Cepas clínicas de SARM de líneas genéticas asociadas a animalesepidemiología, genómica, proteómica y nuevos antimicrobianos stars

  1. Sara Ceballos Marcaida
Supervised by:
  1. Carmen Torres Manrique
  2. Myriam Zarazaga Chamorro

Defense university: Universidad de La Rioja

Year of defense: 2019

Committee:
  1. Francisco Javier Castillo García Chair
  2. Rosa del Campo Moreno Secretary
  3. Patrícia Alexandra Curado Quintas Dinis Poeta Committee member
Thesis with
  1. Mención internacional
Department:
  1. Agriculture and Food

Type: Thesis

Summary

Staphylococcus aureus is part of the normal microbiota of humans and animals, although it can also be an important opportunistic pathogen. S. aureus frequently acquires antimicrobial resistance mechanisms, being methicillin resistance (MRSA) one of the most relevant. MRSA dissemination is increasing due to its capability of establishing new reservoirs, with three main groups: HA-MRSA (hospital associated), CA-MRSA (community associated) and LA-MRSA (livestock associated). The most important LA-MRSA genetic lineage is the clonal complex CC398, present in farm animals, mostly pigs, as well as in humans in contact with those animals (as colonizers or causing infection). The MRSA CC398 phenotypic marker is tetracycline resistance (TetR). Another emerging LA-MRSA genetic lineage is CC130, generally carrying the methicillin resistance gene mecC, and characteristic for having a non β-lactam susceptible phenotype (NBLS). CC130 has been found in animals (farm, domestic or wildlife animals), and sporadically causing human infections, although its prevalence is unknown. For these reasons, all TetR–MRSA isolates obtained during a six-month period in 2016 (total S. aureus: 11,405; total MRSA: 3,383) from 20 hospitals located in 13 regions with different pig-farming density were analyzed in order to establish the MRSA CC398 prevalence in Spain. In addition, it was determined if pig density could influence in the prevalence of CC398. The global MRSA prevalence in the analyzed hospitals (n=20) was 30% (range 12-71%), and 7% were TetR–MRSA. The MRSA CC398/MRSA rate was 4% (range 0%-31%). The association between pig density and MRSA CC398/ MRSA was corroborated in this study (p<0.001), with higher prevalence in those hospitals located in regions with higher number of pigs/km2. Moreover, regression statistic models were created in order to predict the MRSA CC398/ MRSA ratio depending on pig density. Most of CC398 strains belonged to samples of skin and soft tissue infections and respiratory tract infections (74%), according to the usual means of transmission of this lineage (direct contact and air). The predominant spa-type of CC398 strains was t011 (72%), while 40% of the remaining non-CC398 strains belonged to the t127/CC1 clone, also related to livestock. The human immune evasion cluster (IEC) was present in only two out of the 137 MRSA CC398 strains, suggesting an animal origin for the IEC-negative isolates. The IEC positive CC398 strains could be part of an adaptive process of this clone to humans. The multidrug-resistant phenotypes were frequent in MRSA CC398 strains (79%), with a total absence of virulence mechanisms. The next objective was to determine the MRSA CC130 prevalence in 12 out of the 20 previous hospitals, through the analysis of all NBLS-MRSA strains obtained during a sixmonth period in 2016. This phenotype was infrequent in the analyzed hospitals (range 0.3%-7.7%), obtaining a collection of 45 mecC-negative isolates (all mecA-positive). However, the NBLS phenotype was a good marker for the CA-MRSA t008/agr I clone. In addition, more than a third of these NBLS strains produced one of the most clinically relevant virulence factors of S. aureus, the Panton-Valentine Leucocidin. The need of new therapeutic strategies to fight MRSA led to the analysis of the antimicrobial activity of new synthetic compounds belonging to two antibiotic classes, the oxadiazoles and the quinazolinones. Four oxadiazoles and five quinazolinones were tested in a 210 MRSA strains collection of different genetic lineages and origins, as well as in different staphylococci and enterococci species with various resistance mechanisms. A compound of each class stood out for its good antimicrobial activity indicating a great therapeutic potential against MRSA. Deeper knowledge of the physiology of MRSA is necessary for the development of new therapeutic strategies. Proteomic studies are valuable tools for ascertaining changes in protein expression levels in certain physiological circumstances. To this end, the proteomes of two MRSA mecC strains with the same genetic characteristics, but different origin (human and animal), were analyzed at basal state and after exposure to a stress factor, such as the presence of cefoxitin at subinhibitory concentrations. There were clear differences in the number of proteins expressed in the two-dimensional profile of both strains (higher in the human strain). In this human strain, changes in the proteins involved in bacterial metabolism were observed, as well as in those that are part of responses to stress and pathogenicity, after exposure to the antibiotic cefoxitin. Therefore, MRSA CC398 is a frequent and emergent lineage in Spanish hospitals, especially in areas with high pig density, but not so MRSA CC130 (with mecC mechanism), which to date is very rare in our environment. S. aureus is evolving in the animal-human interface and the strategies of genomics and proteomics are necessary to understand these processes.