Risk factors associated with the occurrence of multiresistant Staphylococcus spp. isolated from bovine subclinical mastitis in northern Brazil

Staphylococcus bacteria are often associated with subclinical bovine mastitis. This study aimed to identify multiresistant Staphylococcus spp. associated with subclinical mastitis and the associated risk factors. Twenty-three dairy farms with a history of decrease in milk production, located in the lower Acre region, Brazil, were selected. An epidemiological questionnaire was provided in all farms. All animals were examined using the California Mastitis Test (CMT) and their milk samples were collected for bacterial culture. After isolation and identification, the disk diffusion antimicrobial susceptibility test was performed against nine classes of antimicrobials. Of the 339 cows examined using the CMT, 108 had mastitis. A total of 229 milk samples were collected from individual teats. MALDI-TOF MS found isolates belonging to eight species of Staphylococcus, in 101 of these samples. S. chromogenes (58.4%) demonstrated strongest resistance to the nine classes of antimicrobial active principles. Nineteen isolates with multidrug resistance phenotypic profile were identified. This phenotypic expression indicates wide circulation of resistant genes in this species. The presence of multidrug resistance in Staphylococcus spp. in this study was correlated with lack of water for cleaning the corral, which is a preventive factor, minimizing the transmission and persistence of pathogens in the farms.

Isolation and Identification of Lactic Acid Bacteria from Natural Whey Cultures of Buffalo and Cow Milk

In southern Italy, some artisanal farms produce mozzarella and caciocavallo cheeses by using natural whey starter (NWS), whose microbial diversity is responsible for the characteristic flavor and texture of the final product. We studied the microbial community of NWS cultures of cow’s milk (NWSc) for the production of caciocavallo and buffalo’s milk (NWSb) for the production of mozzarella, both from artisanal farms. Bacterial identification at species and strain level was based on an integrative strategy, combining culture-dependent (sequencing of the 16S rDNA, species/subspecies-specific Polymerase Chain Reaction (PCR) and clustering by Random Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) and culture-independent (next-generation sequencing analysis, NGS) approaches. Results obtained with both approaches showed the occurrence of five species of lactic acid bacteria in NWSb (Lactococcus lactis subsp. lactis, Lactobacillus fermentum, Streptococcus thermophilus, Lactobacillus delbrueckii, and Lactobacillus helveticus) and five species in NWSc (Lc. lactis subsp. lactis, Enterococcus faecium, and S. thermophilus, Lb. helveticus, and Lb. delbrueckii), with the last two found only by the NGS analysis. Moreover, RAPD profiles, performed on Lc. lactis subsp. lactis different isolates from both NWSs, showed nine strains in NWSb and seven strains in NWSc, showing a microbial diversity also at strain level. Characterization of the microbiota of natural whey starters aims to collect new starter bacteria to use for tracing microbial community during the production of artisanal cheeses, in order to preserve their quality and authenticity, and to select new Lactic Acid Bacteria (LAB) strains for the production of functional foods.

The Isolation of Vibrio crassostreae and V. cyclitrophicus in Lesser-Spotted Dogfish (Scyliorhinus canicula) Juveniles Reared in a Public Aquarium

The genus Vibrio currently contains 147 recognized species widely distributed, including pathogens for aquatic organisms. Vibrio infections in elasmobranchs are poorly reported, often with identifications as Vibrio sp. and without detailed diagnostic insights. The purpose of this paper is the description of the isolation and identification process of Vibrio spp. following a mortality event of Scyliorhinus canicula juvenile reared in an Italian public aquarium. Following investigations aimed at excluding the presence of different pathogens of marine fish species (parasites, bacteria, Betanodavirus), several colonies were isolated and subjected to species identification using the available diagnostic techniques (a biochemical test, MALDI-TOF MS, and biomolecular analysis). Discrepancies were observed among the methods; the limits of biochemistry as a unique tool for Vibrio species determination were detected through statistical analysis. The use of the rpoB gene, as a diagnostic tool, allowed the identification of the isolates as V. crassostreae and V. cyclotrophicus. Although the pathogenic role of these microorganisms in lesser-spotted dogfish juveniles has not been demonstrated, and the presence of further pathogens cannot be excluded, this study allowed the isolation of two Vibrio species in less-studied aquatic organisms, highlighting the weaknesses and strengths of the different diagnostic methods applied.

Bioassay Guided Fractionation of Senna singueana and Its Potential for Development of Poultry Phytogenic Feed Additives

There has been burgeoning interest in plant-based feed additives following restrictions placed on the use of antibiotic feed additives in many countries. Phytogenic feed additives are recommended to have a range of useful properties to support the growth and development of poultry to a similar level as that obtained by supplementing feed with antibiotics. The aim of this study was to evaluate the antibacterial, anti-lipoxygenase and antioxidant activity, and in vitro safety of fractions and isolated compounds from leaves of Senna singueana. Antibacterial activities of the fractions and isolated compounds were determined against a panel of bacteria using a two-fold serial microdilution assay and qualitative bioautography assays. Anti-lipoxygenase activity was evaluated using the ferrous oxidation-xylenol orange (FOX) method. Antioxidant activity was assessed qualitatively and quantitatively using radical scavenging assays. Dichloromethane and ethyl acetate fractions from solvent-solvent partitioning had the best antibacterial activity with MIC values ranging from 156 to 313 μg/ml. Fractions obtained from column chromatography had significant to weak antibacterial activity with MIC values ranging from 50 to 1,250 μg/ml. Bioautography showed clear bands of bacterial inhibition, indicating the presence of a number of active compounds in several fractions. The ethyl acetate fraction and all the tested column fractions had potent anti-lipoxygenase activity with IC50 values of ≤2.5 μg/ml which were lower than that of quercetin (positive control), indicating anti-inflammatory potential. The ethyl acetate fraction and several column fractions had powerful antioxidant activity with IC50 values of ≤5 μg/ml in the ABTS assay. Cytotoxicity values against Vero kidney cells ranged from LC50 = 40.0–989.3 μg/ml. Bioassay-guided fractionation led to the isolation and identification of a known bioactive compound, luteolin. S. singueana is a promising candidate for the development of poultry phytogenic feed additives.

Impacts of Angelica Polysaccharide on Proliferation and Differentiation of Mesenchymal Stem Cells of Rat Bone Marrow

In literature, antiosteoporotic effects of Angelica sinensis root have been confirmed, but the impact of Angelica sinensis polysaccharide (ASP) on osteoblastic or adipogenic distinction of BMSCs is limited. This paper aimed to explore the role of ASP on proliferation and differentiation of rat BMSCs. Rat BMSCs were subjected to isolation and identification through flow cytometry. The proliferation of rat BMSCs under ASP was performed by CCK-8 kit. Measures of osteogenesis under different concentrations of ASP were detected by using alizarin red staining for mesenchymal cells differentiation and ALP activity assay to identify ALP activity. Quantitative RT-PCR was selected to identify osteoblastic or adipogenic biomarkers from a genetic perspective. Likewise, we have evaluated measures of indicators of Wnt/β-catenin signal. ASP significantly promoted the proliferation, increased osteogenesis, and decreased adipogenesis of rat BMSCs within the limit of 20–60 mg/L in a dose-dependent manner but was suppressed at 80 mg/L. The expression of cyclin D1 and ß-catenin showed a considerable rise over the course of ASP induced osteogenesis. Dickkopf 1 (DKK1) suppressed the regulation of rat BMSCs differentiation through the mediation of ASP. We have observed that ASP upregulated the osteogenic but downregulated adipogenic differentiation of BMSCs, and our findings help to contribute to effective solutions for treating bone disorders.

Isolation and Identification of a Rare Spike Gene Double-Deletion SARS-CoV-2 Variant From the Patient With High Cycle Threshold Value

Coronavirus disease 2019 (COVID-19) is an emerging life-threatening pulmonary disease caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which originated in Wuhan, Hubei Province, China, in December 2019. COVID-19 develops after close contact via inhalation of respiratory droplets containing SARS-CoV-2 during talking, coughing, or sneezing by asymptomatic, presymptomatic, and symptomatic carriers. This virus evolved over time, and numerous genetic variants have been reported to have increased disease severity, mortality, and transmissibility. Variants have also developed resistance to antivirals and vaccination and can escape the immune response of humans. Reverse transcription polymerase chain reaction (RT–PCR) is the method of choice among diagnostic techniques, including nucleic acid amplification tests (NAATs), serological tests, and diagnostic imaging, such as computed tomography (CT). The limitation of RT–PCR is that it cannot distinguish fragmented RNA genomes from live transmissible viruses. Thus, SARS-CoV-2 isolation by using cell culture has been developed and makes important contributions in the field of diagnosis, development of antivirals, vaccines, and SARS-CoV-2 virology research. In this research, two SARS-CoV-2 strains were isolated from four RT–PCR-positive nasopharyngeal swabs using VERO E6 cell culture. One isolate was cultured successfully with a blind passage on day 3 post inoculation from a swab with a Ct > 35, while the cells did not develop cytopathic effects without a blind passage until day 14 post inoculation. Our results indicated that infectious SARS-CoV-2 virus particles existed, even with a Ct > 35. Cultivable viruses could provide additional consideration for releasing the patient from quarantine. The results of the whole genome sequencing and bioinformatic analysis suggested that these two isolates contain a spike 68-76del+spike 675-679del double-deletion variation. The double deletion was confirmed by amplification of the regions spanning the spike gene deletion using Sanger sequencing. Phylogenetic analysis revealed that this double-deletion variant was rare (one per million in public databases, including GenBank and GISAID). The impact of this double deletion in the spike gene on the SARS-CoV-2 virus itself as well as on cultured cells and/or humans remains to be further elucidated.

Anti-infection roles of miR-155-5p packaged in exosomes secreted by dendritic cells infected with Toxoplasma gondii

Background Toxoplasma gondii is a zoonotic intracellular protozoon that is estimated to infect about 30% of the world’s population, resulting in toxoplasmosis in immunocompromised patients and adverse outcomes in cases of primary infection during pregnancy. Exosomes are tubular vesicles secreted by cells, and function in intercellular communication. It has been reported that the exosomes secreted by T. gondii-infected immune cells transmit infection signals to the uninfected cells. However, the mechanism and effect of the exosome transmission are still vague. We therefore investigated the function of the exosomes transmitted from DC2.4 cells infected with the T. gondii RH strain (Tg-DC-Exo) to the uninfected cells, as well as their roles in anti-infection. Methods We conducted exosome isolation and identification with ultracentrifugation, transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blot (WB) analysis. Exosome uptake by recipient cells was identified by PKH67 assay. The signal transmission and the abundance of miR-155-5p were determined using transwell assay and qRT-PCR. For detection of immune responses, cytokine secretion was evaluated. The T. gondii B1 gene was determined to evaluate tachyzoite proliferation. Results We observed that Toxoplasma infection upregulated miR-155-5p expression in DC2.4 cell-secreted exosomes, and those exosomes could be ingested by murine macrophage RAW264.7 cells. Tg-DC-Exo and miR-155-5p stimulated host proinflammatory immune responses including increased production of proinflammatory cytokines IL-6 and TNF-α, and proinflammatory marker-inducible nitric oxide synthase (iNOS). The NF-κB pathway was activated by downregulation of SOCS1, leading to inhibition of T. gondii tachyzoite proliferation in RAW264.7 cells. Conclusions Our findings provide a novel mechanism for how infected cells transmit infection signals to the uninfected cells through exosome secretion after T. gondii infection, followed by inflammatory responses and anti-infection reactions, which may help us develop a new strategy for toxoplasmosis prevention, especially in immunocompromised patients. Graphical Abstract