Microbiota do queijo Minas Frescal: diversidade bacteriana e resistência aos antimicrobianos

Abstract

The cheese microbiota is highly diverse, and its characterization may provide knowledge about microbiological quality and sanitary safety issues. Of special interest concerns are rising on the role of cheeses as reservoir of bacteria resistant to antimicrobial drugs as the resistance phenomena gain visibility in the One Health approach. Considering economic and cultural relevance of Minas Frescal Cheese (MFC), the aim of this study was to elucidate the identity of dominant bacterial populations in its microbiota through high-throughput sequencing, and to investigate the involvement of MFC in the ecology of antimicrobial drug-resistance. For microbiota sequencing, the workflow consisted of sample processing, DNA extraction, 16S rRNA amplicon library preparation, sequencing on the Illumina MiSeq platform and bioinformatics data analysis. In a second moment, antimicrobial susceptibility patters were evaluated for Gram-negative rods isolated in QMF samples on MacConkey Agar. DNA extraction and class 1 integron screening using the polymerase chain reaction (PCR) technique were performed for isolates that showed resistance to any of the antimicrobials tested. Three non-fermenting Gram negative (NFGN) rods were selected, based on the relevance of their resistance phenotype and presence of class1 integrons, for whole genome sequencing. In high-throughput sequencing analsys, it was observed that cheeses were brand-clustered, exhibiting unique bacterial profiles which varied in diversity and abundance of taxa. Proteobacteria dominated in all samples, corresponding to a mean of 88.7% from all sequences. Bacteria groups associated with low sanitary quality such as, as Pseudomonas, Acinetobacter and Vibrio were highly frequent in most of brands. The core microbiome shared by 100% of the samples showed predominance of enterobacteria. The susceptibility profile of the recovered isolates demonstrated that, among enterobacteria, 37% were resistant to ampicillin, 10% to cefoxitin and 16% to amoxicillin. Among NFGN rods, 76% were resistant to ampicillin, 71% to sulfazotrim and 6% to ceftazidime. The class 1 integron gene was detected in three NFGN rods resistant to ampicillin and sulfazotrim, of which two belonged to the same strain. The isolates selected for whole genome sequencing were identified as Acinetobacter ursingii, Pseudomonas juntendi and Pseudomonas ssp. Preliminary analyzes indicated the presence of genes associated to efflux pumps and resistance to aminoglycosides in the genomes. In general, theses data highlight to a concern regarding the occurrence of opportunistic pathogens in QMF, which can act as hosts for antimicrobial resistance genes, posing an additional risk for consumers.