The relationship between phylogenetic classification, virulence and antibiotic resistance of extraintestinal pathogenic Escherichia coli in İzmir province, Turkey

Abstract

Background: Extraintestinal pathogenic Escherichia coli (ExPEC) is an important bacterium and responsible for many bloodstream infections, including urinary tract infections and even fatal bacteremia. The aim of this research was to investigate whether ExPEC strains isolated from Turkish blood cultures have a relationship between 16S rRNA based phylogenetic clusters and antibiotic resistance profiles, virulence factors or clonal lineages.

Methods: Phenotypically identified ExPEC blood culture isolates (n = 104) were included in this study. The 16S rRNA partial sequence analysis was performed for genotypic identification of ExPEC isolates. Antibiotic susceptibility and Extended-Spectrum β-Lactamase testing of isolates were performed. Phylogenetic classification (A, B1, B2 and D), Multi Locus Sequence Typing analysis and virulence-associated genes were investigated.

Results: Based on 16S rRNA partial sequence analysis, 97 out of 104 (93.26%) ExPEC isolates were confirmed as E. coli. Ampicillin (74.22%) and cefuroxime axetil (65.97%) resistances had the highest frequencies among the ExPEC isolates. In terms of phylogenetic classification of ExPEC, D (38.14%, 37/97) was the most prevalent group after A (29.89%, 29/97), B2 (20.61%, 20/97), and B1 (11.34%, 11/97). The sequence types of the 20 ExPEC isolates belonging to the B2 phylogenetic group were analyzed by Multi Locus Sequence Typing. Ten isolates out of 20 (50.0%) were identified as ST131. The other STs were ST95 (n = 1), ST14 (n = 1), ST10 (n = 1), ST69 (n = 1), ST1722 (n = 2), ST141 (n = 1), ST88 (n = 1), ST80 (n = 1), and ST998 (n = 1). Of the ST131 strains, six (60%, 6/10) represented serogroup O25. The most common virulence factor genes were serum resistance factor gene, traT (55.7%) aerobactin siderophore receptor and yersiniabactin encoding genes iutA (45.3%) and fyuA (50.5%), respectively. In addition, PAI (41.2%), iroN (23.7%), hlyA (15.4%), kpsII (13.4%), ompT (13.4%), papG (12.4%), iss (9.3%), cnf1 (7.2%), ibeA (2.06%), and sfaS (2.06%) genes were present in the ExPEC isolates.

Conclusion: The 16S rRNA-based phylogenetic relationship tree analysis showed that a large cluster was present among 97 ExPEC isolates along with related reference strains. There were 21 main clusters with 32 closely related subclusters. Based on our findings, different clonal lineages of ExPEC can display different antibiotic susceptibilities and virulence properties. We also concluded that virulence factors were not distributed depending on phylogenetic groups (A, B1, B2, and D). The ExPEC isolates belonging to the same phylogenetic group and sequence type could display different resistance and virulence characteristics.

Keywords: 16S rRNA; Escherichia coli; MLST; ST131; Virulence; antibiotic resistance.

Figure 1. Distribution of hospital departments among ExPEC isolates.

The number of departments where ExPEC isolates (n = 97) originated was shown on the pie chart. Newborn-ICU, Newborn- Intensive Care Unit

Figure 2. Distribution of virulence genes among ExPEC isolates.

The bar plot was constructed for 97 ExPEC isolates to illustrate virulence genes distribution. The numbers presented at the top of bars demonstrated the number of virulence genes. PAI: pathogenicity-associated island (PAI) marker (n = 40), ibeA, Invasion of brain endothelium (n = 2); hlyA, hemolysin (n = 15); cnf1, cytotoxic necrotizing factor (n = 7); iutA, Aerobactin siderophore receptor (n = 44); fyuA, Yersiniabactin (n = 49); iroN, Samochelin siderophore receptor (n = 23); papG, P fimbriae (n = 12); sfaS, Sfa fimbriae (n = 2); iss, Episomal increased serum survival; traT, serum resistance (n = 54), (n = 9); kpsII, Capsule synthesis K1 and K5 (n = 13); ompT, Episomal outer membrane protease (n = 13).

Figure 3. Phylogenetic tree of ExPEC blood culture isolates based on 16S rRNA analysis.

The evolutionary history was inferred by using the Maximum Likelihood method based on the Hasegawa–Kishino–Yano model (Hasegawa, Kishino & Yano, 1985). The bootstrap consensus tree inferred from 1,000 replicates (Felsenstein, 1985) is taken to represent the evolutionary history of the taxa analyzed (Felsenstein, 1985). Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. Initial tree(s) for the heuristic search were obtained by applying the Neighbor-Joining method to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach. The analysis involved 111 nucleotide sequences (97 ExPEC isolate sequences and of the 14 related ExPEC strain sequences fetched from the NCBI GenBank database). All positions containing gaps and missing data were eliminated. There were a total of 382 positions in the final dataset. Evolutionary analyses were conducted in MEGA7 (Kumar, Stecher & Tamura, 2016). The symbols shown in the tree indicative for the sub-clusters that were closely related (blue filled circle, green filled circle, and red filled triangle)-(empty blue square and filled yellow square)-(empty lilac circle and aqua filled circle)-(purple filled triangle and lilac filled rhomb)-(empty black triangle)-(empty red circle)-(black filled square)-(red filled square)-(grey filled triangle)-(lime filled triangle)-(dark green filled circle)-(orange filled triangle)-(empty black rhomb)-(black filled circle)-(yellow filled triangle)-(empty orange square)-(empty black square)-(blue filled rhomb)-(empty black circle).