The ability of bacteria to produce a biofilm is considered an important virulent property in pathogenesis of mastitis. The purpose of studies is to investigate the ability to form biofilms, their density, to determine and compare the sensitivity to antibacterial drugs of planktonic and biofilm forms of the main bovine mastitis pathogens on dairy farms of the Western region of Ukraine.Diagnosis of bovine mastitis, selection of milk samples and secretions of the mammary gland, microbiological studies were carried out in accordance with generally accepted methods. The performed studies have established that among pathogens, both acute and chronic forms of mastitis, the most productive film-forming ability had S. aureus strains, which on average 1.5 times more often formed the biofilm than Str. agalactiae and Str. dysgalactiae strains. It was revealed that S. aureus strains, isolated from cows under the subclinical form of mastitis and at carriage, 2.0 times (p <0.05) more often formed biofilms than in the clinical form of mastitis. The highest sensitivity of planktonic bacteria to pathogens of mastitis of streptococci and staphylococci was to ceftriaxone and doxycycline (100-80.9%). The least susceptible streptococci and staphylococci were to benzylpenicillin 32.3-45.4%, and the susceptibility of S. aureus strains was 19.0%.When determining the influence of antibiotics on biofilm forms of bacteria found that cells in the biofilm are more resistant to antibacterial drugs. It was found that antibiotic enrofloxacin completely inactivated streptococci and staphylococci in biofilms. Also, antibiotics ceftriaxone and doxycycline were also effective on bacteria in biofilms. At the same time, under the action of antibiotics penicillins, aminoglycosides and macrolides, the amount of microbial cells that survived in a biofilm was about lg 5.3 CFU/cm2 of area. Consequently, studies have shown that it is necessary to seek effective methods and develop new drugs that would influence the bacteria in biofilms to effectively treat bovine mastitis.
The article presents the results of the research of the new detergent agent “San-active” for meat processing enterprises. It was established that “San-active” in the concentration from 0.3 to 2.0% is moderately alkaline (concentration of hydrogen ions is 11.44–12.7), at a concentration of 2.5% and above, with very alkaline pH ≥ 13.11 units. In the “San-active” detergent, at the concentration from 0.3 to 2.5%, the surface tension is 34.97–28.24 mN/m. The absorbability of the parts of the technological equipment with the solutions of the “San-active” means sharply increases with increasing concentration. At the temperature of solutions of the medium 19.0 ± 1.0 °С the angle of wetting decreases from 69.8 degrees. at a concentration of 0.3% to 50.5 degrees. at a concentration of 2.5% (in 1.4 times). It has been established that “San-active” in 0.5% concentration provides the bactericidal effect on test cultures of conditionally pathogenic bacteria, spore-forming microorganisms and fungi. The “San-active” agent at 0.5% concentration is bactericidal to S. aureus and E. faecalis cells that are in a biofilm in 10 minutes of exposition. For the inactivation of E. coli and P. aeruginosa cells in a biofilm, it is necessary that the “San-active” acts in a concentration not lower than 0.5% and not less than 30 minutes. The agent shows a washing effect on the evaluation of “good” at 0.5% concentration, and 1.0% and above the concentration on the score “excellent”. “San-active” in the concentration from 1.0 to 2.0% shows very weak corrosion activity on stainless steel. The use of “San-active” detergent for the sanitary treatment of equipment surfaces in the intestinal workshop at the concentration of the working solution 1.0–2.0% and the temperature 60 ± 5 °C for 20 minutes provides 99.9–100% efficiency of sanitary treatment.
Bacteriophages may be an alternative method of treatment for antibiotic-resistant bacteria, including mastitis in cows. Our study describes the initial isolation and bacteriological activity of bacteriophages, circulating on dairy farms, the against S. aureus var. bovis. Samples of cow’s milk secretions with signs of mastitis and sewage water were used as the study material. The isolation and production of pure bacteriophage lines were performed according to the double agar method. The method of studying a single cycle of phage reproduction was used to determine the duration of the latency period. Determination of the spectrum of the lytic activity of bacteriophages against the clinical isolates of the microorganisms was carried out by the drop method. As a result of the research, four phages, specific for S. aureus var. bovis were isolated: Phage SAvB07, Phage SAvB08, Phage SAvB12 and Phage SAvB14. The negative colonies of the isolated phages were 1–2 mm in size, rounded with clear edges, with varying degrees of transparency. The latency period of Phage SAvB14 was 35 min, with the number of active virions increasing by 8 orders. In the study on growth curves of other bacteriophages, taken in the experiment, the latency period was more than 35 min, and their titre increased by only two orders. Phage SAvB07, Phage SAvB08 and Phage SAvB12 were able to lyse the bacterial strains of S. aureus var. bovis in 25–45.6% of the cases (low lytic activity), whereas Phage SAvB14 lysed 94.1% of S. aureus strains were isolated from the cows. Studies have shown that among the bacteriophages we have studied, Phage SAvB14 with a short latency period has the best lytic action on the culture S. aureus var. bovis. The resulting bacteriophage strain can be used to create a bacteriophage-based drug for the treatment of mastitis in cows.
Methicillin-resistant staphylococci can often asymptomatically colonize animals and humans and are capable of causing disease in them. Therefore, their identification and species identification are important for establishing the source of zoonotic infection and the reservoirs of antimicrobial resistance genes. The purpose of the search was to study the spread of methicillin-resistant staphylococci on dairy farms in the Western region of Ukraine. BD Baird-Parker Agar (HiMedia, India) was used to isolate staphylococci. Specific identification of pure cultures was performed using “RapID Staph Plus” kits (Oxord, UK). Staphylococcus sensitivity to methicillin was determined by inoculum application on Muller-Hinton agar with oxacillin (HiMedia, India). The sensitivity of the isolates to antibacterial preparations was determined by disco-diffusion method. The results of our searches show that Staphylococcus aureus is virtually identical in the amount both from cows (50.1 %) and from humans (62.4 %). In this case the frequency of its isolation among other species was 20.3 %. Along with Staphylococcus aureus there are such species as: S. haemolyticus (20.3 %), S. saprophyticus (13.6 %), S. xylosus (14.0 %), S. chromogenes (11.1 %), S. sciuri (8.8 %), S. epidermidis (4.8 %), S. hominis (3.4 %), S. cohnii (2.6 %) and S. warner (0.7 %). In this case, approximately the same irradiation of cows, humans and the environment by species S. haemolyticus (44.5:70.8:58.8 %), S. epidermidis (12.7:16.6:9.1 %), S. xylosus (26.0:37.4:52.9 %) is observed. The share of S. aureus strains on methicillin-resistant dairy farms in the Western Ukraine is 26.8 %. The proportion of S. aureus strains on methicillin-resistant dairy farms in the Western Ukraine is 26.8 %. Methicillin resistance is also shown S. haemolyticus, S. saprophyticus, S. xylosus та S. chromogenes. In this case their number is 1.1, 1.3, 1.6 and 5.5 times lower, respectively, and S. hominis 1.2 times higher than S. aureus. In addition, the selected cultures simultaneously show resistance to two or more antibiotics. Thus, staphylococci circulating on dairy farms are a large reservoir of resistance genes of antimicrobial preparations. Therefore, it is necessary to establish a constant control of the secretion of staphylococci resistant to β-lactam antibiotics.
A significant element of the prophylaxis of nosocomial infection in veterinary clinics is monitoring ambient objects, air, equipment, and instruments. In order to determine the role of boxes for keeping ill animals as a source of transmission of pathogens of nosocomial infections in veterinary clinics, we studied the microflora of surfaces of boxes and bioaerosol prior and after sanitation. For this purpose, we collected rinses from the surfaces of plastic and steel boxes, air samples prior to morning sanitation, after cleaning and wiping the surfaces with water and detergents and after disinfection. From the surfaces of the boxes for holding animals, we mostly isolated bacteria of Staphylococcus spp., Streptococcus spp., Micrococcus spp., Corynebacterium spp., Enterococcus spp. and Bacillus spp. Gram-negative species we found were bacteria of Escherichia spp., Acinetobacter spp. and Enterobacter spp. After wet cleaning and disinfection of plastic boxes, we detected species of Staphylococcus spp. and Enterococcus spp. in 5.4% of the samples, Micrococcus spp. in 8.1% and Bacillus spp. in 2.7%. Gram-negative bacteria of Enterobacter spp. were found in 2.7% of the samples. At the same time, the number of microorganisms in samples in which the bacteria were found after disinfection on the surfaces of stainless-steel boxes was 2.0 times lower than in such from the surfaces of plastic boxes. We determined that after wet disinfection of boxes’ surfaces, there occurred decrease in the microbial number in the air, equaling 3.7 times on average, compared with prior to disinfection. The basis of the air microflora after disinfection comprised species of Micrococcus spp., Corynebacterium spp. and Staphylococcus spp., which can be airborne-transmitted. Bacteria that were isolated from the boxes after disinfection (Micrococcus spp., Staphylococcus spp.) formed highly dense biofilms, which probably ensure the survival of the microbial cells, thus making the boxes a probable source of nosocomial infection.
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