ANTIBACTERIAL SUSCEPTABILITY OF E. COLI STRAINS ISOLATED FROM RAW MILK Текст научной статьи по специальности «Биологические науки»

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ANTIBACTERIAL SUSCEPTABILITY OF E. COLI STRAINS ISOLATED FROM RAW MILK

Valerii USHKALOV1, Vyacheslav DANCHUK1, Artem USHKALOV2, Aidyn SALMANOV3, Yuriy VISHOVAN1, Sergiy BOIANOVSKIY1, Sergiy TERESHCHENKO1, Liliana DAVYDOVSKA1

1National University of Life and Environmental Sciences of Ukraine, Ukrainian Laboratory of Quality and Safety of Agricultural Products

2Main administration of state service of Ukraine on food safety and consumer protection in Kharkiv reg. 3Shupyk National Medical Academy of Postgraduate Education, Ukrainian National Committee of Infection Control and Antimicrobial Resistance

Corresponding author: Valerii Ushkalov, e-mail: [email protected] DOI: 10/38045/ohrm.2021.3.08 CZU: 637.12:579.842.11+615.33.015.8

Keywords: Escherichia coli, strains, microorganisms, raw milk, antibiotic resistance, susceptibility.

Cuvinte cheie:

Escherichia coli, tulpini, microorganisme, lapte crud, re-zistentâ la antibio-tice, sensi-bilitate.

Introduction. 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 ofE. 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/sulbac-tam, 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.

SENSIBILITATEA LA ANTIMICROBIENE A TULPINILOR DE E. COLI ISOLATE DIN LAPTE CRUD Introducere. Prelucrarea produselor din lapte crud poate duce la contaminarea cu microflora nedorita din cauza conditiilor sanitaro-igienice precare. Utilizarea inadecvatá a antibioticelor in ultimele decenii a dus la aparitia si ráspándirea pe scará larga a populatiilor bacteriene, in special a Escherichia coli, care a dezvoltat rezistentá la preparatele antibacteriene. Material metode. Probele de lapte crud au fost obtinute de la vaci sánátoase, din punct de vedere clinic, de la fermele din regiunile Kiev si Polta va, din care au fost isolate pi identifícate E. coli, Staphylococcus spp., Enterococcus spp. Testarea sensibilitápii la antimicrobiene a fost efectuatá utilizánd metoda disc difuziometricá reco-mandatá de EUCAST si MU privind „Determinarea sensibilitápii microbiene la preparatele antibacteriene". Rezultate. Probele de lapte examinate au relevat prezenta izolatelor de E. coli, Staphylococcus spp. si Enterococcus spp., ceea ce elucideazá conditiile sanitaro -igienice precare ale procesului de productie a laptelui. Izolatele de E. coli au fost sensibile la Ampici-lin/sulbactam, Cefoxitin (100%), Meropenem, Tobramicin (100%), Netilin, Tigeciclin, Ni-troxolin (100%), Gatifloxacin si Nitrofurantoin (100%). Izolatele studiate de E. coli au pre-zentat rezistenpá la Ampicilin (100%), Imipenem, Tetraciclin si Doxiciclin (100%). 41,7% din izolatele de Staphylococcus epidermidis, Staphylococcus aureus au fost rezistente la Oxa-cilin, dintre care 90% au fost rezistente la Benzilpenicilin si 20% la Rifampicin. Concluzii. Circulatia tulpinilor de Enterobacteriaceae rezistente la antibiotice printre animalele de fermá prezintá o problemá majorá care necesitá dezvoltarea strategiei menitá sá previná aparitia si ráspándirea rezistentei la antibiotice in intreaga lume.

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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 zoosan-itary 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 p-lactams, tetracyclines, aminoglycosides, lincosa-mides, macrolides, and sulfonamides (5, 6). Close to ANVISA (7). Microorganisms isolated from lactating cows show resistance to both natural and synthetic antibiotics. Escherichia coli isolated from cattle rectum exhibited high resistance to ampi-cillin (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-glucuroni-de. Isolation and identification of Staphylococcus spp. was carried out in accordance with ISO 68881: 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 En-terococci). 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 INTERSCIENCE.

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,

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Staphylococcus spp. isolates - in 100%, including 87.5% of Staphylococcus epidermis and 12.5% of coagulase-positive Staphylococcus aureus. It should be noted that among Staphylococcus

strains, 41.7% of isolates were resistant to Oxacillin, of which 90% were resistant to Benzylpenicillin and 20% to Rifampicin (fig. 1).

Figure 1. Antibiotic susceptibility of Staphylococcus aureus strains isolated from raw milk. ANT001 Benzylpenicillin (r), ANT012 Oxacillinum (r), ANT040 Levofloxacin (ATU), ANT043 Norfloxacin (s), ANT044 ofloxacin (ATU), ANT045 Amikacin (s), ANT053 Vancomycin (s), ANT056 Erythromycin (r), ANT063 Tetracyclinum (r), ANT068 Chloramphenicol (s), ANT074 Nitrofurantoin (s), ANT083 Kanamycin (r); «s» - sensitive; «r» - resistant; « ATU » - Area of Technical Uncertainty.

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, Ti-carcillin, Ampicillin/sulbactam, Ticarcillin/ clavulanic acid was also studied. The research results showed that 100% of the studied cultures showed resistance to Ampicillin (fig. 2). 57.1% of strains were resistant to Piperacillin, 14.3% were moderately resistant, and 28.6% of the studied cultures were sensitive. The studied cultures exhibited resistance to Ticarcillin in 50%, 14.3% and 35.7% respectively.

Most of the studied isolates showed sensitivity to Ampicillin/sulbactam viz. in 95.8% of cases, thus showing resistance in 4.2% of E. coli isolates. 75%

of isolates showed resistance to Ticarcillin/ clavulanic acid, 4.2% - moderate resistance, and 20.8% - sensitivity (tab. 1).

The antibiotic susceptibility of E. coli isolates to the group of cephalosporins was studied, namely to Cefalotin, Cephalexin, Cefazolin (1st generation); to Cefaclor, Cefoxitin, Cefuroxime, Cefaman-dole (2nd generation); to Cefixim, Cefoperazone, Cefotaxim, Ceftriaxone, Ceftazi-dime (3rd generation); to Cefepim (IV generation) (tab. 1). No susceptible E. coli isolate was identified to Cefalotin from first-generation cephalosporins; 37.5% of isolates were moderately resistant and 62.5% of strains were found resistant. However, 70.8% and 71.4% were sensitive to Cephalexin and Cefazolin, respectively; 29.2% and 7.2% of E. coli isolates showed resistance; and 21.4% of E. coli isolates were moderately resistant to Cefazolin.

The E. coli isolates showed ambiguous sensitivity to the second- generation cephalosporins. 100% of E. coli isolates were susceptible to Cefoxitin. 25%, 83.3% and 70% of E. coli isolates exhibited

Figure 2. Antibiotic susceptibility of E. coli strains isolated from raw milk. ANT002 Ampicillin (r), ANT003 Ampicillin/sulbactam (s), ANT009 Ticarcillin/clavulanic acid (r), ANT017 Cefaclor (r), ANT019 Cefalexin (r), ANT021 Cefepim (r), ANT022 Cefixim (s), ANT061 Doxycycline (r), ANT063 Tet-racyclinum (r), ANT064 Tigecycline (r), ANT068 Chloramphenicol (s), ANT074 Nitrofurantoin (s), ANT075 - Nitroxoline (s); «s» - sensitive; «r» - resistant; « ATU » - Area of Technical Uncertainty.

Table 1. Antimicrobial susceptibility of E. coli isolates (n=24).

Antibiotic Cultures

s ATU r

Penicillins

Ampicillin - - 100%

Ampicillin/sulbactam 95.8% - 4.2%

Ticarcillin 35.7% 14.3% 50%

Ticarcillin / clavulanic acid 20.8% 4.2% 75%

Piperacillin 28.6% 14.3% 57.1%

Cephalosporins

Cefalotin (I) - 37.5% 62.5%

Cephalexin (I) 70.8% - 29.2%

Cefazolin (I) 71.4% 21.4% 7.2%

Cefaclor (II) 25% 37.5% 37.5%

Cefoxitin (II) 100% - -

Cefuroxime (II) 83.3% - 16.7%

Cefamandole (II) 70 % 10 % 20%

Cefixim (III) 87.5% - 12.5%

Cefoperazone (III) 40% 20% 40%

Cefotaxim (III) 58.3% 16.7% 25%

Ceftriaxone (III) 83.4% 8.3% 8.3%

Ceftazidime (III) 64.3% 28.6% 7.1%

Cefepim (IV) 8.4% 20.8% 70.8%

Carbapenems

Imipenem 20% - 80 %

Meropenem 90% - 10%

Note: «s» - sensitive; «r» - resistant; « ATU » - Area of Technical Uncertainty

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sensitivity to Cefaclor, Cefuroxime, Cefamandole, respectively, and were resistant in 37.5%, 16.7%, 20% of isolates. 37.5% of isolates were moderately resistant to Cefaclor and 10% to Cefaman-dole.

The isolates showed sensitivity to Cefixim, Cefoperazone, Cefotaxim, Ceftriaxone, Ceftazidime in 87.5%, 40%, 58.3%, 83.4%, and 64.3%, respectively; resistance - in 12.5%, 40%, 25%, 8.3%, and 70.8% of cases. 8.4% strains were susceptible to Cefepim, moderately resistant -20.8%, and resistant in 70.8% of E. coli isolates.

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.

Table 2. Antimicrobial susceptibility of E. coli isolates (n=24).

Antibiotic Cultures

s m R

Aminoglycosides

Gentamicin (I) 75% - 25%

Kanamycin (I) 16.6% 41.7% 41. 7%

Tobramycin (II) 100% - -

Netilin (II) 95.8% - 4.2%

Amikacin (III) 66.7% - 33.3%

Tetracyclines

Tetracycline

16.7%

83.3%

Doxycycline - - 100%

Tigecycline 80% - 20%

Note: «s» - sensitive; «r» - resistant; «ATU» - Area of Technical Uncertainty

83.3%, 100% and 20.0% of the investigated E. coli isolates were resistant to the drugs of the tetracyclines group (Tetracycline, Doxycycline and Tigecycline), respectively. 16.7% of them showed

sensitivity to Tetracycline and 80% of strains to Tigecycline (tab. 2).

The investigated E. coli isolates were sensitive to quinolones (tab. 3).

Table 3. Antimicrobial susceptibility of E. coli isolates (n=24).

Antibiotic Cultures

s m R

Quinolones

Nalidixic acid (I) 83.33% 8.33% 8.33%

Nitroxoline (I) 100 % - -

Norfloxacin (II) 87.5% - 12.5%

Ciprofloxacin (II) 83.3% 12.5% 4.2%

Ofloxacin (II) 62.5% 25% 12.5%

Lomefloxacin (II) 41.7% 45.8% 12.5%

Pefloxacin (II) 60% - 40%

Gatifloxacin (IV) 95.8% - 4.2%

Other antibiotics

Nitrofurantoin_100%_-_

Chloramphenicol 50% - 50%

Note: «s» - sensitive; «r» - resistant; «ATU» - Area of Technical Uncertainty

100% of the tested cultures were susceptible to 83.3%, 62.5%, 41.7%, 60% of E. coli isolates were Nitroxoline and 83.33% to Nalidixic acid 87.5%, susceptible to fluoroquinolones of the II genera

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tion, viz. Norfloxacin, Ciprofloxacin, Ofloxacin, Lomefloxacin, Pefloxacin, respectively. 95.8% of the studied E. coli isolates were susceptible to the fourth-generation fluoroquinolone, namely Gat-ifloxacin.

Sensitivity to Nitrofurantoin was proved in 100 % and to Chloramphenicol in 50% of E. coli isolates tested (tab. 3).

DISCUSSIONS

Therefore, the present research revealed the presence of Escherichia coli, Staphylococcus spp. (Staphylococcus epidermidis, Staphylococcus aureus) Enterococcus spp. isolates in all the samples of raw milk, which proved the non-compliance with the sanitary and hygienic conditions for milk production processing.

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/sulb-actam and 20.8% of the cultures were sensitive to Ticarcillin / clavulanic acid. Escherichia coli isolates were predominantly susceptible to cephalo-sporins.

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

CONCLUSIONS

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-mi-nantly susceptible to quinolones and fluoroquin-olones, particularly to the high activity of Nitrox-oline, Nalidixic acid, and Gatifloxacin. Nitrofu-rantoin 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.

1. The study results revealed the presence of E. coli, Staphylococcus spp., and Enterococcus spp. isolates in 100% of milk samples obtained from clinically healthy cows on livestock farms from Kiev and Poltava regions of Ukraine.

2. Escherichia coli isolates were predominantly resistant to semi-synthetic penicillins.

3. High percentage of Escherichia coli isolates were found resistant to Tetracycline (80%) and Doxycy-cline (100%).

4. Escherichia coli isolates exhibited susceptibility to Ampicillin / sulbactam, Cefoxitin (100%), Mero-penem, Tobramycin (100%), Netilin, Tigecycline, Nitroxoline (100%), Gatifloxacin, and Nitrofurantoin (100%). The tested Escherichia coli isolates were resistant to Ampicillin (100%), Imipenem, Tetracycline, and Doxycycline (100%).

5. Staphylococcus spp. strains included Staphylococcus epidermidis (87.5%) and coagulase-positive Staphylococcus aureus (12.5%); 41.7% of Staphylococcus spp. isolates were resistant to Oxacillin, of which 90% were resistant to Benzylpenicillin and 20% to Rifampicin.

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CONFLICT OF INTERESTS

All authors declare no competing interests.

ACKNOWLEDGMENT

Research was carried out with the financial support of the Ministry of Education and Science of Ukraine.

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Valerii USHKALOV, ORCID ID: 0000-0001-5694-632X, SCOPUS Author ID 36130483300 Vyacheslav DANCHUK, ORCID ID: 0000-0003-2156-1758 Artem USHKALOV, ORCID ID: 0000-0001-8317-7909

Aidyn SALMANOV, ORCID ID: 0000-0002-4673-1154, SCOPUS Author ID 56964145900

Yuriy VISHOVAN, ORCID ID: 0000-0003-1128-593X

Sergiy BOIANOVSKIY, ORCID ID: 0000-0002-4621-5192

Sergiy TERESHCHENKO, ORCID ID: 0000-0002-5786-8711

Liliana DAVYDOVSKA, ORCID ID: 0000-0003-5385-4500

I