Tese
Fatores de patogenicidade, perfil de resistência a antimicrobianos e diversidade clonal de Staphylococcus aureus isolados de leite cru, soro fermento, manipuladores e queijo Minas artesanal da região de Campo das Vertentes, Brasil
Fecha
2019-04-15Autor
Renata Dias de Castro
Institución
Resumen
Staphylococcus aureus is one of the main pathogens found in cheeses produced with raw milk,
including Minas artisanal cheese from Brazil. However, information on S. aureus isolated from
artisanal cheeses and its sources of production in small-scale dairies is very limited. We aimed to
characterize the virulence aspects and clonal diversity of S. aureus isolated from Minas artisanal
cheese, raw milk, endogenous starter culture and handlers from the region of Campo das
Vertentes, Minas Gerais State, Brazil. The samples were identified by MALDI-TOF MS. Biofilm
production was evaluated on Congo Red Agar (CRA) and polystyrene plates. PCR was used to
detect icaA, icaB, icaC, sea, seb, sec, sed, see, tsst-1, agr and mecA. Expression of staphylococcal
toxin genes in PCR positive samples was evaluated by qRT-PCR and the production of these
toxins as well as hemolytic activity were in vitro evaluated. We also evaluated the antimicrobial
resistance profile of the samples. For statistical analysis, cluster, chi-square test and
correspondence were used. The study of the clonal diversity of the samples was performed by the
spa typing, rep-PCR ((GTG)5) and MALDI-TOF MS techniques. The discriminatory power, the
typability and the concordance between the typing techniques were evaluated. A total of 76
Staphylococcus aureus samples were analyzed. In the PCR, 18.42%, 18.42%, 2.63% and 77.63%
of the samples were positive for sea, tsst-1, sec and agr, respectively. Staphylococcal toxin genes
were of low expression in qRT-PCR and only two samples were TSST-producing. Most of the
samples (61.84%) had hemolytic activity and were biofilm forming in CRA (69.73%) and in
polystyrene plates (81.58%). None of the samples presented mecA neither presented a
multiresistance pattern, with the highest resistance observed for Penicillin G (67.11%) and
Tetracycline (27.63%). The most frequent genotype in the study was t605 (27%), followed by
t002 (10.8%) and t521 (9.5%). Spa typing and (GTG)5 presented high discriminatory power, in
contrast to MALDI-TOF MS, which presented low discriminatory power and low concordance
with typification genomic techniques. In conclusion, the samples presented toxigenic potential,
with a higher prevalence of sea and tsst-1. We found that the biofilm production was the main
virulence factor of the samples. Six clusters were formed whose distribution frequencies differed
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for hemolytic activity, biofilm formation (qualitative and quantitative analyses) and resistance to
Penicillin, Tetracillin and Erythromycin (P <0.05). Staphylococcus aureus samples showed high
genetic diversity, and there are evidences that the presence of this microorganism in the final
product is the result of cross-contamination of raw milk, serum-yeast (pingo) and asymptomatic
manipulators of this bacterial agent. These findings emphasize the need for effective measures to
prevent staphylococcal food poisoning by limiting S. aureus growth and enterotoxin formation, ,
as well as reinforcing the importance of implementing good manufacturing practices to reduce
the risk of transmission of potentially pathogenic bacteria along the cheesemaking chain.