In Vitro Sensitivity Research Concerning Some Microorganisms at Hydroxyquinoline and Cupric Derivatives Deposited onto Hydroxyapatite

ABSTRACT Introduction: The preparations based on hydroxyquinoline, in various combinations, are used in medicine, being shown to have an inhibitory effect against bacteria, molds, fungi, parasites, and viruses, but also having other beneficial effects mentioned in other medical conditions (anti-cancer, anti-degenerative, anti-inflammatory). Aims: In vitro susceptibility testing of microorganisms: bacteria (Gram positive and Gram negative), yeast (Candida spp.,) and unicellular algae (Prototheca spp.) at the preparations based on hydroxyquinoline (HQ) and its cupric derivatives deposited on hydroxyapatite (HAP). Materials and methods: There were tested microbial strains of the following genera: Escherichia, Staphylococcus, Micrococcus, Bacillus, Candida, and Prototheca. The tested products (developed in the Laboratory for Nanobiomaterials Synthesis, Center of Physical Chemistry, Faculty of Chemistry and Chemical Engineering, UBB Cluj-Napoca) were developed in three versions: 1) HQ-Cu2+-HAP1; 2) HQ-Cu2+-HAP2; and 3) NHQ-Cu2+-HAP2, where NHQ stands for nitro hydroxyquinoline. Determination of the inhibitory effect was conducted by diffusion technique in nutrient agar, according to CLSI 2013 standards, with necessary adaptations for testing of products made in the form of suspensions. Results: Following interpretation, it was found that the inhibition zones, arising from the antimicrobial effect of the tested products showed variability in size, depending on the test product and the microbial strain: Escherichia coli (8-10 mm), Staphylococcus sp. (17.6 - 23.2 mm), Micrococcus spp. (24.4 - 27.6 mm), Bacillus spp. (14.0 - 16.0 mm), Candida spp. (20.4 - 25.2 mm), Prototheca spp. (20.8 - 30.0 mm). From the three tested products, the best efficacy was found at the product no. 3 (NHQ - Cu2+ - HAP2), followed by no. 1 (HQ- Cu2+-HAP1) and no. 2 (HQ-Cu2+-HAP2). Conclusions: The inhibitory effect was bactericidal, manifested more intensively against Gram-positive bacteria, yeasts, and prototheca. Such products, prepared in the form of suspensions, may have practical application in the prevention and treatment of skin or hooves disorders. No resistance phenomena are recorded. Keywords: copper, hydroxyapatite, hydroxyquinoline, microorganisms, sensitivity

In Vitro Preparation And Testing Of Anti-Salmonella Vaccine Against Abortion In Sheep

Abstract: In March 2016, the microbiology laboratory of the Faculty of Veterinary Medicine in Cluj-Napoca, 3 type of antisalmonella vaccine for sheep were prepared. For type 1, 24 hours salmonella on brain heart infusion (BHI) broth culture, heat inactivated for 1 hour at 60°C, then with formaldehyde in a concentration of 3 ‰. Variant 2 - the culture supernatant obtained on solid BHI medium, washed with PBS, frozen-thawed 6 times, centrifuged at 4000 rpm for 15 minutes, filtered through 0.2 μm Millipore filter Orange Scientific. Variant 3 - suspension of cell walls remaining after centrifugation suspended in PBS and inactivated with formaldehyde.After the bacterial and fungal sterility control, the three types of vaccine were administered in rabbits by the subcutaneous route at a dose of 1 ml/individual along with 1 ml of plant extract adjuvant (decoction of Ewernia prunastri) sterilized by filtration.There have been two booster inoculations of the initial administration, 7 and 14 days after. Before each dose of vaccine, blood was sampled from the marginal auricular vein in order to control the immunogenicity by anti-somatic ˮOˮ serum antibody (Ab) titration using slow microplate agglutination test (Widal reaction). After three inoculations with the vaccine variant 1, Ab serum titer reached 1/128, and in types 2 and 3, 1/512 after 2 inoculations, decreasing to 1/256 after the second booster administered with no immunomodulator

Morphological and Cultural Characterization of some Strains of Unicellular Algae of the Genus Prototheca Sampled from Mastitic Cow Milk

Prototheca is a unicellular, achlorophillous, ubiquitary and saprophytic alga spread in the surrounding environment mostly in vastly damp locations in the presence of the organic matters. This is deemed to be pathogenic to a low degree, taking immediate advantage of the environment and triggering diseases at a time when the immunological capability of defense of the organisms is low or, when favorable factors come about. Prototheca is capable to induce disease in man as well as in several other animal species. Twenty strains of Prototheca sp, were sampled from cows with mastitis. The strains are characterized morphologically and developmentally on various culture media: glucose media (broth and agar), blood agar, potato medium, glucose medium of various pH values, medium containing antibiotics, Mac Conkey agar, Smith Baskerville medium (without antibiotics), and engine-oil medium. Growth took place within conditions of aerobic at 37 C and the culture became visible within 36-48 hours. On liquid media there has been noticed that the medium keeps settled and the tube bottom shelters granular drags, that lend to slight homogenization. On solid media the colonies become visible within 36-48 hours. By continuing to maintain them at room temperature, they increase in size. First, they are small, irregular in shape resembling ice crystals reflecting shiny irisations. With lapse of time, they increase in size reaching 3-5 mm, displaying pale-white colour. They also increase in height and the surface takes a mulberry or cauliflower shape. Morphological assessment was carried out on wet smear in Lol solution or in thinned staining solutions (methylene blue, malachite green, Congo red or fuchsine), allowing for good differentiation in the inner structures. The prevailig shape of Prototheca is that of an egg, seldom round or, that of a kidney with variation in size between 9.5/10.5?m up to 27.5/30.4?m. With some cells 6 to 8 endospores were noticed. Based on morphological and cultural characters there was set down that the strains under surveillance belonged to the species of Prototheca zopfii.</span

Prevalence and Antimicrobial Susceptibility Profiles of Pathogen Isolated from Bovine Mastitis Milk in Transylvania, Romania

Mastitis in cows, one of the most common and economically important infectious diseases of dairy cattle, all over the world, with significant impact due to economic losses, occurs when the udder becomes inflamed because the leukocytes are released into the mammary gland usually in response to bacteria invasion of the teat canal. The main objective of this study was to evaluate the in vitro antimicrobial susceptibility of bacteria isolated from milk in order to design specific control programs for bovine mastitis in this area. A total of 204 milk samples aseptically collected both from farms and private owners were processed during May 2014 and March 2016 within the Microbiology Laboratory of the Faculty of Veterinary Cluj-Napoca, Romania. The microbiological examination was carried out by inoculation on blood agar and MacConkey medium. After the overnight incubation in aerobic conditions, the identification of the isolates was performed using microscopic, cultural and biochemical methods. Biochemical identification was based on API 20 Biomerieux system. Susceptibility to antibiotics was evaluated using Kirby Bauer disk diffusion method on Mueller-Hinton agar; the antibiotics were represented by Amoxicillin and Clavulanic Acid, Ceftiofur, Florfenicol, Mastidiscs, Enrofloxacin, Penicillin and Tetracycline. Staphylococcus spp. was the most common isolated pathogen, in 54.9% of the specimens, followed by Streptococcus spp. in 20.1%, Escherichia coli in 10.78%, Klebsiella spp. in 8.34%, Bacillus spp. in 5.88%. The most frequent associations were represented by staphylococci-streptococci in 62.7% of the samples, followed by streptococci-bacillus in 19.8% of the samples. The most important etiological agents identified were Staphylococcus aureus, S uberis, Streptococcus agalactiae, and Escherichia coli. Antimicrobial susceptibility test for the total isolates revealed good sensitivity to Enrofloxacin, Mastidiscs and Amoxicillin and Clavulanic Acid. Resistance was observed for Penicillin and Tetracycline. The major mastitis pathogens identified was Staphylococcus aureus, while recurrent mastitis treatment was based on systemic and local administration of Enrofloxacin and Mastidiscs respectively