Staphylococcus spp. from animals intended for human consumption, animalderived food and humans in Spain and Senegalgenetic lineages, antibioresistance and virulence stars

Abstract

The first antimicrobial resistance global report on surveillance of the World Health Organization (WHO) in 2014 pointed out the need to deal with the problem under a “One Health” approach which focusses on the interactions between humans, animals and their environments, in order to enhance health and welfare. Staphylococcal species (both coagulase-positive (CoPS) and -negative (CoNS)) are normal colonizers of skin and mucous of healthy humans and animals, but also opportunistic pathogens, that can cause skin and soft tissues infections, mastitis, food intoxication, bacteraemia etc. This thesis aimed to analyse the diversity of staphylococcal species in different niches, including wild and farm animals destined for human consumption, animal-derived food and humans, to determine the antimicrobial resistance phenotype/genotype of recovered isolates, and to evaluate the occurrence of livestock-associated lineages among S. aureus isolates. The first chapter studied the molecular epidemiology of staphylococcal species in nasal and/or faecal samples of non-conventional food animals such as wild boar and horses in Spain. A great diversity of staphylococcal species was observed in wild boar, being S. aureus (76.1%) and S. hyicus (16.4%) the predominant CoPS, and S. sciuri (39.7%) and S. xylosus (13%) the predominant CoNS. In horses, the predominant species were S. aureus (37%), S. delphini (21%) and S. sciuri (21%). Most isolates recovered from wild boar were pan susceptible (73.1% of CoPS and 77.6% of CoNS). However unusual genes involved in macrolide, lincosamide and phenicol resistance were detected among CoNS. Regarding horses’ isolates, 88.7% of CoPS were pan susceptible while 48.6% of CoNS showed resistance to at least one of the antimicrobial agents. A multidrug resistance phenotype (MDR) was only observed among CoNS from both animal species. The predominant S. aureus lineage in wild boar was t3750-ST2328/CC133 (29.4%) and one isolate of the livestock-associated methicillin resistant S. aureus lineage (LA-MRSACC398) (t011-ST398) was identified. S. aureus lineage ST1640 (61.7% of S. aureus), is associated with horses for the first time in this study. All S. aureus isolates detected from horses, except those of lineage ST1460, carried the genes encoding the equid-adapted leukocidin (LukPQ) and the blocker of equine complement system activation (eqSCIN). The second chapter focussed on staphylococci from common food-producing animals from two countries with different antibiotic use policy (calve, lamb, goat (Spain); cow and chicken (Senegal)). In Spain, the prevalence of staphylococci was higher in lamb (54%) and goat (50%) than in cow (21%) samples. In Senegal, staphylococci prevalence was higher in cow (26.8%) than in chicken (3%). In Spain, the most frequent lineage was CC133 while the ST291 was dominant in Senegal isolates. Six species were identified among CoNS in each country, with prevalent species being S. sciuri and S. simulans. CoPS isolates from Spain were mostly pan susceptible, while those from Senegal were mostly penicillin resistant. In both countries, a relatively high percentage of CoNS isolates (32.5% (Senegal), 47% (Spain)) was resistant to at least one of the antimicrobials tested. Resistance to penicillin, tetracycline and SXT was observed among CoNS isolates of both countries. Additionally, resistance to methicillin, streptomycin, clindamycin, erythromycin, chloramphenicol and tobramycin was observed among Spain isolates. The Panton-Valentine Leukocidin (PVL) encoding genes were present in isolates of both countries, and the human virulent clone USA300 was identified among Spain isolates. In the chapters three and four, the lineage CC398 was screened for pig-derived food S. aureus isolates on one hand, and among patients’ blood culture S. aureus isolates from Spanish hospitals located in regions with different pig density degrees, on the other hand (multicentre study). CC398 was the predominant lineage among S. aureus isolates of pig products (64.1%; n=23). The prevalence of MRSA-CC398 was 20.8%, observing differences in products with skin (76.5% (ear/snout)) and without skin (3.3% (fillet), 14.8% (chopped meat)); this clone was mostly typed as t011. All MRSA-CC398 isolates were mecA-positive, tetracycline resistant (tet(M) and tet(K)+/- tet(L)) and scn-negative. 82.6% were MDR and 17.4% harboured virulence genes. Two methicillin susceptible S. aureus (MSSA)-CC398 isolates were detected; both being resistant to penicillin and erythromycin/clindamycininducible (erm(T) gene). They were typed as t5452 and were scn-positive. Concerning the clinical isolates, the prevalence of CC398 isolates among S. aureus was 4.3%. MSSA-CC398 represented 90.2% of total CC398 isolates, corresponding to 5.2% of MSSA and 3.9% of S. aureus. t571 and t1451 were the predominant spa-types among MSSA-CC398 isolates. Resistance to erythromycin/clindamycin-inducible, mediated by erm(T) gene, was only observed among MSSA-CC398 isolates (72.9%). All MSSA-CC398 isolates, but three, were scn-positive. No obvious correlation was observed between MSSA-CC398 prevalence at hospital level and the pig density degree in surrounding areas. LA-MRSA-CC398 and LA-MRSA-CC1 were detected in hospitals located in high pig density areas.