analysis (498, 406, 306, 172, 96, 87, 39, 36, 20 bp, Additional file 1: Fig. S1). Consequently, we verified that the newly identified mutS-rpoS chromosomal region—referred to as “Different A”—corresponds to one of the analyzed o454-nlpD patterns.

The pattern distribution of the o454-nlpD region for the 124 tested UPEC strains was determined and compared with the phylogroups, virulence factors, and TRS-PCR fingerprints.

Pattern III was overrepresented, constituting 39% (49 strains) of the tested isolates. Patterns named different A and pattern I constituted 30% (37 strains) and 21% (26 strains), respectively. Only 9% (11 strains) had pattern IV, and one strain had a different B pattern. Our collection had no strains with pattern II (Fig. 1).

Distribution of o454-nlpD region patterns among UPEC strain collections

The phylogenetic structure of UPEC strains for obtained o454-nlpD region patterns was tested. The results are presented in Fig. 2. The UPEC strains with pattern III all belonged to phylogenetic group B2. The phylogenetic groups D, B1, and F were represented at similar levels among strains with pattern I (31%, 31%, and 27%, respectively). Strains representing phylogenetic group A predominated among strains with a pattern different A (59%). In the case of strains with pattern IV, more than half of this group belonged to phylogenetic group B1 (55%).

Distribution of phylogroups of UPEC strains with different o454-nlpD region patterns

Our laboratory UPEC collection of strains had the virulence factor repertoire tested as published elsewhere [12, 26]. The distribution of virulence factors in strains with particular patterns of the o454-nlpD region was determined (Table 1). Genes related to siderophore systems, such as fyuA, iutA, and iroN, were detected among all strains with the pattern I. Twenty-five of 26 strains also encoded fimG/fimH, one strain had Sat, and two had Tsh—serine-protease autotransporter toxins of Enterobacteriaceae (SPATE). Six strains from this group encoded papC, two encoded sfa, two encoded cnf1, one encoded usp, and three encoded hlyA.

The pattern III strains had at least two virulence profiles associated with uropathogenicity. Moreover, the pattern III strains had the most variable profiles of virulence genes. The gene fyuA, which is related to the siderophore system, was detected in all isolates from this group. The genes iutA, iroN, and sat were present in approximately 59%, 92%, and 29% of isolates, respectively.

Only 2% of strains with the pattern different A possessed the papC gene. Each gene, such as sfaD/sfaE, cnf1, usp, and hlyA, was present in 1% of these strains. However, genes associated with siderophore systems, such as fyuA, iutA, iroN, as well astA, were identified in approximately 32%, 24%, 35%, and 24% of isolates, respectively.

Only three strains with pattern IV encoded the astA gene, and one strain encoded the tsh gene. Most of them had iutA (approx. 73%), iroN (approx. 82%), and fyuA (approx. 45.5%).

None of the tested strains had virulence genes such as bfpB, invE, elt, escV, stx1, stx2, estIa, or estIb, and they were not included in statistical analyses associated with the o454-nlpD region.

Next, Fisher’s exact test was used to estimate the correlation between the presence of o454-nlpD patterns and the identified virulence factors. The relationship was statistically significant when P values were smaller than 0.05. Pattern I: no positive associations were detected; negative association with cnf1, hly1, sfaD/sfaE, and usp; no association with papC, fimG/fimH, fyuA, iutA, iroN, sat. Pattern III: positive association with papC, sfaD/sfaE, cnf1, usp, hly1, fyuA, and iroN; negative association with fimG/fimH and sat; no association was observed with iutA. Pattern IV: no positive and no negative associations were detected; no association with papC, sfaD/sfaE, cnf1, usp, hly1, fimG/fimH, iutA, fyuA, iroN, and sat. Pattern Different A: no positive associations were detected; negative associations with papC, sfaD/sfaE, cnf1, usp, hly1, fimG/fimH, fyuA, iutA, iroN, and sat.

For virulence factors such as tsh, astA, aggR, and pic, no association was observed with any tested pattern of the o454-nlpD region. Detailed information is gathered in Table 2.

A collective comparative analysis showing the clustering of E. coli strains in the averaged TRS-PCR analysis and the relationships between the o454-nlpD profile, phylotype, and virulence factor repertoire are presented in Fig. 3.

A collective comparative analysis of 124 E. coli strains showing clustering in the average CGG-, GTG-, and CAC-PCR fingerprint analyses and the relationships between the o454-nlpD profile, phylotype, and virulence factor repertoire

The cluster grouping mainly highly pathogenic phylogroup B2 and type III o454-nlpD strains is marked with a black dot. Strains with profile III o454-nlpD are marked with a blue square; pathogenicity factors with a positive correlation with the o454-nlpD profile type are marked in green; pathogenicity factors with a negative correlation to the o454-nlpD profile type are marked in red.