EST. DA INTERACÇÃO ENTRE A BACTÉRIA MULTIRRESISTENTE STAPHYLOCOCCUS PSEUDINTERMEDIUS E O HOSPEDEIRO CANINO NUM MODELO EX VIVO CUTÂNEO

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Insights into the dynamics of methicillin-resistant staphylococci in animals : a focus on Staphylococcus pseudintermedius in dogs

Publication . Couto, Natacha; Pomba, Maria Constança Matias Ferreira; Coelho, Ana Maria de Jesus Bispo Varela

Staphylococci are a group of bacteria with clinical, agricultural, and economic importance because of their wide range of virulence factors and ability to become resistant to antimicrobials. This thesis has pursued three main objectives: I. Determine the frequency of methicillin-resistant S. aureus (MRSA) strains in several animal species, identify the characteristics of strains present in animals and comparison with human strains MRSA nasal screening was performed in 71 horses and 307 calves, and the observed frequencies were 3% and 2%, respectively. Seventy-four MRSA isolated from 2001 to 2014 were characterized: fourteen spa types, three SCCmec types and three clonal complexes (CC) 5, CC22 and CC398, were found. Most isolates were multidrug-resistant. Fourteen MRSA CC398 strains had qac genes (13 qacG and 1 qacJ), while 4 isolates (three CC5 and one CC22) had insertions in the norA promoter gene. MRSA linages from pets (CC5 and CC22) harboured specific sets of virulence genes and a lower number of resistance genes than CC398 from livestock-animals. II. Reveal antimicrobial/biocide susceptibility patterns/trends and resistance genes in methicillin-resistant staphylococci (MRS) Several antimicrobial resistance patterns and genes were found in MRS from horses. Minimum bactericidal concentrations of biocides chlorhexidine acetate, benzalkonium chloride, triclosan and glutaraldehyde were lower than the recommended in-use concentrations for veterinary medicine, although two MRS carried plasmid-borne qacA and sh-fabI or qacB and qacH-like genes. An investigation on the evolution of resistance to 38 antimicrobials, corresponding mechanisms and molecular characteristics of 644 clinical Staphylococcus spp. isolates obtained from companion animals between 1999-2014 revealed resistance to the majority of antimicrobials and the number of mecA-positive strains increased significantly over time. Considering S. pseudintermedius, the methicillin-susceptible (MSSP) were genetically more diverse than methicillin-resistant (MRSP). All MRSP and two MSSP strains were multidrug- resistant, with several antimicrobial resistance genes identified. One MSSP isolate harbored a qacA and another a qacB gene. Three biocide products had high bactericidal activity (Otodine®, Clorexyderm Spot Gel®, Dermocanis Piocure-M®), while Skingel® failed to achieve a five log reduction in the bacterial counting. III. Study of the pathogenesis of S. pseudintermedius in dogs The agr type III predominated in MRSP. Five virulence genes were found in all strains and only spsO gene was significantly associated with MSSP. MSSP produced more biofilm on BHIB and BHIB+1% glucose than MRSP isolates. Several virulence genes encoding surface proteins and toxins were highly expressed in the MRSP strain (compared to MSSP). By whole proteome characterization of S. pseudintermedius through 2DE MALDI-TOF/TOF MS approach we were able to identify 367 unique proteins, of which 39 were surface proteins. By subsequent use of the serological proteome analysis (SERPA) approach we identified 4 antigenic proteins with promising features for vaccine development. These results indicate that MRS were widely disseminated in the studied animal population, the environment and people in contact with these animals. The resistant trends and mechanisms detected in MRS strains are worrying and make animals a reservoir of important MRS clones and genes. Biocides are still a good therapeutic choice, even in the presence of efflux genes. Higher expression of virulence genes may play a role in the rapid and widespread of MRSP clones. Dogs are able to mount an IgG-response against S. pseudintermedius and the proteins identified by the immune system can in the future be used as vaccine candidates.

2016-01-13Doctoral thesis

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