Vinícius da Silva Duarte

Use of phages against antibiotic-resistant Staphylococcus aureus isolated from bovine mastitis1

Bovine mastitis is the primary disease of dairy cattle worldwide and it causes large economic losses. Among several microorganisms that are the causative agents of this disease, Staphylococcus aureus is the most prevalent. Although antibiotic therapy is still the most widely used procedure for the treatment of bovine mastitis, alternative means of treatment are necessary due to the presence of antibiotic residues in milk, which is a growing concern because of its interference with the production of milk derivatives and the selection of resistant bacterial strains. The use of bacteriophages as a tool for the control of pathogens is an alternative treatment to antibiotic therapy. In this work, to obtain phages with the potential for use in phage therapy as a treatment for mastitis, we isolated and identified the bacteria from the milk of mastitis-positive cows. A total of 19% of the animals from small and medium farms of the Zona da Mata Mineira, Brazil, was positive for bovine mastitis, and bacteria of the genus Staphylococcus were the most prevalent pathogens. The majority of the S. aureus isolates tested was resistant to penicillin and ampicillin. In parallel, we isolated 10 bacteriophages able to infect some of these S. aureus isolates. We determined that these phages contained DNA genomes of approximately 175 kb in length, and the protein profiles indicated the presence of 4 major proteins. Electron microscopy revealed that the phages are caudate and belong to the Myoviridae family. The isolates exhibited interesting features for their use in phage therapy such as a high lytic potential, a wide range of hosts, and thermostability, all of which favor their use in the field.

Short communication: Comparison of growth kinetics at different temperatures of Streptococcus macedonicus and Streptococcus thermophilus strains of dairy origin

Within the genus Streptococcus, S. thermophilus and S. macedonicus are the 2 known species related to foods. Streptococci are widely used as starter cultures to rapidly lower milk pH. As S. macedonicus has been introduced quite recently, much less information is available on its technological potential. Because temperature is an important factor in fermented food production, we compared the growth kinetics over 24 h of 8 S. thermophilus and 7 S. macedonicus strains isolated from various dairy environments in Italy, at 4 temperatures, 30°C, 34°C, 37°C and 42°C. We used the Gompertz model to estimate the 3 main growth parameters; namely, lag phase duration (λ), maximum growth rate (µmax), and maximum cell number at the stationary phase (Nmax). Our results showed significant differences in average growth kinetics between the 2 species. Among the strains tested, 37°C appeared to be the optimal temperature for the growth of both species, particularly for S. macedonicus strains, which showed mean shorter lag phases and higher cell numbers compared with S. thermophilus. Overall, the growth curves of S. macedonicus strains were more similar to each other whereas S. thermophilus strains grew very differently. These results help to better define and compare technological characteristics of the 2 species, in view of the potential use of S. macedonicus in place of S. thermophilus in selected technological applications.

Draft Genome Sequence of the Yeast Starmerella bacillaris (syn., Candidazemplinina) FRI751 Isolated from Fermenting Must of Dried Raboso Grapes

ABSTRACT Starmerella bacillaris is an ascomycetous yeast commonly present in enological environments. Here, we report the first draft genome sequence of S. bacillaris FRI751, which will facilitate the study of the characteristics of this interesting enological yeast.

Complete Genome Sequence of vB_EcoM-UFV13, a New Bacteriophage Able To Disrupt Trueperella pyogenes Biofilm

ABSTRACT vB_EcoM-UFV13, a member of the T4virus genus, shows lytic activity against Escherichia coli and effectiveness in controlling the biofilm formed by Trueperella pyogenes, which qualifies it as a promising component of phage cocktails for mastitis and metritis control.

A T4virus prevents biofilm formation by Trueperella pyogenes

Trueperella pyogenes is an opportunistic pathogen of many animal species. It causes economic losses worldwide, through mastitis, metritis and mainly endometritis in dairy cows. The ability of this bacterium to form biofilms is implicated in chronic infections through hampering immune system recognition and antibiotic penetration. Since it is difficult to eradicate T. pyogenes infections with antibiotics, phage therapy presents itself as a non-toxic, effective and economically viable alternative. The present study evaluated the use of the bacteriophage vB_EcoM-UFV13 (UFV13) in the prevention of T. pyogenes biofilm development. Based upon two different approaches (crystal violet and sessile cell counting) we observed that only a multiplicity of infection (MOI) of 10 showed a statistically significant reduction in biofilm formation. Although the exact mechanisms of biofilm disruption and cell-adhesion inhibition have not been determined, genome sequence analysis of the Escherichia phage UFV13 revealed a repertoire of virion-associated peptidoglycan hydrolases (VAPGHs). The present study presents new findings regarding the disruption of biofilm formation of a Gram-positive bacterium. Subsequent transcriptomic and proteomic research will help us to understand the exact interaction mechanisms between UFV13 and T. pyogenes.

Genome Sequence of Enterococcus mundtii EM01, Isolated from Bombyx mori Midgut and Responsible for Flacherie Disease in Silkworms Reared on an Artificial Diet

ABSTRACT The whole genome sequence of Enterococcus mundtii strain EM01 is reported here. The isolate proved to be the cause of flacherie in Bombyx mori. To date, the genomes of 11 other E. mundtii strains have been sequenced. EM01 is the only strain that displayed active pathological effects on its associated animal species.

Comparative Transcriptomic Analysis of Streptococcus thermophilus TH1436 and TH1477 Showing Different Capability in the Use of Galactose

Streptococcus thermophilus is a species widely used in the dairy industry for its capability to rapidly ferment lactose and lower the pH. The capability to use galactose produced from lactose hydrolysis is strain dependent and most of commercial S. thermophilus strains are galactose-negative (Gal−), although galactose-positive (Gal+) would be more technologically advantageous because this feature could provide additional metabolic products and prevent galactose accumulation in foods. In this study, a next generation sequencing transcriptome approach was used to compare for the first time a Gal+ and a Gal− strain to characterize their whole metabolism and shed light on their different properties, metabolic performance and gene regulation. Transcriptome analysis revealed that all genes of the gal operon were expressed very differently in Gal+ and in the Gal− strains. The expression of several genes involved in mixed acid fermentation, PTS sugars transporter and stress response were found enhanced in Gal+. Conversely, genes related to amino acids, proteins metabolism and CRISPR associated proteins were under-expressed. In addition, the strains showed a diverse series of predicted genes controlled by the transcriptional factor catabolite control protein A (CcpA). Overall, transcriptomic analysis suggests that the Gal+ strain underwent a metabolic remodeling to cope with the changed environmental conditions.

In vitro Probiotic Potential and Anti-cancer Activity of Newly Isolated Folate-Producing Streptococcus thermophilus Strains

Most probiotic strains commercially available today are lactic acid bacteria. Within this functional group, Streptococcus thermophilus is a thermophilic species widely used as starter culture for a huge number of dairy products. Besides being rapid acidifiers, many S. thermophilus strains are able to produce and release folate during growth but, unfortunately, they are seriously impaired during passage through the human gastrointestinal tract. In this work, we studied eight S. thermophilus strains isolated from dairy environments in Italy, which already had shown good technological properties, to evaluate their possible probiotic potential and cytotoxicity against cancer cells in vitro. All strains were also evaluated for some health-related properties such as susceptibility to most common antibiotics, hemolytic activity, resistance to simulated gastrointestinal conditions, bile salts hydrolytic activity, production of folate, adhesion to HT-29 human colorectal adenocarcinoma cells and cytotoxic activity against cancer cells and production of biogenic amines. Results revealed that two fast acidifying S. thermophilus strains were found to possess in vitro probiotic properties along with anticancer activity and production of folate. These properties resulted similar and, in some cases, superior to those of Lactobacillus rhamnosus GG, a well-known commercial probiotic strain. These findings encourage further in vivo studies to evaluate the actual health benefits of these strains on the human host.

Whole genome comparison of two Starmerella bacillaris strains with other wine yeasts uncovers genes involved in modulating important winemaking traits

&NA; Starmerella bacillaris is an osmotolerant yeast with interesting winemaking traits such as low‐ethanol and high‐glycerol production, previously considered as wine spoilage and recently proposed to improve the sensory quality of wine. This is the first work performing a whole‐genome analysis of the variants identified by comparing two S. bacillaris strains (PAS13 and FRI751). Additionally, an extensive search for orthologous genes against Saccharomyces and non‐Saccharomyces yeasts produced a detailed reconstruction of the pan‐genome for yeast species used in winemaking. Starmerella bacillaris PAS13 was able to produce 36% more glycerol than S. bacillaris FRI751 without increasing ethanol level over 5% (v/v). Orthologous genes revealed new insights in the response to osmotic stress determined by the mitogen‐activated protein kinase (MAPK) from S. bacillaris strains. The comparison between the two S. bacillaris genomes revealed 33 771 high‐quality variants that were ranked considering their predicted impact on gene functions. Furthermore, analysis of structural variations in the genome revealed five translocations. The absence of some transcriptional factors involved in the regulation of GPD (glycerol‐3‐phosphate dehydrogenase), like the protein kinases YpK1p and YpK2p, and the identification of a tandem duplication increasing the GPP1 (glycerol‐3‐phosphate phosphatase) gene copy number suggest a remarkably different regulation of the glycerol pathway for S. bacillaris in comparison to S. cerevisiae.

Differences in Carbohydrates Utilization and Antibiotic Resistance Between Streptococcus macedonicus and Streptococcus thermophilus Strains Isolated from Dairy Products in Italy

Streptococcus thermophilus and S. macedonicus are the only two species of the genus related to food productions so far known. In the present study, eight S. thermophilus and seven S. macedonicus strains isolated from dairy environments in Italy were compared in order to evidence possible species-specific technological characteristics. Their capability to use lactose, galactose, fructose, and glucose, sugars commonly present in foods and two carbohydrates considered as prebiotics, xylose and inulin, along with the respective growth kinetics were studied. Results showed a luxuriant growth on lactose and different behaviors on galactose, glucose, and fructose. No growth on inulin and xylose was recorded, which is a positive feature for strains intended to be used as starter cultures. Growth parameters, namely, λ, µmax, and Nmax, were estimated by using the Gompertz model. Antibiotic resistance to 14 drugs revealed an overall similar behavior between the two species with only a marked difference regarding gentamycin. Antimicrobial activity was also tested against six deleterious bacterial strains, but none of the strains evidenced inhibitory capabilities. The results presented here could be helpful to compare technological potentialities of the two species and to choose strains of the most suitable species for selected microbiological food transformations.