ANTIBACTERIAL SUSCEPTABILITY OF E. COLI STRAINS ISOLATED FROM RAW MILK

ntroduction. The processing of most raw milk products can lead to contamination with unwanted microflora due to poor sanitation and hygienic conditions. The inadequate antibiotic use over the past decades has led to the emergence and wide spread of bacterial populations, particularly of Escherichia coli, which developed resistance to antibacterial drugs.Material and methods. Raw milk samples were obtained from clinically healthy cows on farms from Kiev and Poltava regions to identify E . coli, Staphylococcus spp., Enterococcus spp. isolates. Antimicrobial susceptibility testing was performed using the EUCAST disk diffusion method and MU on “Determination of microbial susceptibility to antibacterial drugs”. Results. The examined milk samples revealed the presence of E . coli, Staphylococcus spp. and Enterococcus spp. isolates, which proves poor sanitary and hygienic conditions of milk production process. Escherichia coli isolates were found susceptible to Ampicillin/sulbactam, Cefoxitin (100%), Meropenem, Tobramycin (100%), Netilin, Tigecycline, Nitroxoline (100%), Gatifloxacin, and Nitrofurantoin (100%). The studied E. coli isolates were found resistant to Ampicillin (100%), Imipenem, Tetracycline, and Doxycycline (100%). 41.7% of isolates of Staphylococcus epidermidis, Staphylococcus aureus were found resistant to Oxacillin, of which 90% were resistant to Benzylpenicillin and 20% to Rifampicin. Conclusions.The circulation of antibiotic-resistant Enterobacteriaceae strains among farm animals is a major problem requiring a strategy development aimed to prevent the emergence and spread of antibiotic resistance worldwide.


INTRODUCTION
The production of high-quality raw milk depends on various factors related to both genetics and physiological condition of the dairy cattle, as well as on the product manufacturing technology. Moreover, individual factors might have a remote impact on milk quality and safety. Thus, the use of antibiotics for therapeutic purposes in lactating animals can significantly affect the antibiotic-resistant properties of microorganisms found in milk and serve as one of the pathways for the spread of antibiotic-resistance genes in the environment (1). Furthermore, the antibiotic resistance of the same microbial strains, isolated from animals kept in the same room, may differ depending on the type of antibiotics used to treat cows at different stages of production. A research on raw drinking milk on retail sale in England revealed pathogenic agents or signs of poor zoosanitary guidelines in almost half of the samples studied (2). This problem occurs regard-less of the level of livestock farming and dairy industry development (3). More than 150 antibiotics are used in the production of livestock products used for human consumption and 90% of them are natural products of bacteria, fungi and semi-synthetic substances obtained as a result of natural products modification or even synthesis (4). The most widely used antimicrobial agents used in treatment of productive animals are β-lactams, tetracyclines, aminoglycosides, lincosa-mides, macrolides, and sulfonamides (5,6). Close to AN-VISA (7). Microorganisms isolated from lactating cows show resistance to both natural and synthetic antibiotics. Escherichia coli isolated from cattle rectum exhibited high resistance to ampicillin (59.09%) and tetracycline (43.43%) (8). Special attention should be paid to the commensal microbiota (Escherichia coli, enterococci). These bacteria can also acquire antimicrobial resistance due to the selective pressure and may act as reservoirs for antimicrobial resistance and virulence genes within the environment, as well as in food and agricultural animals, which are likely to transmit resistance to pathogenic bacteria (9). Previous researches suggest that E. coli may generally enhance the mutation rates of target cells contributing to antibiotic resistance (10). Staphylococci were found the most common pathogens isolated from milk samples taken from cows with clinical and subclinical mastitis across several countries. Staphylococcus aureus is the main pathogen of this genus, being responsible for up to 40% of all mastitis cases in some geographic regions (11). A thorough under-standing on antibiotic resistance mechanisms is paramount to developing new strategies for preventing the emergence of resistance (12).

MATERIAL AND METHODS
Milk samples (32) were obtained from clinically healthy cows from the farms of the Kiev and Poltava regions. Culture media were prepared and controlled according to ISO 11133:2014 Microbiology of food, animal feeding stuffs and water. Preparation, production, storage and performance testing of culture media. The nutrient media, commercial tests, and discs with antimicrobial drugs manufactured by HiMedia were used within the study. Isolation and identification of E. coli used the appropriate ISO 16649-2:2014 (ISO 16649-2:2001, ITD) Microbiology of food and animal feeding stuffs. Horizontal method for the enumeration of -glucuronidase-positive Escherichia coli. Part 2. Colony-count technique at 44°C using 5-bromo-4-chloro-3-indolyl-D-glucuronide. Isolation and identification of Staphylococcus spp. was carried out in accordance with ISO 6888-1: 1999 / Amd 1: 2003. Microbiology of food and animal feeding stuffs -Horizontal method for the enumeration of coagulase-positive staphylococci (Staphylococcus aureus and other species) -Part 1: Technique using Baird-Parker agar medium -Amendment 1: Inclusion of precision data. Isolation and identification of Enterococcus spp. was carried out in accordance with SSU 8534: 2015 Food products. Method for detection and determination of Enterococci (8534: 2015 Food products Method for detection and enumeration of Enterococci). Antimicrobial susceptibility testing was performed using the EUCAST disk diffusion method and MU on "Determination of microorganisms susceptibility to antibacterial drugs" (MHU 2009) (13,14). The study results were recorded and interpreted via an Automatic Colony Counters Scan® 500 manufactured by INTER-SCIENCE.

RESULTS
The present study examined milk samples obtained from clinically healthy cows from livestock complexes located in the Kiev and Poltava regions. The results of bacteriological studies showed that E. coli and Enterococcus spp. strains were found in 100% of raw milk samples; thus,  Antibiotic sensitivity in 24 E. coli strains isolated from raw milk was studied to beta-lactams from the groups of penicillins (semi-synthetic and inhibitor-protected drugs), cephalosporins (I-IV generations), carbarpenems; as well as the E. coli sensitivity to aminoglycosides (I-III generations), tetracyclines, quinolones (I-IV generations), Nitrofurantoin and Chloramphenicol.
E. coli sensitivity to the group of semi-synthetic penicillins, namely to Ampicillin, Piperacillin, Ticarcillin, Ampicillin/sulbactam, Ticarcillin/ clavulanic acid was also studied. The research results showed that 100% of the studied cultures showed resistance to Ampicillin ( fig. 2)   The studied isolates also showed ambiguous sen sitivity to carbapenems viz. 20% of isolates were sensitive to Imipenem and 90% -to Meropenem, being resistant in 80% and 10% of strains, respectively (tab. 1).
E. coli isolates were found sensitive to Gentamicin, Kanamycin, Tobramycin, Netilin, Amikacin from the aminoglycoside group in 75%, 16.6%, 100%, 95.8% and 66.7%, respectively (tab. 2). It should be noted the high occurrence of isolates susceptible to Tobramycin (100%) and Netilin (95.8%); whereas a greater amount of isolates were resistant (41.7%) and moderately resistant (41.7%) to Kanamycin.  Most Escherichia coli isolates were found resistant to semisynthetic penicillins; 100% of the isolated cultures were resistant to Ampicillin. Escherichia coli isolates were selectively susceptible to inhibitor-protected penicillins, thus 95.8% of the cultures were sensitive to Ampicillin/sulbactam and 20.8% of the cultures were sensitive to Ticarcillin / clavulanic acid. Escherichia coli isolates were predominantly susceptible to cephalosporins.
Furthermore, the isolates showed no sensitivity dependence to the group of cephalosporins, belonging to specific generation type. At the same time, 100% of the studied isolates were susceptible to Cefoxitin and no susceptible E. coli isolates were detected to Cefalotin. It should also be noted the low percentage of strains sensitive to Cefepim (IV).
Moreover, there was no clear sensitivity proved to antimicrobial drugs from the carbapenem group. Most isolates showed resistance to Imipenem and were sensitive to Meropenem.
The tested E. coli isolates were generally susceptible to Aminoglycosides, viz. to Tobramycin (second-generation aminoglycosi-des) in 100% and to Netilin in 95.8% of isolates. At the same time, 16.6% of isolates were susceptible to Kanamycin.
The studied E. coli isolates were predominantly resistant to Tetracyclines and only 16.7% of the isolates were susceptible to Tetracycline. At the same time, 80% of the studied strains showed sensitivity to Tigecycline (the first-generation antibiotic of the glycylcycline group).
The tested E. coli isolates were also predo-minantly susceptible to quinolones and fluoroquinolones, particularly to the high activity of Nitroxoline, Nalidixic acid, and Gatifloxacin. Nitrofurantoin was also highly active against E. coli isolates, while only 50% of the studied E. coli isolates were susceptible to Chloramphenicol. 41.7% of Staphylococcus epidermidis isolates were resistant to Oxacillin, of which 90% were resistant to Benzylpenicillin, -20% to Rifampicin, thus indicating an inappropriate use of antibacterial drugs in animals for disease control, prevention, and treatment.