CLINICAL ISOLATES OF E.COLI IN PIGS – ANTIMICROBIAL RESISTANCE AND PERSPECTIVES TO OPTIMIZE ANTIBIOTIC ADMINISTRATION

Modern livestock production inevitably involves the use of antimicrobial drugs. Adequate use thereof depends on the application of appropriate biosecurity measures and timely and accurate diagnostics of diseases. Administration of antimicrobial drugs without previous identifi cation of “zootechnical issues” or relevant laboratory analyses may lead to the development of antimicrobial resistance (AMR). Surveillance and monitoring of AMR is conducted according to prescribed procedures and includes sampling at slaughter line. Development of antimicrobial resistance (AMR) and occurrence of resistance gene may be a result of inadequate use of antibiotics and uncontrolled trading of antibiotics. In this research, we monitored the presence of specifi c bacterial species belonging to the Enterobacteriaceae family and their sensitivity to particular antibiotics in diverse animal categories on pig farms over the breeding period. Th e aim of the study was to establish the following: development of antimicrobial resistance by isolated bacteria, resistance to several diverse groups of antibiotics, and potential alternatives to antibiotics in the cases when therapy is required. Th e research confi rmed the development of AMR during pig production process, which is oft en manifested as multiple resistance (group of penicillin and synthetic penicillin drugs, aminoglycosides, fl uoroquinolones, tetracyclines).


INTRODUCTION
Competitive interactions between microbes in natural environment resulted in the development of antimicrobial compounds as a necessary "weapon" aimed at limiting the presence and growth of specifi c organisms that make the ecosystem microfl ora. Th e creation of such substances has enabled the pro-ducer-organisms to eff ectively inhibit the growth of the competitor-microfl ora and thus provide favorable conditions to disperse in the environment (Huttner et al., 2013). Th e presence of antimicrobial substances inevitably instigated the development of microbial defense mechanisms adopted by bacteria to overcome the cidal and static eff ects of antimicrobials and survive in natural environments (Hibbing et al., 2010;Kassen et al., 2004;Boles et al., 2004;Kirisits et al., 2005). From the perspective of the nature, the described mechanisms cannot be considered a problem for antimicrobial resistance. Since the discovery of antibiotics and their use for therapy and later for preventive purposes or for the boost of growth in livestock production, they have been putting tremendous pressure on all microorganisms in all of their habitats and contact sites. Th us, in order to survive in the environment, the microbes have developed diverse forms of resistance. It could be concluded that from the moment of producing antimicrobial substances in surplus, the response of the nature was to develop and spread antimicrobial resistance.
Application of antibiotics in livestock production involves therapeutic use for treating infections, the use for prophylactic purposes and growth promotion to improve production potential and decrease undesirable eff ects of bacteria (Jarlier et al., 2012) and stimulate productive and genetic potential of animals (Kittitat et al., 2018). Administration of antibiotics over a short or prolonged period might result in development of antimicrobial resistance, which reduces and/or completely eliminates the eff ectiveness of antibiotics. Th e consequences of antimicrobial resistance are associated not only with decreased production results but also with poor therapy prospects in diseased animals.
Antimicrobial resistance nullifi es the eff ects of antibiotics in preventing adverse eff ects of existing bacterial fl ora or limits the presence of pathogenic organisms through static/cidal eff ects. Transfer of microbial resistance gene poses a particular problem. Both humans and animals can be exposed to highly resistant bacteria through diff erent transmission routes, even if they are not close to the carriers. Resistant strains of enteric bacteria pose the most serious threat to human health (Huttner et al., 2013). Th is problem is of major signifi cance, since pathogens that have acquired resistance are able to colonize animals and/or humans who had previously not been exposed to antibiotics, resulting in therapeutic failure.
Th e control of antimicrobial resistance in domestic animals is related to the examination of susceptibility of isolated microorganisms at slaughter line (Commission Implementing Decision, 2013). Such monitoring practices can bring about diff erent results on antimicrobial resistance as compared to the fi ndings obtained during grower and fattening phase. Namely, during grower and fattening stage, the animals are exposed to antibiotics, which results in development of antimicrobial resistance. In the period before slaughtering, the use of antibiotics is prohibited in order to eliminate antibiotic residues from meat and contribute to reduction or elimination of the resistance (Commission Implementing Decision, 2013).
In this research, we monitored the presence of particular bacterial species from the Enterobacteriaceae family in diff erent production categories of pigs on farms and their susceptibility to specifi c antibiotics. Th e aim of the study was to determine the following: development of antimicrobial resistance in isolated bacteria, resistance to several diff erent groups of antibiotics, and potential alternatives to antibiotics in the cases when therapy is necessary.

MATERIAL AND METHODS
Th e material for examination was obtained from eight pig farms. Th e samples included rectal swabs from piglets, parenchymatous organs and intestines of dead animals. All the animals selected for the sampling were subjected to antibiotic therapy due to diff erent health problems (respiratory and digestive diseases). A total of 26 rectal swabs and 9 samples from dead pigs were examined. Antibiograms were prepared from 28 isolated bacterial strains. Before collecting rectal swabs from piglets, the swabs were immersed into sterile saline and transported to the laboratory in cooling boxes on the same day. Th e swabs and organs of dead animals were inoculated onto the MacConkey (Biokar), XLD (Biokar) and blood agar (TSA (Biokar) +5% defi brinated sheep blood). Identifi cation of isolated bacteria from the Enterobacteriaceae family was performed using biochemical tests (oxidase and catalase test, indole, methyl red, urea, citrate).
Suspension for determination of antimicrobial susceptibility of isolated bacteria was prepared according to CLSI procedure (CLSI, 2016). Microbial suspension was prepared from 24-hour old cultures matching McFarland 0.5 standard. Using a swab, the suspension was transferred to a Mueller-Hinton agar (Biokar, France), and antibiotic discs (Bioanalyse, Turkey) were placed onto the cultures. Inhibition zone readings were performed according to manufacturer's instructions.
Antibiotic disks and method were controlled according to manufacturer's instruction. Reference strain (Escherichia coli ATCC 25922) was used as control test. Suspension of E. coli ATCC 25922 was tested on three antibiotics (Tetracycline 30 μg, Gentamicin 10 μg and Ceft riaxone 15 μg).

RESULTS
During the investigation period, rectal swabs of piglets or organs of dead piglets at nursery/growing stage (from the pre-weaning period at 4-5 weeks of age to the pre-fattening stage at 10-12 weeks of age) were analyzed. Considering that this is the most critical period of piglet breeding cycle, the animals received diverse antibacterial therapies. Drug administration was indicated according to clinical fi ndings of digestive disorders manifested by various forms of diarrhea with lethal outcomes. Besides digestive disorders, respiratory symptoms were recorded and relevant antibacterial therapy was introduced. Administration of antimicrobial drugs was not indicated by previous laboratory examination. Th e therapy was applied without a prior evaluation of microbiological status and antibiotic susceptibility of bacterial fl ora. Table 1 illustrates the data on susceptibility/resistance of bacteria isolated from piglets. Th e data are expressed as a percentage, according to each individual farm.

DISCUSSION
Th e control of antimicrobial resistance is carried out according to the Directive on the monitoring and reporting of antimicrobial resistance in zoonotic and commensal bacteria and pertains to determination of susceptibility and/or changes in the susceptibility of specifi c bacterial species that may be isolated from food-producing animals or from foods (Salmonella spp., Campylobacter jejuni, Campylobacter coli, Escherichia coli, Enterococcus faecium and Enterococcus faecalis). Sampling procedures and monitoring of bacterial species, which are potential carriers of the resistance, are laid down in relevant legislation and regulations on the collection of materials for examination from the slaughterhouses, the amount which will be proportional to the annual production output in the country (Commission Regulation, 2005). Our research was aimed at monitoring the occurrence of resistance in strains isolated from animals at the beginning of production cycle. Th e isolated strains of E. coli, Salmonella enetrica serovar infantis and Enterobacter spp. are part of bacterial fl ora commonly found in pigs. Confi rmed presence of resistant bacterial strains strongly indicated previous application of substantial amounts of antibiotics. During our research, a high percentage of resistance of isolated strains was monitored with regards to bacterial species and the farm. Our investigation of sensitivity of some bacteria from the Enterobacteriaceae family revealed high resistance rates in piglets at grower stage. Th e highest resistance was determined for penicillin, tetracycline and doxycycline, whereas resistance to amoxicillin, neomycin and streptomycin was somewhat lower. Multiple resist-ance was observed on all farms, except on farm I. On almost all farms, multiple resistance to three or more diff erent antibiotic groups was recorded. Th e study from 2018 that included two farms -the fi rst one used in-feed tiamulin and amoxicillin while the second one used feed without antibiotic supplementsrevealed the presence of multiple resistance of isolated enterobacterial strains on both farms. However, the prevalence was somewhat higher on the farm where the animals were fed antibiotics. In the same research, the authors established the highest resistance rate of enterobacteria in the post-weaning period and during growerstage (Kittitat et al., 2018). Our results correspond to the results of this research, especially for the resistance in piglets at grower stage. Investigation of antimicrobial resistance of E. coli strains isolated on pig farms revealed multiple resistance resulting from inadequate administration of antimicrobial drugs (Kallau et al., 2018). According to the available data (Lagha et al., 2017), tetracycline is most commonly used antibiotic in pig production and thus one of the causes of resistance of some strains (especially E.coli) to this antibiotic, which was also confi rmed in our study. Th e development of resistance by isolated E. coli strains is of importance not only for pathogenic strains. Since this is a commensal organism and most important bacterium residing in digestive system, the resistance of these strains and potential transfer of resistance gene poses the highest risk for the entire natural environment Addressing the problem of antimicrobial resistance and prevention of its transfer within human population includes several approaches. Prevention of transmission of resistant bacterial strains among humans (horizontal -via immediate contact) through improved hygiene is one of the fi rst steps. Limiting antibiotic administration by avoiding their imprudent use as therapeutics in cases when they are not appropriately indicated and stimulating the development of novel antibiotic drugs are of crucial importance (Carlet et al., 2011;Jarlier et al., 2012). Th e aforementioned measures for preventing transmission of resistance gene carrier strains are also applicable in pig industry. Considering the specifi cities of livestock production providing relevant zoohygienic and biosecurity measures as well as the costs of novel antibiotic drugs, the implementation of such measures is quite a challenging task. Th e use of feed supplements that aff ect the intestinal pH level (prebiotics) or the gut microbiota composition (probiotics) are potential alternatives to antibiotic administration.
Bacteriocins are peptides synthesized mostly by Gramm positive organisms. Th e eff ects thereof are bacteriostatic and/or bactericidal (Lagha et al., 2017). Th e majority of bacteriocins shows static and/or cidal eff ects limited to the bacteria closely related to producer -species. However, antibacterial eff ects of some bac-teriocins extends to a wider range of diff erent bacterial species (Riley and Wertz, 2002). Some bacteriocins act in synergy with conventional antibiotics, thus enabling reduction of bacteriostatic concentrations (Cavera et al., 2015). Th e mechanisms of action of bacteriocins are diverse. Th ey can target bacterial cell wall and stimulate cell lysis or aff ect protein synthesis inside the cell, i.e. bacterial gene expression (Cotter et al., 2013). Bacteriocins are divided into four classes including (I) lantibiotics, (II) non-lantibiotics or unmodifi ed peptides, (III) high molecular mass peptides, and (IV) circular peptides (Heng and Tagg, 2006).
Th e options for bacteriocins application are determined by the specifi c purpose of their use, i.e. whether they are used in food industry or in livestock production. In general, purifi ed bacteriocins can be used for both purposes and directly aff ect the microfl ora (food or digestive system) or as bacteriocin-producing strains which inhibit the growth of pathogenic bacteria. In pig production, bacteriocins (nisin or enterocin) are combined with an antibiotic to improve its eff ectiveness or can be used as specifi c bacteriocins-producing strains manifesting static eff ects on Streptococcus suis, E. coli, Haemophilus parasuis, Treponema spp., Bacteroides spp. (Lagha et al., 2017).

CONCLUSION
Th e development of resistance is apparent in piglets at grower stage, and our research indicated the necessity of monitoring bacterial susceptibility throughout all the stages of livestock production. Such fi nding strongly suggests that transmission of resistant strains to humans does not occur only through food (examination at slaughterhouses and in the market) but also through direct contact of farm staff .
To avoid the overuse of antibiotics and reduce and/or eliminate antimicrobial resistance, it is necessary to implement all biotechnical measures to prevent stress and immune decline as well as to prevent introduction of pathogenic strains in farms. Moreover, the application of bacteriocin-producing strains could complement or completely replace antibiotics. Administration of probiotics along with bacteriocin-producing strains can be considered potential sound alternative to antibiotics and contribute to reduction of antimicrobial resistance.

ACKNOWLEDGEMENT
Th is article is based on the work from scientifi c and technological cooperation between the Republic of Serbia and the Slovak Republic, bilateral project number: 337-00-107/2019-09/14 and SK-SRB-18-0001, project title: Bacteriocins, a safe way to inhibit antibiotic resistant bacteria from pigs for healthy farm-ing, supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia, and by APVV agency of Slovak Republic.