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Base teórica: A Fibrose Cística (FC) é uma doença genética que acomete diversos órgãos, entretanto, as infecções crônicas das vias aéreas são uma das principais comorbidades que afetam estes indivíduos. A determinação do perfil microbiológico destas infecções crônicas é realizada através de técnicas tradicionais de cultivo. Dentre as bactérias comumente isoladas nas técnicas de cultivo predominam de forma mais significativa a Pseudomonas aeruginosa, o Staphylococcus aureus e espécies do complexo Burkholderia cepacia. Essas bactérias provocam infecções persistentes no pulmão do paciente com FC e são de difícil erradicação. Com base em tecnologias moleculares, sugere-se que há uma diferença na diversidade bacteriana nas vias aéreas e a qual pode estar associada a uma melhor evolução clínica da doença. As metodologias moleculares independem do cultivo bacteriano em meios de cultura e tem como base o sequenciamento do gene 16S rRNA que é bastante conservado em bactérias e permite identificar diferentes espécies. O sequenciamento do gene 16S rRNA tem a potencial capacidade de apresentar maior sensibilidade que as técnicas de cultivo tradicional, o que pode antecipar a detecção de um patógeno ou indicar a persistência deste no pulmão de pacientes com FC. Objetivo: Determinar o microbioma de pacientes com FC em amostras de escarro, afim de correlacionar a microbiota de pacientes jovens com o resultado da cultura bacteriológica tradicional, mutações em CFTR e presença de leucócitos no escarro. Métodos: Amostras de escarros de pacientes com FC foram coletadas para dois estudos distintos. No primeiro estudo uma amostra de escarro de 27 pacientes diferentes foram obtidos e, em um segundo estudo, foi determinado a presença do gênero Burkholderia através do microbioma em 22 amostras de escarros de 9 pacientes diferentes. Para ambos os trabalhos a biblioteca do 16S rRNA foi preparada usando a região V3V4 do gene e o produto de amplificação foi submetido a sequenciamento usando Illumina MiSeq. A determinação dos táxons presente na amostra foi realizado por Amplicon Sequence Variants (ASV). Resultado: Em relação ao primeiro estudo, a análise do microbioma detectou os gêneros Staphylococcus e Pseudomonas em todas as amostras de escarro. Na técnica de cultivo tradicional a espécie mais prevalente foi Staphylococcus aureus presente em 70,4% (19/27) das amostras de escarros analisadas, seguida por Pseudomonas aeruginosa em 33,3% (9/27) e complexo Burkholderia cepacia em 29,63% (8/27). Foi possível observar pelo microbioma uma diminuição significativa da diversidade bacteriana nas amostras de escarro com presença de leucócito e o mesmo ocorreu nas amostras oriundas de pacientes internados. Entretanto não foi observado variações estatisticamente significativas entre os microbiomas de pacientes com diferentes mutações no gene CFTR. No segundo estudo, através do microbioma foi possível detectar o gênero Burkholderia em pelo menos uma amostra dos 9 (100%) pacientes analisados, o que representou 63,64% (14/22) dos escarros analisados. Em contrapartida, na cultura bacteriológica foi identificada a Bcc em apenas 4 amostras de escarro de 3 diferentes pacientes. Cabe mencionar que os estudos acima foram os primeiros a determinar o microbioma pulmonar de pacientes com FC através da análise por ASV. Conclusão: Os resultados de ambos estudos indicaram que o microbioma pôde antecipar a detecção de gêneros de importância clínica (Pseudomonas, Staphylococcus e Burkholderia) em comparação com técnicas de cultivo laboratorial, e, com isso contribuir para um tratamento mais precoce. Além disso, a menor diversidade do microbioma pode ser relacionada a aumento do processo inflamatório (presença de leucócitos) bem como a necessidade de internação hospitalar. Backgorund: Cystic Fibrosis (CF) is a genetic disease that affects different organs; however, the chronic airway infection is the main comorbidity of these individuals. The evaluation of the microbiological profile of these chronic infections is usually performed by bacteriological culture. Among the bacteria commonly isolated by culture-dependent technique, the main species are Pseudomonas aeruginosa; Staphylococcus aureus and members of Burkholderia cepacia complex (Bcc). These bacteria cause persistent infections in the lung of CF patients which are difficult to eradicate. Based on molecular technologies, it has been suggested that there is a difference in the bacterial diversity from the airways which may be associated with a better prognosis of the disease. These molecular methods are independent of bacteriological culture as they are based on sequencing of the 16S rRNA gene, which is highly conserved in bacteria and allows to differentiate the species. The 16S rRNA sequencing has the potential to be more sensitive than bacteriological culture; and it may anticipate the detection of a pathogen or indicate its persistence in the lung of patients with CF. Objective: To evaluate the microbiome in the respiratory tract of patients with CF in order to correlate the airways microbiota of young patients with the result of bacteriological culture; CFTR mutations and the presence of leukocytes in sputum. Methods: Sputum specimens from patients with CF were collected for two different studies. For the first study, 27 sputa from different patients were obtained; for the second study, the presence of the Burkholderia genus was determined in the microbiome of 22 sputum specimens from 9 different patients. For both studies, library of 16S rRNA was prepared according to a standard protocol using a V3V4 region; the product of amplification was submitted to sequencing into Illumina MiSeq. The determination of the taxa in the specimens was performed using the amplicon sequence variants (ASV). Results: In relation to the first study, microbiome was capable to detect the genera Staphylococcus and Pseudomonas in all sputum samples. In the traditional culture technique, the most prevalent species were Staphylococcus aureus present in 70.4% (19/27) of the sputum specimens analyzed, followed by Pseudomonas aeruginosa in 33.3% (9/27) and Burkholderia cepacia complex in 29, 63% (8/27). It was possible to observe, by microbiome analysis, a significant decrease in bacterial diversity in sputum specimens; lower alpha diversity was directly correlated to the presence of leukocytes and hospitalized patients. There was no significant difference in alpha diversity and CFTR mutations. In the second study, according to the microbiome analysis, it was possible to detect the genus Burkholderia in at least one specimen of all 9 (100%) patients; which represented 63.64% (14/22) of the sputum analyzed. Conversely, the bacteriological culture was capable to detect Bcc in only 4 sputa (18.2%) from 3 different patients. Noteworthy, the studies above were the first to determine the lung microbiome of patients with CF using ASV analysis. Conclusion: The results of both studies indicates that the microbiome could anticipate the detection of clinically important genera (Pseudomonas, Staphylococcus e Burkholderia) compared to bacteriological culture results, and this may contribute to earlier treatment. In addition, the lower alpha diversity may be related to an increase in the inflammatory process (presence of leukocytes) and with hospitalization status.

Routine evaluation of the slaughter process is performed by the enumeration of the aerobic colony count, Enterobacteriaceae and Salmonella spp. on the carcass through destructive or non-destructive methods. With non-destructive methods, bacteria are counted from a minimum area of 100 cm2 in different sampling sites on the pork carcasses, and the results of these investigated areas are pooled to one value for the complete carcass evaluation (a total of 400 cm2). However, the composition of the bacterial community present on the different sampling areas remains unknown. The aim of the study was to characterize the microbial population present on four areas (ham, back, jowl and belly) of eight pork carcasses belonging to two different slaughterhouses through culture-dependent (Matrix-assisted laser desorption/ionization time-of-flight Mass Spectrometry MALDI-TOF MS, combined with 16S rRNA gene sequencing) and complementary culture-independent (16S rRNA amplicon sequencing) methods. The presence of Salmonella spp. and Y. enterocolitica was additionally assessed. Using MALDI-TOF MS, Staphylococcus, Pseudomonas, and Escherichia coli were found to dominate the bacterial cultures isolated from the 8 carcasses. Based on the 16S rRNA amplicon sequencing analyses however, no specific genus clearly dominated the bacterial community composition. By using this culture-independent method, the most abundant genera in microbial populations of the ham, back, jowl and belly were found to be similar, but important differences between the two slaughterhouses were observed. Thus, present data suggests that the indigenous bacterial population of individual animals is overruled by the microbial population of the slaughterhouse in which the carcass is handled. Also, our data suggests that sampling of only one carcass area by official authorities may be appropriate for the evaluation of the hygienic status of the carcasses and therefore of the slaughter process.