2.1 Samples collection and preliminary characterization
A total of 95 isolates were subjected to preliminary characterization including serogrouping, antimicrobial resistance (AMR) testing and PCR detection of virulence and antibiotic resistance genes. The disc diffusion test showed that 69.5% were resistant to three or more groups of antimicrobials, which we considered as a criterion of multiresistance. Resistance to ampicillin was recorded in 78 isolates (82.0%), followed by resistance to nalidixic acid (62 isolates; 65.3%), sulphonamides (45; 47.4%) and sulphonamides-trimethoprim (28; 29.5%). Nineteen isolates (20.0%) showed reduced susceptibility to ciprofloxacin (additional file 1 – Figure 1). Using four antisera (O1, O8, O18 and O78), 49 isolates (52%) were typeable, with predominant serogroups O1 (30; 32%) and O8 (13; 14%). Four isolates reacted to O78 and two against O18 antisera.
The blaTEM gene was detected in 45 isolates (47.4%). Other prevalent genes detected by PCR included tet(A) (26 isolates; 27.4%) and sul2 (28.4%). As for virulence genes, most isolates carried typical APEC plasmid-associated genes iss (75; 78.9%), iroN (73; 76.8%), iut(A) (68; 71.6%), whereas others, plasmid- or chromosome-associated genes, e.g. cvaC (49; 51.6%), frzorf4 (44; 46.3%), tsh (32; 33.7%) and felA (7; 7.4%) were less prevalent.
2.2 In silico serotyping, MLST and phylogenetic analysis of 32 selected isolates
We found a diversity of serogroups in the collection of 32 isolates subjected to whole genome sequencing. Overall 14 different O types and 16 H types were identified and 10 isolates failed to be typed by WGS (the results are summarized in Table 1, additional file 1). Except for serogroup O8 (7 isolates), the remaining serogroups were only represented by one or two isolates. The predominance of O8 serogroup appeared as a selection bias since only Czech isolates were selected for sequencing. Of the O8 serogroup, six isolates belonged to the O8:H9 serotype, most of them to ST23 type.
The isolates were derived from all phylogroups according to the Clermont scheme (29) except for the group E: group F (3 isolates), B2 (9 isolates), D (2 isolates), clade I (1 isolate), A (4 isolates), C (6 isolates), B1 (7 isolates).
The MLST analysis identified 22 distinct sequence types (please see Table 1 in the supplementary material and Figure 1), most of them represented only by a single isolate (ST352, ST95, ST140, ST354, ST93, ST4110, ST1249, ST1914, ST770, ST2223, ST746, ST1249, ST162, ST1157, ST602, ST1841, ST533, ST7104). Two isolates were typed as ST117 belonging to the phylogenetic group F; ST429 of the B2 group and ST23 of the C group were detected in 4 and 6 isolates, respectively.
The core genome consisted of 2763 genes (55.28 kbp). The phylogenetic tree based on the core SNPs corresponded to the structure of E. coli phylogeny. Groups F, D and clade I were represented by only a few isolates and did not form any distinct clusters except the minor subcluster of the two group D isolates; two ST117 isolates from the F phylogroup were unrelated to other isolate of F phylogroup (ST354) and formed their own distinct clade. In the B2 cluster two subclusters (B2a, B2b) were found; B2b subcluster was formed by four closely related ST429 isolates and one ST4110. Another cluster included isolates from phylogroups A, C and B1. Interestingly, all isolates of the C group belonged to ST23, O8:H9 serotype (with one exception of O78:H9 serotype).
2.3 Identification of resistance genes
blaTEM-1 (9/32; 28.1%) and a combination of blaTEM-106, 135 (6/32; 18.8%) belonged amongst the most prevalent resistance genes. A combination of plasmid-mediated β-lactamase genes blaCMY-2, -59 was detected in four isolates (12.5%), three of them belonging to the ST429, the remainig one to ST354. PMQR (plasmid-mediated quinolone resistance) gene qnrS1 was carried by seven isolates (21.8%) within sequence types 23 and 429. Other identified AMR genes were sul1 (7/32; 21.8%), sul2 (8/32; 25.0% ), dfrA14 (1/32; 3.0%), dfrA15 (4/32; 12.5%), dfrA5 (1/32; 3.0%), tet(A) (12/32; 37.5%), tet(B) (2/32; 6.3%), aadA (6/32; 18.8%), aac(3)-VIa (3/32; 9.4%), ant(2)-1a (1/32; 3.0%), aph(3)-1b (6/32; 18.8%), aph(3)-1a (2/32; 6.3%), aph(6)-1d (5/32; 15.6%), catA1 (2/32; 6.3%), floR (1/32; 3.0%) and blaTEM-30 (1/32; 3,0%). In addition, all isolates showed the presence of genes encoding components of various multidrug efflux pumps, participating in resistance to aminoglycosides, macrolides and fluoroquinolones. Except for qnrS1, which is associated with partial resistance to fluoroquinolones, no other PMQR gene was detected. Reduced susceptibility to quinolones in most isolates appeared to be due to chromosomal mutations, especially in the gyrA gene (21; 65.6%), to lesser extent also in parC (5; 15.6%) and parE (1; 3%). In five ST23 isolates (15.6%), a mutation in the ampC promoter was detected. (For overview of resistance genes, please see the Table 1, additional file 1.)
2.4 Identification of virulence genes
The genomic analysis confirmed a great diversity of selected isolates (see figure 2 and supplementary material, file 3). Overall, factors associated with adhesion and invasion, as well as siderophores were found in most isolates; more than 90 % of isolates encoded F1 fimbriae, curli, E. coli common pilus and enterobactin. All but one isolate carried ibeB gene, while ibeA was present mostly in B2 and F phylogenetic groups, but not in isolates from other groups. A siderophore system salmochelin (81%), haemolysin F (90.6%) and serum-resistance associated proteins, Iss (87.5%) and TraT (78%) were present in most isolates with generally equal distribution in all phylogenetic groups. Full SitABCD iron transport system was detected in 78% isolates, outer membrane protease (OmpT) and colicin V synthesis protein (CvaC) in 68.8% and 59% isolates, respectively.
Several virulence- genes were associated with particular branches of the phylogenetic tree. For example, Stg fimbriae, Ycb fimbriae, CFA/1 fimbriae and genes associated with ETT2 (E. coli type III secretion system 2) were common in B1 and C groups, but almost or entirely absent in B2 phylogenetic group. In contrast, yersiniabactin, aerobactin and hemin receptor (chuA), as well as OmpA, capsular antigens (kpsD, kpsT, kpsM), pathogenicity-island marker malX, uropathopathogenic-specific protein (usp) and afore-mentioned brain-endothelium invasin (ibeA) appeared to be B2 group-associated.
Lastly, some well known virulence-associated genes were detected uniquely amongst our isolates. For example, complete pap operon was present only in the one ST95 isolate, as well as the intimin-like adhesin (fdeC). Similarly, neuC gene was found in ST95, ST140 and two ST429 isolates (all B2 phylogroup) and K99 (F5) fimbriae only in ST354 isolate of the F phylogroup. For overview of all virulence-associated genes detected please see the Table 2, additional file 2.
2.5 Identification of plasmid replicons
All but one isolate harboured a replicon of the F incompatibility group, FIB replicon being the most commonly detected (31/32 isolates; 96.9%). At the same time, Col replicons were detected in most (22/32; 68.8%) isolates. Groups IncB/O/K/Z (10; 31.3%) and IncX1 (6; 18.8%) also appeared relatively frequently, while others were identified only in individual isolates. The IncHI1B replicon was identified in two isolates of the ST429 and ST23 cluster, respectively. Overall, types and number of replicons varied greatly even within the two closely related clusters. (For overview of replicons, please see the Table 1, additional file 1.)