Epidemiología molecular en Escherichia coli procedente de fauna salvajeresistencia antimicrobiana, virulencia y diversidad y diversidad genética stars

  1. Carla Andrea Alonso Arribas
Supervised by:
  1. Carmen Torres Manrique

Defense university: Universidad de La Rioja

Year of defense: 2019

Committee:
  1. José Leiva León Chair
  2. Yolanda Sáenz Domínguez Secretary
  3. Patrícia Alexandra Curado Quintas Dinis Poeta Committee member
Thesis with
  1. Mención internacional
Department:
  1. Agriculture and Food

Type: Thesis

Summary

In 2014, the World Health Organization published the first global report on Surveillance of Antimicrobial resistance. It addressed the urgent need to come up with a coordinated set of strategies to fight the problem in a One Health approach, which contributes to protect the public health through the control of antibiotic resistance at the interface between humans, animals and the environment. According to the World Organisation for Animal Health, about 60% of human pathogens are of animal origin. However, antimicrobial resistance is not limited to bacteria causing infectious diseases, it also affects those non-pathogenic ones occupying diverse ecological niches. Thus, commensal bacteria are also a threat, since they can act as important reservoirs of resistance and virulence genes. This thesis aims at exploring the extent of antimicrobial resistant E. coli in wildlife, the major circulating clones, the mechanisms and genetic platforms involved in the flow of resistance genes between humans, animals, and the environment, and potential wild reservoirs of zoonotic E. coli pathotypes. In the first and second chapters, population structure and antimicrobial resistance of E. coli from wildlife were investigated. A low to moderate frequency of antibiotic resistant strains was observed in mammals (11.1%), predominantly against old agents such as tetracyclines, penicillins and sulfonamides. The prevalence of ESBL/AmpC-producing E. coli was high among birds (16%), with SHV-12 as the main enzyme variant, followed by CTX-M-1, CTX-M- 14 and CMY-2. Eighty-two percent of the ESBLs from wildlife showed a multi-resistant genotype, often in relation to the carriage of class 1 integrons containing long arrays of gene cassettes. Although quinolone resistance was mainly due to chromosomal mutations in GyrA/ParC, a qnrS1 gene was identified in an E. coli from a barn owl. All the ESBL and plasmid-mediated AmpC encoding genes were transferable by conjugation, and we observed some associations like blaCTX-M-1/IncN, blaSHV-12/IncI1 or blaCMY-2/IncI1. Although some ESBL/AmpC-producing E. coli lineages were first described in wildlife (ST4564, ST4954, ST4996, ST7624, ST7629, ST7630, ST7631, ST7632), most of the isolates belonged to clones frequently detected among humans, domestic animals and food (ST131, ST10, ST155, ST224, ST38, ST57), which support the existence of successful lineages strongly associated with the bidirectional dissemination of ESBL/AmpC genes. Cryptic Escherichia clades were identified in various wild mammals, highlighting the detection of a clade V member carrying blaCTX-M-14 and different virulence factors. The third chapter focused on the analysis of distinct genetic elements involved in the selection, persistence and spread of resistance determinants among E. coli from different ecosystems (humans, domestic animals, wildlife, food). The first paper provides insights into the molecular background of plasmids and additional genetic platforms associated with the dissemination of SHV-12 encoding gene. The horizontal transfer of blaSHV-12 was mainly driven by IncI1 plasmids, with the pST3 subtype prevailing in poultry and the pST26 (and other CC26 associated subtypes) being equally distributed among isolates from various origins. The complete sequencing of an IncI1 plasmid revealed the presence of a Tn21-blaSHV-12-ΔTn1721 resistance complex, containing the atypical intI1-estX-psp-aadA2-cmlA1-aadA1-qacI-IS440- sul3 integron and the tet(A) gene, which seems to play an important role in the spread of SHV- 12. Restriction analysis of IncK plasmids suggest the occurrence of horizontal blaSHV-12 events from local non-ST131 isolates to the pandemic ST131 clone. We identified an IncX3 plasmid, not typeable by PBRT, co-harbouring qnrS1 and blaSHV-12 genes, the latter one integrated in a composite transposon structure that could facilitate the en-bloc mobilization of the ESBL. The second paper aimed at gaining knowledge about the location and genetic organization of class 2 integrons in E. coli from different hosts and countries. Although a low diversity of gene cassettes was shown, many novel structures were identified due to the integration of IS at different sites. These IS elements might modulate the expression and movilization of adjacent genes, or even define the genomic location and dissemination capability of class 2 integrons. Most class 2 integrons were chromosomally inserted at the attTn7 site, adjacent to the essential glmS gene. Only a few were transferable by conjugation and the comobilization of BLEE genes was never involved in the process. Preliminary functional assays showed that class 1 integrase was capable of efficiently excising the aadA1 cassette found in the variable region of class 2 integrons. In the fourth chapter, the epidemiological role of wildlife in the maintenance and dissemination of enteropathogenic (EPEC) and Shiga toxin-producing E. coli (STEC) was investigated. Among STEC recovered from wild animals, the stx2b/subAB2/ehxA virulence profile was the most common and 9 isolates belonged to seropathotypes frequently associated with hemolytic uremic syndrome (seropathotypes B -O145:[H28] - and C -O22:H8, O128:[H2] -) or diarrhea (seropathotype D -O110:H28, O146:H21, O146:[H28], ONT:H8-) in humans.We first reported a wild boar as carrier of a bfpA-positive O49:[H10] eae-κ strain of the same characteristics as tEPEC isolated from human diarrhea. Wild ruminants (deer and mouflon), as well as wild boar, act as important reservoirs of potentially pathogenic STEC and EPEC. In the final chapter, we studied by WGS the accessory and core genome of 38 commensal E. coli strains from wildlife. We analyzed the CRISPR/Cas systems as well as the resistance and virulence modules, which show similar genetic structures and organizations than those reported in strains from the human setting. WGS enabled a more consistent characterization of the composition and diversity of the plasmidome, facilitating the study of small replicons, the differentiation of extra-chromosomal phages or phage-like elements and the detection of non-typeable plasmids. Our results suggest an ongoing flow of both mobile elements and E. coli lineages between human and natural ecosystems, rather than the occurrence of a parallel microevolution in the gut of wild animals.