Epidemiología molecular de Staphylococcus spp. desde un enfoque One Healthgenes emergentes e inusuales de resistencia a antibióticos y de virulencia

Abstract

The human, animal and environmental health are interconnected, since all of them can contribute to the emergence, evolution and spread of antibiotic resistance. Hence, this problem must be addressed through a One Health approach. Staphylococcus spp., both coagulase-positive (CoPS) and coagulase-negative (CoNS), are commensal bacteria of the skin and mucous membranes of healthy humans and animals, but they can also act as opportunistic pathogens, causing diseases of diverse severity. The objective of this thesis was to analyze the frequency and diversity of species, and to perform the molecular characterization of Staphylococcus spp. of diverse origins, including animals and the environment, with special attention to emergent and unusual antibiotic resistance and virulence genes. Staphylococcus spp. isolates recovered from tracheal samples of 242 wild birds were characterized. SCoP were recovered from 8.3% of wild birds tested, and the isolates were identified as S. aureus (n=9) and S. delphini (n=12). Most of S. aureus isolates (n=7) were methicillin-resistant (MRSA), mecC-positive and belonged to the lineage CC130, but a mecA-carrying CC398 isolate was also identified. The virulence gene etd2 was present in all MRSA-mecC isolates, and the human immune evasion cluster (IEC) type E was identified in two of them. S. delphini isolates were methicillin-susceptible but showed resistance to at least one of the antimicrobials tested, with high penicillin (75%) and tetracycline (58%) resistance rates. All S. delphini isolates carried the virulence genes lukS-I, siet and se-int. The 60% of birds tested were CoNS-carriers, and 173 isolates were recovered, which belonged to 11 different species with predominance of S. sciuri (68.2%) and S. lentus (14.5%). The 34% of CoNS showed a multidrug resistance phenotype, and 42 mecA-carrying methicillin-resistant CoNS (MRCoNS) were detected. Two S. sciuri isolates harbored the virulence gene tst. Seventy-two staphylococci recovered from environmental samples (air and liquid manure tank) of two pig farms, with intensive production (IP) and semiextensive production (SEP), were analyzed. Four MRSA and MSSA (methicillin-susceptible S. aureus) isolates of lineage CC398 were recovered from the air of the PI farm. Regarding CoNS, multidrug resistance was observed in 59 of the 68 isolates (80%), and eight mecAMRCoNS isolates were identified. The rates of resistance for five groups of antimicrobial agents were significatively higher in CoNS isolates recovered from the IP farm in relation to those of SEP farm. One S. simulans isolate carrying the genes encoding the Panton-Valentine leukocidin (PVL) and three S. cohnii harboring the virulence gene eta were identified. Two S. equorum or S. arlettae isolates recovered from the IP farm showed linezolid resistance and carried the cfr gene. The genetic lineages and the antibiotic resistance and virulence genes of 56 CoPS recovered from animals with signs of infection (pigs and companion animals), were investigated. Among the isolates recovered from diseased pigs (23 S. aureus), a great prevalence of MRSA CC398 was detected, but other livestock associated genetic lineages, such as MRSA CC9, were also identified, most of them (91%) multiresistant isolates. One MRSA CC398 isolate of porcine origin showed linezolid resistance mediated by the cfr gene, which was co-located with the fexA gene in a Tn558 variant. Among the isolates recovered from companion animals (five S. aureus and 28 S. pseudintermedius), nine methicillinresistant isolates (27%) carrying the mecA gene were detected, and the eight methicillin-resistant S. pseudintermedius (MRSP), belonged to the epidemic clones ST71, ST68 and ST258. The S. pseudintermedius isolates showed high penicillin, tetracycline and trimethoprimsulfamethoxazole resistance rates (>50%), and all of them carried the siet, se-int and lukS/F-I virulence genes. Among the S. aureus isolates, a MRSA isolate ascribed to CC398 and MSSA isolates of human-associated genetic lineages (CC22 and CC5) carrying the PVL and IEC encoding genes were detected; a t10576-ST1660 SASM isolate of potential equine origin, harboring the lukPQ and scneq genes, was also identified. Lastly, a comparative study on the mechanisms of linezolid resistance among the animal and environmental isolates, and those recovered from clinical samples of different Spanish hospitals, was performed. The cfr gene was detected in two clinical mecA-MRSA isolates: 1) one MRSA CC398 isolate, recovered from the nasal sample of a pig farmer, with the cfr and fexA genes located in a Tn558 variant; 2) one MRSA isolate, from a pharyngeal sample, with the cfr gene identified together with the fexA gene in a conjugative 38864 bp plasmid. In the remaining clinical isolates (one MSSA and 17 CoNS), linezolid resistance was mediated by point mutations in the rRNA 23S (G2576T or C2534T) and/or amino acid changes in the ribosomal proteins L3 and L4. This thesis brings a One Health approach in order to understand the population dynamics of Staphylococcus spp., the antibiotic resistance and virulence genes flow, as well as the interaction among the bacteria of this genus in the human, animal and environmental settings.