Evolutionary and dissemination aspects that explain the current architecture of class 2 integrons

MASSÓ, MG; ALONSO, CA; ALVAREZ, VA; PIEKAR, MARÍA; TORRES, C; QUIROGA MARÍA PAULA; RAMIREZ MS; CENTRON D

Viena

Congreso; 27th European Congress of Clinical Microbiology and Infectious Diseases (ECCMID); 2017

Background: Class1 and 2 integrons are associated with multidrug-resistance due to theircapability to mobilize and express multiple gene cassettes (GC). Class 2integrons usually carry in the variable region (VR) the dfrA1-sat2-aadA1 array, followed by a putative GC called orfX (ybeA, unknown function) and three additional genes (ybfA, ybfB, ybgA), embedded in the Tn7 transposition module (TnsEDCBA). The intI2 is frequently interrupted by a prematurestop codon, rendering a non-functional integrase. Here, we characterized thegenetic location and organization of class 2 integrons carried by E. coli isolates from Spain, Tunisia andMexico. Material/methods:Class 2 integrons of 34 E. coli isolates from different origins (15 poultry meat, 6 chickens,4 wild animals, 5 pets, 5 humans) were fully characterized. E. coli from wild animals (gull, stork,genet, deer), pets (dogs, turtles) and chickens were recovered from faeces. Human-sourcestrains were isolated from hemocultures (n=2) and healthy donors? faeces (n=3).PCR cartography was performed with different primer combinations to determinethe VR and the presence of tns genes.In integrons lacking ybeA-ybfA-ybfB-ybgA and transposition module, the 3?ends of classic and unusual class 1 integrons were screened. Newly designedprimers were used to determine novel arrangements. Integron?s location at Tn7 transposon and its chromosomal insertion was confirmed by PCR. The intI2 gene was sequenced in integronsshowing structures different to the classical one. Clonal relatedness wasdetermined by MLST and/or OD-PCR.  Results:Diversity of the VR among the studied integronsincluded: a) The classical GC arrangement (dfrA1-sat2-aadA1-ybeA-ybfA-ybfB-ybgA)(n=27), b) Excisions/substitutions of common GCs (sat2-aadA1-ybeA-ybfA-ybfB-ybgA,n=1; estX-sat2-aadA1-ybeA-ybfA-ybfB-ybgA,n=2), c) IS-element insertions (IS10-dfrA1-sat2-∆aadA1-ybeA-ybfA-ybfB-ybgA,n=1; dfrA1-sat2-aadA1-ybeA-ISSen4-ybfA-ybfB-ybgA,n=2), d) Absence of the ybeA-ybfA-ybfB-ybgA module (dfrA1-sat2-aadA1). Another feature of this last integron, detected in turtle?s faeces,was the lack of tns genes. Although novelrearrangements and the occurrence of hybrid class2/class1 integrons were tested,none of the PCRs were successful. Among integrons harboring the classicalarrangement, mosaic structures of the tnsmodule were detected. The integron containing an ISSen4-element (IS3family), frequently identified next to blaNDM-1/blaKPC genes, has been firstdescribed here. It was found in two E.coli from Mexico, assigned to ST57 and showing indistinguishable OD-PCR-pattern,but from distinct sources (dog/meat). Ninety-one percent of the integrons wereembedded in Tn7 transposons chromosomallylocated adjacent to glmS gene. Conclusions:Genetic background of class 2 integrons is highlyconserved among E. coli fromdifferent ecosystems, being novel arrangements mainly driven by the insertionof IS-elements or the activity of a functional integrase. We described a novel class2 integron carrying an ISSen4 in two ST57E. coli from Mexico. Also, the novelstructures evidenced how class 2 integrons evolved by the activity of integronintegrases and invasions of a wide variety of transposons and insertionsequences.