The World Health Organization characterized COVID-19 as a pandemic in March 2020, the second pandemic of the twenty-first century. Expanding virus populations, such as that of SARS-CoV-2, accumulate a number of narrowly shared polymorphisms, imposing a confounding effect on traditional clustering methods. In this context, approaches that reduce the complexity of the sequence space occupied by the SARS-CoV-2 population are necessary for robust clustering. Here, we propose subdividing the global SARS-CoV-2 population into six well-defined subtypes and 10 poorly represented genotypes named tentative subtypes by focusing on the widely shared polymorphisms in nonstructural (nsp3, nsp4, nsp6, nsp12, nsp13 and nsp14) cistrons and structural (spike and nucleocapsid) and accessory (ORF8) genes. The six subtypes and the additional genotypes showed amino acid replacements that might have phenotypic implications. Notably, three mutations (one of them in the Spike protein) were responsible for the geographical segregation of subtypes. We hypothesize that the virus subtypes detected in this study are records of the early stages of SARS-CoV-2 diversification that were randomly sampled to compose the virus populations around the world. The genetic structure determined for the SARS-CoV-2 population provides substantial guidelines for maximizing the effectiveness of trials for testing candidate vaccines or drugs.

16The World Health Organization characterized the COVID-19 as a pandemic in March 2020, the second 17 pandemic of the 21 st century. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a 18 positive-stranded RNA betacoronavirus of the family Coronaviridae. Expanding virus populations, as 19 that of SARS-CoV-2, accumulate a number of narrowly shared polymorphisms imposing a 20 confounding effect on traditional clustering methods. In this context, approaches that reduce the 21 complexity of the sequence space occupied by the SARS-CoV-2 population are necessary for a robust 22 clustering. Here, we proposed the subdivision of the global SARS-CoV-2 population into sixteen well-23 defined subtypes by focusing on the widely shared polymorphisms in nonstructural (nsp3, nsp4, nsp6, 24 nsp12, nsp13 and nsp14) cistrons, structural (spike and nucleocapsid) and accessory (ORF8) genes. 25Six virus subtypes were predominant in the population, but all sixteen showed amino acid 26 replacements which might have phenotypic implications. We hypothesize that the virus subtypes 27 detected in this study are records of the early stages of the SARS-CoV-2 diversification that were 28 randomly sampled to compose the virus populations around the world, a typical founder effect. The 29 genetic structure determined for the SARS-CoV-2 population provides substantial guidelines for 30 maximizing the effectiveness of trials for testing the candidate vaccines or drugs. Main 32In December 2019, a local pneumonia outbreak of initially unknown etiology was detected in 33 Wuhan (Hubei, China) and quickly determined to be caused by a novel coronavirus 1 , named Severe 34 acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 2 and the disease as COVID-19 3 . SARS- 35CoV-2 is classified in the family Coronaviridae, genus Betacoronavirus, which comprises enveloped, 36 positive stranded RNA viruses of vertebrates 2 . Two-thirds of SARS-CoVs genome is covered by the 37 ORF1ab, that encodes a large polypeptide which is cleaved into 16 nonstructural proteins (NSPs) 38 involved in replication-transcription in vesicles from endoplasmic reticulum (ER)-derived 39 membranes 4,5 . The last third of the virus genome encodes four essential structural proteins: spike (S), 40 envelope (E), membrane (M), nucleocapsid (N) and several accessory proteins that interfere with the 41 host innate immune response 6 . 42Populations of RNA viruses evolve rapidly due to their large population sizes, short generation 43 times, and high mutation rates, this latter being a consequence of the RNA-dependent RNA 44 polymerase (RdRP) which lacks the proofreading activity 7 . In fact, virus populations are composed of 45 a broad spectrum of closely related genetic variants resembling one or more master sequences [8][9][10] . 46 Mutation rates inferred for SARS-CoVs are considered moderate 11,12 due to the independent 47 proofreading activity 13 . However, the large SARS-CoV genomes (from 27 to 31 kb) 14 provide to them 48 the ability to explore the sequence spa...

Antibiotic resistance has become a major concern for human and animal health. As fluoroquinolones have been extensively used in human and veterinary medicine, there has also been the rapid emergence and spread of antimicrobial resistance around the world. Here, we analysed the microbiome of goat milk using samples from healthy goats and those diagnosed with persistent mastitis and treated using the antibiotic enrofloxacin with 16S rRNA amplicon sequencing. We selected a group of 11 goats and 22 samples of milk that did not respond clinically to enrofloxacin treatment. Milk samples were evaluated before and after treatment to verify changes of the microbiota; the three first lactating goats were selected from the healthy control group. The milk samples from the healthy control animals presented a larger abundance of different species of bacteria of the Staphylococcus genus, but a smaller number of different genera, which indicated a more specific niche of resident bacteria. The Firmicutes phylum was predominantly different between the studied groups. Samples from before-treatment animals had a higher number of new species than those from the control group, and after being treated again. These microbiota received new bacteria, increasing the differences in bacteria even more in relation to the control group. Genotypes such as Trueperella and Mannheimia, between other genera, had a high abundance in the samples from animals with persistent mastitis. The dysbiosis in this study, with marked evidence of a complex microbiota in activity in cases of the failure of antimicrobial treatment for persistent chronic mastitis, demonstrates a need to improve the accuracy of pathogen identification and increases concern regarding antibiotic treatments in milk production herds. Antimicrobial therapies have been shown to be increasingly problematic due to the development multiple types of antimicrobial resistance (AMR) mechanisms, and for that reason, therapeutic alternatives to treat multidrug-resistant microorganisms are rapidly dwindling. Fluoroquinolones have been extensively used in human and veterinary medicine as they are considered among the most effective drugs for the treatment of bacterial infections 1,2. Enrofloxacin, is a fluoroquinolone exclusively developed for use in veterinary medicine 1,3,4. This drug is a potent inhibitor of bacterial DNA Topoisomerase II (Gyrase) and the DNA Topoisomerase IV (Topo IV), which are essential enzymes involved in key cellular processes including DNA replication 5-10. The drug has a broad spectrum of activity, being active against major pathogenic bacteria (both Gram-positive and Gram-negative), mycoplasmas 11 , and also mycobacteria 12 , but is ineffective against obligate anaerobes 13. Furthermore, in both mammalian and non-mammalian species, enrofloxacin is partially metabolised in the liver to ciprofloxacin, a primary metabolite of which is cyclopropyl, a potent antimicrobial agent itself 14. The active substance is characterised by a low host toxicity, being non-mutagenic with a...

Background: Staphylococcus aureus is one of the main causative agents of mastitis in small ruminants. Antimicrobial use is the major treatment, but there are many flaws linked to resistance, tolerance or persistence. This study aimed to verify changes in resistance, virulence and clonal profiles of S. aureus isolated from persistent mastitis goat milk before and after enrofloxacin treatment. Results: MIC increased to at least one antimicrobial in S. aureus isolates after enrofloxacin treatment compared to before. The most detected resistance genes before and after treatment were tetK, tetM, and blaZ, with more resistance genes detected after enrofloxacin treatment (p < 0.05). Occasional variations in efflux system gene detection were observed before and after treatment. Nine virulence genes (hla, fnbA, fnbB, eta, etb, sea, sec, seh, and sej) were detected at both times, and between these, the hla and eta genes were detected more in isolates after treatment. All isolates of S. aureus belonged to the same sequence type (ST) 133, except for two S. aureus isolates prior to enrofloxacin treatment which were classified as ST5 and the other as a new one, ST4966. Isolates of S. aureus 4, 8, and 100 from before and after treatment had identical pulse types, while others obtained from other animals before and after treatment were classified into distinct pulse types. Conclusion: There were occasional changes in the studied profiles of S. aureus isolated before and after treatment of animals with enrofloxacin, which may have contributed to the permanence of bacteria in the mammary gland, even when using traditional treatment, resulting in persistent mastitis.

Two experiments were conducted to evaluate different digestible isoleucine/lysine ratios on diets for broiler chickens in the starter (7 to 21 days) and finishing (30 to 43 days) phases. For the tests, the experimental design was of randomized blocks with seven treatments (six different digestible isoleucine/lysine ratios and a control treatment) and eight repetitions of 25 and 20 birds (COBB males) per experimental unit in the starter and finishing phases, respectively. The diets met the requirements, except for isoleucine and lysine. To avoid excess lysine in the experimental diets, the digestible lysine content was calculated to be 87% and 89% of the recommended for the starter and finishing phases, respectively. The control treatment was adequate in lysine and isoleucine. Feed intake, weight gain, feed conversion and carcass yield in the two phases were evaluated. There was quadratic effect of different ratios on feed intake in the finishing phase and on weight gain and feed conversion rate in both phases. There was quadratic effect on breast meat yield and breast fillets in the starter phase, but there was no significant effect on carcass yield in the finishing phase. The digestible isoleucine/lysine ratio recommended for broilers in the starter phase (7 to 21 days) is 66% and for the finishing phase (30 to 43 days), it is 68%