Adherent/invasive Escherichia coli (AIEC) isolates from asymptomatic people: new E. coli ST131 O25:H4/H30-Rx virotypes

Barrios‑Villa et al. Ann Clin Microbiol Antimicrob https://doi.org/10.1186/s12941-018-0295-4 (2018) 17:42 Annals of Clinical Microbiology and Antimicrobials Open Access RESEARCH Adherent/invasive Escherichia coli (AIEC) isolates from asymptomatic people: new E. coli ST131 O25:H4/H30‑Rx virotypes Edwin Barrios‑Villa1, Gerardo Cortés‑Cortés1, Patricia Lozano‑Zaraín1, Margarita María de la Paz Arenas‑Hernández1, Claudia Fabiola Martínez de la Peña1, Ygnacio Martínez‑Laguna1, Carmen Torres2 and Rosa del Carmen Rocha‑Gracia1* Abstract Background: The widespread Escherichia coli clone ST131 implicated in multidrug-resistant infections has been recently reported, the majority belonging to O25:H4 serotype and classified into five main virotypes in accordance with the virulence genes carried. Methods: Pathogenicity Islands I and II (PAI-I and PAI-II) were determined using conventional PCR protocols from a set of four E. coli C TXR ST131 O25:H4/H30-Rx strains collected from healthy donors’ stool. The virulence genes pat‑ terns were also analyzed and compared them with the virotypes reported previously; then adherence, invasion, macrophage survival and biofilm formation assays were evaluated and AIEC pathotype genetic determinants were investigated. Findings: Non-reported virulence patterns were found in our isolates, two of them carried satA, papA, papGII genes and the two-remaining isolates carried cnfI, iroN, satA, papA, papGII genes, and none of them belonged to classical ST131 virotypes, suggesting an endemic distribution of virulence genes and two new virotypes. The presence of PAI-I and PAI-II of Uropathogenic E. coli was determined in three of the four strains, furthermore adherence and invasion assays demonstrated higher degrees of attachment/invasion compared with the control strains. We also amplified intI1, insA and insB genes in all four samples. Interpretation: The results indicate that these strains own non-reported virotypes suggesting endemic distribution of virulence genes, our four strains also belong to an AIEC pathotype, being this the first report of AIEC in México and the association of AIEC with healthy donors. Keywords: AIEC, IBD, Crohn’s disease, Virotype Background Escherichia coli is one of the predominant Gram negative bacterial species of the intestinal microbiota. It mainly colonizes the gastrointestinal tract but also extra intestinal environments. Among E. coli strains there are some considered pathogens and others pathobionts, this *Correspondence: 1 Benemérita Universidad Autónoma de Puebla, Posgrado en Ciencias Microbiológicas, Centro de Investigaciones en Ciencias Microbiológicas, Instituto de Ciencias, Puebla, Mexico Full list of author information is available at the end of the article depending on the virulence factors that they expressed. E. coli genetic variability is caused mostly by horizontal gene transfer, acquiring virulence factors and antibiotic resistance genes. This acquisition is mediated by mobile genetic elements (MGEs), such as transposons, plasmids, bacteriophages and Pathogenicity Islands (PAI) [1]. These elements enhance bacterial capacity to survive in the host environment and to adapt to it. A well accepted infectious disease paradigm indicates that the development of antibiotic resistance allows susceptible species to overtake resistant species [1]. Nevertheless, the increasing evidence of the rising threat of antibiotic resistant © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Barrios‑Villa et al. Ann Clin Microbiol Antimicrob (2018) 17:42 bacteria suggests that the evolution of resistance may be more associated with a fitness advantage, including enhanced virulence [2, 3]. Extended-spectrum β-lactamases (ESBL) are enzymes that hydrolyze penicillins by disruption of β-lactam ring and also third generation cephalosporins [4]. The largest group of ESBL are the cefotaximases (CTX-Ms), which have become globally disseminated, being blaCTX-M-15 and blaCTX-M-14 the predominant genotypes. This group of ESBL restricts treatment options, increasing the use of carbapenems, and leading to the emergence and spread of carbapenemase-producing Enterobacteriaceae [5, 6]. There is an increasing prevalence of β-lactamase resistance due to ESBL, particularly the presence of CTX-M enzymes, and associated fluoroquinolone resistance in MGEs in Extra-Intestinal Pathogenic Escherichia coli (ExPEC), being a serious global clinical problem during the last decade [7]. The Type I fimbrial adhesin FimH has been associated with E. coli pathogenicity because some fimH variants enhance uroepithelial colonization [8]. Furthermore, the fimH-30 variant has been linked with high fluoroquinolone resistance levels simultaneously with ESBL CTXM-15 production (H30-Rx) [9, 10]. Additionally, E. coli isolates can be classified using the multilocus sequence typing (MLST) technique, sequencing seven housekeeping genes (adk, fumC, gyrB, icd, mdh, purA, recA). Using the MLST scheme, ST131 clones have been classified, which have been identified worldwide spread [11, 12]. Increasing prevalence of antibiotic resistance and ESBL CTX-M-15 production in UPEC strains has been linked to this sequence type [13–18]. Clermont et al. [19], established a method based on multiplex PCR for chuA, yjaA, TspE4.C2, arpA and trpA genes, classifying E. coli strains into seven phylogroups and one clade. E. coli ST131 strains belong to phylogenetic group B2 in subgroup I, [19, 20] and they belong mostly to the O25:H4 serotype, although some strains have been found to be O16:H5 serotype [21–27]. It is well known that B2 strains harbor several virulence factors and there is a scheme that classifies the E. coli ST131 into five virotypes (A to E). These virotypes depend on the presence or absence of pap (adhesin-encoding P fimbriae), cnfI (cytotoxic necrotizing factor), sat (secreted autotransporter toxin), kpsMII (group 2 capsule synthesis), iroN (catecholate siderophore receptor), afa/draBC (Afa/Dr adhesins), ibeA (invasion of brain endothelium), hlyA (alpha-hemolysin) and cdtB (cytolethal distending toxin) genes (Table 3). This scheme has been useful to infer virulence in strains isolated worldwide and to determine intercontinental spread [11, 14, 16, 28, 29]. ST131 strains have been linked with community- and hospital-acquired urinary tract infections (cystitis and Page 2 of 11 pyelonephritis) worldwide, but also have been reported (...truncated)