Search Results (11)

Cryptococcus neoformans (C. neoformans) is a capsulated yeast that enters the body through inhalation and migrates via the bloodstream to the central nervous system, causing cryptococcal meningitis. Diagnosis methods are culture, serology, and India ink staining, which require cerebrospinal fluid (CSF) or whole blood. Molecular methods are used for epidemiological studies and require expensive commercial DNA extraction kits. This study aimed to develop an economical in-house method for extracting C. neoformans DNA from whole blood. C. neoformans cells of varying McFarland standards were spiked into expired blood, then lysed using laboratory-prepared lysis buffer and ox-bile solution, followed by organic DNA extraction. Ordinary PCR targeting the CNAG 04922 gene was performed. To determine the limit of detection, serial dilutions of C. neoformans were made, and DNA extraction was performed on other parts cultured on yeast extract peptone dextrose agar to determine colony-forming units (CFU). The lysis buffer method successfully extracted DNA from as low as the average of 62 CFU in 0.9 mL of expired blood with superior quality and high yield compared to ox-bile. The lysis buffer method yielded higher DNA quality and quantity than ox-bile and detected low concentrations of C. neoformans in expired blood. This method presents a cost-effective alternative for molecular diagnosis in resource-limited settings. Full article

This work aimed to evaluate some of the probiotic features and safety of the bacteriocin-producing Latilactobacillus sakei subsp. sakei 2a. The effect of selected commercial drugs from different generic groups and antibiotics on the growth of Ltb. sakei subsp. sakei 2a was also determined. The presence of virulence factors was determined based on PCR with total DNA from Ltb. sakei subsp. sakei 2a. Good growth of Ltb. sakei subsp. sakei 2a was recorded in MRS broth supplemented with 0.2% or 0.4% oxbile or in MRS broth adjusted to a pH from 5.0–9.0. Auto-aggregation of Ltb. sakei subsp. sakei 2a was 62.59%. Different levels of co-aggregation were recorded between Ltb. sakei subsp. sakei 2a and Enterococcus faecalis ATCC19443, Ltb. sakei ATCC15521 and Listeria monocytogenes ScottA. Growth of Ltb. sakei subsp. sakei 2a was not inhibited by commercial drugs from different generic groups. The inhibitory effect on the growth of Ltb. sakei subsp. sakei 2a was recorded only in the presence of Arotin [selective serotonin reuptake inhibitor antidepressant] Minimal Inhibition Concentration (MIC) 1.0 mg/mL, Atlansil [Antiarrhythmic] MIC 0.625 mg/mL, Diclofenac potassium [non-steroidal anti-inflammatory drug (NSAID)] MIC 2.5 mg/mL and Spidufen [NSAID] MIC 15.0 mg/mL. Only two antibiotics tested in this study, Amoxil and Urotrobel, inhibited the growth of Ltb. sakei subsp. sakei 2a with a MIC of <0.5 mg/mL and 5.0 mg/mL, respectively. However, Ltb. sakei subsp. sakei 2a generated positive PCR results on the DNA level for vanA (vancomycin resistance), hyl (hyaluronidase), esp (enterococcal surface protein), ace (adhesion of collagen) and cilA (cytolisin) and a high virulence profile when examined for the presence of virulence factors. It is important to underline that cytolysis has been described as a virulence and antibacterial factor. Full article

Chronic infections often involve notorious pathogens like Pseudomonas aeruginosa and Staphylococcus aureus, demanding innovative antimicrobial strategies due to escalating resistance. This investigation scrutinized the antibacterial prowess of bile salts, notably taurocholic acid (TCA), ursodeoxycholic acid (UDCA), and ox bile salt (OBS), against these pathogens. Evaluations encompassed minimum inhibitory concentration (MIC) determination, scrutiny of their impact on biofilm formation, and anti-virulence mechanisms. UDCA exhibited the highest efficacy, suppressing S. aureus and P. aeruginosa biofilms by 83.5% and 78%, respectively, at peak concentration. TCA also significantly reduced biofilm development by 81% for S. aureus and 75% for P. aeruginosa. Microscopic analysis revealed substantial disruption of biofilm architecture by UDCA and TCA. Conversely, OBS demonstrated ineffectiveness against both pathogens. Mechanistic assays elucidated UDCA and TCA’s detrimental impact on the cell membrane, prompting the release of macromolecular compounds. Additionally, UDCA and TCA inhibited protease and elastase synthesis in P. aeruginosa and staphyloxanthin and lipase production in S. aureus. These results underscore the potential of UDCA and TCA in impeding biofilm formation and mitigating the pathogenicity of S. aureus and P. aeruginosa. Full article

Rural households all over the world rear backyard chicken mainly for their own consumption and, to a lesser extent, for barter trade. These chickens represent a staple dish with numerous culinary variations and a cheap source of protein. Although some Campylobacter species, and particularly Campylobacter jejuni and Campylobacter coli, have been associated with industrial poultry carcasses, studies concerning the ecology of this genus in rural households do not exist. To assess the prevalence of Campylobacter species in the tissues of backyard chickens, samples were collected from birds Gallus domesticus bred in households in the rural area of Epirus (Greece), and Campylobacter strains were isolated by quantitative methods at 37 °C and 42 °C. In total, 256 strains were identified, belonging to 17 Campylobacter species, with C. jejuni and C. coli being the most prevalent. From the four ecological parameters studied (size of the flock, presence of small ruminants in the same household, presence of other poultry species in the same household, and feeding leftovers of the household), the size of the flock and the presence of small ruminants and/or pigs in the same household mostly affected the distribution of these strains. To study the phenotypical resistance against 14 antibiotics, 215 strains were selected. The results showed a high prevalence of multidrug-resistance (MDR) strains extending to all classes of antibiotics. Further genome analysis revealed the presence of genes coding resistance (bla_OxA-61, tet(O), tet(A) cmeA, cmeB, cmeC, and gyrA (Thr-86-Ile mutation)), with the efflux pump CmeABC being the most prevalent. All antimicrobial resistance-encoded genes co-circulated, except for bla_OXA-61, which moved independently. The minimum inhibitory concentration (MIC) values of two out of three antibiotics (representing different classes) were reduced when the strains tested were exposed to carbonyl cyanide 3-chlorophenylhydrazone (CCCP), a known efflux pump inhibitor. The same result was obtained with the addition of CCCP to the MIC values of bile salts. These results lead to the conclusion that Campylobacter species are present in an impressive diversity in backyard chicken tissues and that they exert a significant resistance to antibiotics, raising a potential danger for public health. Full article

In this study, bile acid-based vesicles and nanoparticles (i.e., bilosomes and biloparticles) are studied to improve the water solubility of lipophilic drugs. Ursodeoxycholic acid, sodium cholate, sodium taurocholate and budesonide were used as bile acids and model drugs, respectively. Bilosomes and biloparticles were prepared following standard protocols with minor changes, after a preformulation study. The obtained systems showed good encapsulation efficiency and dimensional stability. Particularly, for biloparticles, the increase in encapsulation efficiency followed the order ursodeoxycholic acid < sodium cholate < sodium taurocholate. The in vitro release of budesonide from both bilosytems was performed by means of dialysis using either a nylon membrane or a portion of Wistar rat small intestine and two receiving solutions (i.e., simulated gastric and intestinal fluids). Both in gastric and intestinal fluid, budesonide was released from bilosystems more slowly than the reference solution, while biloparticles showed a significant improvement in the passage of budesonide into aqueous solution. Immunofluorescence experiments indicated that ursodeoxycholic acid bilosomes containing budesonide are effective in reducing the inflammatory response induced by glucose oxidase stimuli and counteract ox-inflammatory damage within intestinal cells. Full article

Fourteen lactic acid bacteria (LAB) strains were isolated from the intestinal tract and feces of piglets. Among these isolates, only twelve were biochemically (API 50 CHL and ABIS online for bacterial identification) and genetically (16S rRNA sequencing) confirmed as Limosilactobacillus fermentum, Lactobacillus acidophilus, and Lactiplantibacillus plantarum. Experiments to evaluate the probiotic potential of the isolates including pH tolerance (pH 2.0 and 3.0), bile salts (0.3% ox gall) resistance, hemolysis activity, antibiotic susceptibility, and high-temperature resistance were tested. Only two isolates from identified strains exhibited high survival rates when exposed to low pH and bile salts, these were L. acidophilus IBNA 76 and L. plantarum IBNA 84. The antibiotic test presented 100% resistance of both strains to gentamicin, kanamycin, lincomycin, colistin sulfate, erythromycin, amikacin, oxytetracycline, enrofloxacin, streptomycin, and tilmicosin, lower than the 0.6 mm inhibition zone diameter. Promising isolates (L. acidophilus IBNA 76 and L. plantarum IBNA 84) were exposed to the spray-drying technique based on visible probiotic potential and survival rates. Carrier matrix material was used as a maltodextrin-glucose solution. The encapsulation probiotic isolated survived both over 67% and 77%, corresponding to a decrease in strain viability from 10⁹ to 10⁷ CFU/g. After further in vitro evaluations, the findings of this study showed that, from all LAB strains, L. acidophilus IBNA 76 and L. plantarum IBNA 84 may be considered probiotic candidates for animal nutrition and may have promising performance in piglet feed due to their origin of isolation. Full article

Typhoid fever is caused by Salmonella enterica serovar Typhi (S. Typhi). Around 3–5% of individuals infected become chronic carriers, with the gallbladder (GB) as the predominant site of persistence. Gallstones (GS) aid in the development and maintenance of GB carriage, serving as a substrate to which Salmonellae attach and form a biofilm. This biofilm matrix protects bacteria from the host immune system and environmental stress. This shielded environment is an ideal place for the development of persister cells, a transient phenotype of a subset of cells within a population that allows survival after antibiotic treatment. Persisters can also arise in response to harsh environments such as the GB. Here we investigate if GB conditions affect the number of persisters in a Salmonella population. To simulate the chronic GB environment, we cultured biofilms in cholesterol-coated 96-well plates in the presence of ox or human bile. We then treated planktonic or biofilm Salmonella cultures with high concentrations of different antibiotics. This study suggests that biofilms provide a niche for persister cells, but GB conditions either play no role or have a negative influence on persister formation, especially after kanamycin treatment. The antibiotic target was important, as antimicrobials directed against DNA replication or the cell wall had no effect on persister cell formation. Interestingly, repeated treatment with ciprofloxacin increased the percentage of S. Typhimurium persisters in a biofilm, but this increase was abolished by GB conditions. On the other hand, repeated ciprofloxacin treatment of S. Typhi biofilms in GB conditions slightly increased the fraction of persisters. Thus, while the harsh conditions in the GB would be thought to give rise to increased persisters, therefore contributing to the development of chronic carriage, these data suggest persister cell formation is dampened in this environment. Full article

Hyperlipidemia and oxidative stress with elevated oxidized low-density lipoprotein (ox-LDL) exacerbate hepatic inflammation and fibrosis. The plasma level of low-density lipoprotein (LDL) is controlled by proprotein convertase subtilisin/kexin 9 (PCSK9). Alirocumab is a monoclonal antibody that decreases LDL via inhibiting PCSK9 function. Apart from lipid-lowering effects, alirocumab exerts anti-inflammation, anti-angiogenesis and anti-oxidant effects. This study aims to investigate the impact of alirocumab treatment on common bile duct ligation (BDL)-induced biliary cirrhotic rats. After a 4-week treatment of alirocumab, the hemodynamic data, blood biochemistry, ox-LDL level, oxidative stress markers, severity of hepatic encephalopathy and abnormal angiogenesis of BDL rats were measured and compared to the control group. BDL rats presented cirrhotic pictures and elevated ammonia, total cholesterol, LDL and ox-LDL levels compared to the control group. Alirocumab decreased plasma levels of total cholesterol, LDL, and oxidative stress markers; however, it did not affect the hemodynamics, liver and renal biochemistry, and the plasma levels of ammonia and ox-LDL. The motor activities, portal-systemic collaterals and mesenteric vascular density were not significantly different between alirocumab-treated and control groups. In addition, it did not affect hepatic inflammation, intrahepatic angiogenesis, liver fibrosis and free cholesterol accumulation in the liver of BDL rats. In conclusion, PCSK9 inhibition by alirocumab treatment ameliorates hyperlipidemia and systemic oxidative stress in biliary cirrhotic rats. However, it does not affect the plasma level of ox-LDL, intrahepatic inflammation and fibrosis. In addition, PCSK9 inhibition has a neutral effect on abnormal angiogenesis and hepatic encephalopathy in biliary cirrhotic rats. Full article

Machine perfusion (MP) allows the maintenance of liver cells in a metabolically active state ex vivo and can potentially revert metabolic perturbations caused by donor warm ischemia, procurement, and static cold storage (SCS). The present preclinical research investigated the metabolic outcome of the MP procedure by analyzing rat liver tissue, bile, and perfusate samples by means of high-field (600 MHz) nuclear magnetic resonance (NMR) spectroscopy. An established rat model of normothermic MP (NMP) was used. Experiments were carried out with the addition of an oxygen carrier (OxC) to the perfusion fluid (OxC-NMP, n = 5) or without (h-NMP, n = 5). Bile and perfusate samples were collected throughout the procedure, while biopsies were only taken at the end of NMP. Two additional groups were: (1) Native, in which tissue or bile specimens were collected from rats in resting conditions; and (2) SCS, in which biopsies were taken from cold-stored livers. Generally, NMP groups showed a distinctive metabolomic signature in all the analyzed biological matrices. In particular, many of the differentially expressed metabolites were involved in mitochondrial biochemical pathways. Succinate, acetate, 3-hydroxybutyrate, creatine, and O-phosphocholine were deeply modulated in ex vivo perfused livers compared to both the Native and SCS groups. These novel results demonstrate a broad modulation of mitochondrial metabolism during NMP that exceeds energy production and redox balance maintenance. Full article

Necrotic enteritis (NE), mainly induced by the pathogens of Clostridium perfringens and coccidia, causes huge economic losses with limited intervention options in the poultry industry. This study investigated the role of specific bile acids on NE development. Day-old broiler chicks were assigned to six groups: noninfected, NE, and NE with four bile diets of 0.32% chicken bile, 0.15% commercial ox bile, 0.15% lithocholic acid (LCA), or 0.15% deoxycholic acid (DCA). The birds were infected with Eimeria maxima at day 18 and C. perfringens at day 23 and 24. The infected birds developed clinical NE signs. The NE birds suffered severe ileitis with villus blunting, crypt hyperplasia, epithelial line disintegration, and massive immune cell infiltration, while DCA and LCA prevented the ileitis histopathology. NE induced severe body weight gain (BWG) loss, while only DCA prevented NE-induced BWG loss. Notably, DCA reduced the NE-induced inflammatory response and the colonization and invasion of C. perfringens compared to NE birds. Consistently, NE reduced the total bile acids in the ileal digesta, while dietary DCA and commercial bile restored it. Together, this study showed that DCA and LCA reduced NE histopathology, suggesting that secondary bile acids, but not total bile acid levels, play an essential role in controlling the enteritis. Full article

Isoliquiritigenin (ISL) exhibits antioxidation and anti-inflammation activity. We sought to investigate the effects and mechanism of ISL on the development of atherosclerotic lesions in apolipoprotein E-deficient (apoE^−/−) mice. Firstly, we determined that ISL reduced the mRNA levels of inflammatory factors interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), and monocyte chemotactic protein-1 (MCP-1), while it increased the expression of several lipoprotein-related genes in peritoneal macrophages treated with lipopolysaccharide (LPS). ISL also enhanced peroxisome proliferator-activated receptor gamma (PPARγ) protein levels and reversed the changes of ATP-binding cassette transporter A (ABCA1) and cluster of differentiation 36 (CD36) in macrophages treated with oxidative low-density lipoprotein (ox-LDL). Then, in an in vivo study, female apoE^−/− mice were fed a Western diet with ISL (0, 20, 100 mg/kg/day) added for 12 weeks. We found that ISL decreased the plasma cholesterol levels of very low-density lipoprotein (VLDL)/LDL, promoted plasma superoxide dismutase (SOD) and paraoxonase-1 (PON1) activities, and decreased plasma IL-6, TNF-α, and MCP-1 levels. Moreover, ISL significantly reduced the atherosclerotic lesions and hepatic steatosis in apoE^−/− mice. In the liver, ISL altered the expression of several key genes (such as SRBI, ABCA1, ABCG8, PPARγ, and FASN) involving cholesterol-selective uptake and excretion into bile, triglyceride (TG) biosynthesis, and inflammation. These results suggest that the atheroprotective effects of ISL are due to the improvement of lipid metabolism, antioxidation, and anti-inflammation, which involve PPARγ-dependent signaling. Full article