Staphylococcus aureus en animales de vida libre y medioambiente. Resistencia a antimicrobianos, virulencia, líneas genéticas circulantes y genómica comparativa stars

  1. Paula Gómez Villaescusa
Supervised by:
  1. Carmen Torres Manrique
  2. Myriam Zarazaga Chamorro

Defense university: Universidad de La Rioja

Year of defense: 2019

Committee:
  1. Fernanda Ruiz Larrea Chair
  2. María Isabel Morosini Secretary
  3. L. Pallecchi Committee member
Thesis with
  1. Mención internacional
Department:
  1. Agriculture and Food

Type: Thesis

Summary

Staphylococcus spp., especially S. aureus species, are part of the normal microbiota of healthy humans and animals, but it can also be opportunistic pathogens. Methicillin-resistant S. aureus (MRSA) is a serious health problem since it shows resistance to almost all β-lactam antibiotics. In 2005, for the first time, a genetic MRSA clonal complex CC398 was detected associated with livestock (MRSA-LA), whose main reservoir are pigs. Since then, it has been observed a large dissemination of this genetic lineage, as well as the transmission to people in direct or indirect contact with farm animals. In 2011, a new mec gene (mecC) was described associated with genetic lineages of S. aureus adapted to animals, especially CC130. MRSA-mecC is an emerging problem, detected in production and free-living animals, and also sporadically in people. The human, animal, and environmental ecosystems, are interconnected allowing a flow of bacteria and genes between them. The study of bacterial molecular epidemiology requires a global and integrating approach (one world, one health), such as the one proposed in this thesis. Environmental samples [surface water, treated and untreated sewage water from waste water treatment plants (WWTP)], and free-living animals [small mammals, red deer and white storks] have been studied for the isolation and characterization of Staphylococcus. Likewise, using the whole genome sequencing, a comparison of CC130 strains of different origins and of CC398 strains was made, both clonal complexes being emerging in the animal-human interface. A high variability of species was detected in wastewater and surface waters: S. aureus, S. equorum, S. vitulinis, S. lentus and S. sciuri being the most abundant in WWTP waters, and S. aureus, S. epidermidis, S. vitulinis, S. sciuri and S. fleurettii the most frequent ones in surface waters. A MRSA-ST398-t011 strain which with a multiresistant phenotype (scn-negative) was detected in treated wastewater. The remanding S. aureus strains from WWTP were susceptible to methicillin but some of them contained numerous enterotoxin genes. In surface waters, no MRSA strains were detected, and the isolated S. aureus belonged to CCs associated with humans (CC5) or adapted to animals (CC130, CC133). The 21.8% of coagulase-negative Staphylococcus (CoNS) isolated from the WWTP contained the mecA gene, and 15.6% presented a multiresistance antimicrobial phenotype. These percentages raised to 30.5% and 23.7%, respectively, in CoNS of surface waters. Coagulase positive Staphylococcus (CoPS) strains of free-living animals were characterized, and detected prevalences for S. aureus were as follows: small mammals (13%), red deer (24.6%), and white storks (34.8%); a big difference was detected in storks, depending on where they were fed: areas with human influence (55.8%) or natural areas (16.3%). Moreover, 2% of small mammals and 17% of deers animal samples obtained from the same farm) were carriers of MRSA strains, all of them mecC-positive, typed as ST1945 and carrying genes of IEC system (type E). This is the first description of MRSA-mecC-IEC-positive strains. To date, no IEC-positive strain belonging to CC130 has been described. The similarity of these strains in the analysis carried out suggests an animal-animal transmission. The remaining S. aureus of red deer and small mammals were susceptible to methicillin and to the other antimicrobials tested, being described as CC5 and CC1956 (in small mammals) and CC133 (in red deer). On the other hand, the clonal diversity of S. aureus isolated from white storks was very high, highlighting the detection of three MRSA strains, one mecC-IEC-negative (ST3061-CC130), and two mecA-positive (one CC5 and another CC398-LA). Among the MSSA strains, CC398 lineage with different spa-types was detected (between them: t571, IEC type C, and erm(T)-positive, associated with invasive infections in humans). Other lineages detected were: CC5, CC7, CC22, CC30, CC45, CC59 and CC133. The analysis of the complete genome sequences of 18 S. aureus isolated from multiple animal species belonging to 6 sequences type ascribed to CC130 (14 MRSA and 4 MSSA) allowed to establish possible phylogeny relationships. The obtained results show that, except for the ST700 lineage, the core genome did not present differential characteristics that allowed to group the strains according to the animal species or the geographical origin area, although there seems to be a certain clustering depending on the methicillin resistance phenotype and the sequence type. The study of comparative genomics of two strains ST398-t011, one MSSA and another MRSA, isolated from a swine driving men, showed that both belonged to the animal clade and that they had a very high clonal relationship. The results obtained suggest the evolutionary change from MRSA to MSSA due to the loss of the mecA gene (associated with the loss of a 17342pb fragment), probably due to the recombination of putative primase which caused a SCCmec remnant.