RESISTANCE TO FLUOROQUINOLONES IN ESCHERICHIA COLI FROM PIGS

Th e resistance of Escherichia coli (E. coli) to fl uoroquinolones has become a serious issue on large pig farms worldwide, since these antimicrobial agents are widely used in the control of various diseases such as neonatal diarrhea, post weaning diarrhea, the edema disease as well as others. Being a frequent inhabitant of the digestive tract, E.coli is oft en exposed to antimicrobial agents, which are used to treat various infections. Uncontrolled application of fl uoroquinolones has led to the emergence of resistant pathogenic strains as well as commensals. Th e spread of resistant strains is mostly found in animal and human food production chains, which are potentially huge threat for the general population. Th e resistance to fl uoroquinolones may very oft en be combined with the resistance to other classes of antibiotics. Th erefore, the use of fl uoroquinolones for treating uncomplicated infections in pigs must be under strict control or completely banned. In this paper, we compared the results from available literature addressing the prevalence of antimicrobial resistance to fl uoroquinolones in E. coli strains both worldwide and in countries from the nearby region.


INTRODUCTION
Intensive pig farming sometimes requires the use of antimicrobial agents for the therapy of various bacterial infections.If the antimicrobial treatment begins without the prior bacteriological examination of the diseased pigs, and if the use of antibiotics is excessive, it may lead to resistance in certain strains of bacteria (Henderiksen et al., 2008).Th e widespread use of antimicrobial agents has caused the mobilization of bacterial specifi c mechanisms of resistance in order to survive in the environment (Velhner et al., 2010).In addition to the pathogenic bacteria, commensal microorganisms may also become resistant to antibiotics by creating the possibility for horizontal spread of the resistance genes (Schiearch et al., 2009).Meat and meat products produced for consumption from animals infected with bacteria resistant to antibiotics pre-sent the major threat to public health (Th orsteinsdottir et al., 2008).In addition to this, multidrug resistant microorganisms have a tendency to expand intercontinentally, which means that some of the bacteria have a unique phenotype and genotype, and may be discovered by using molecular typing methods.Th e detection of resistance genes and specifi c mutations on targeted genes could be useful for the epidemiological analysis (Velhner et al., 2010).For example, dihydrofolate reductase genes such as dfrA1 are more frequent in E. coli isolates from Europe while the dfrA17 and dfrA12 are more prevalent in Korea and Australia (Blahna et al., 2006).Th e sequence type ST131 of the E. coli resistant to fl uoroquinolones and the extended spectrum beta lactam drugs with the CTX-M15 gene is widespread around the world causing urinary tract and bloodstream infections (Petty et al., 2014).
E. coli is a Gram negative bacteria, which belongs to the Enterobacteriaceae family and can cause several illnesses in humans and animals.E.coli is normally present in the microbiota of the digestive tract in humans and animals, where it is exposed to the infl uence of various antimicrobial agents, which are used for either therapy or prevention.Consequently, E. coli has developed numerous mechanisms, thanks to which it can withstand their infl uence, where the most important mechanism of resistance is the one towards the fl uoroquinolones, due to their intensive and uncontrollable use (Yue et al., 2008).Fluoroquinolones are antimicrobial agents used for the therapy in both humans and animals.Th eir mode of action is to inhibit topoisomerase enzymes.Th e resistance develops due to the point mutations on genes which encode the topoisomerase (Hopkins et al., 2005).

MUTATIONS ON THE TOPOISOMERASE GENES
Mutations that cause the development of resistance to fl uoroquinolones are located on a protein segment called the quinolone resistance-determining region (QRDR).Th e gyrA and gyrB genes encode the gyrase A enzyme.Th e QRDR is located between amino acids Ala67-Gln106 on the gyrA and Asp426-Lys447 on the gyrB gene.Single or double mutants are oft en found on codons 83 and/or 87.Th e parC and parE genes encoding the enzyme topoisomerase IV, are the secondary target for quinolone in Gram-negative bacteria.In isolates which demonstrate increased resistance to fl uoroquinolones, mutations on all four genes may be found (Hopkins et al., 2005).Resistance to fl uoroquinolones can oft en result in a cross resistance to other antibiotics such as β-lactams, tetracyclines, chloramphenicol and others.Consequently, the health of people and animals may be seriously endangered, if the use of antimicrobial agents should be needed for treatment (Hopkins at al., 2005).

THE SITUATION WORLDWIDE
Th e E coli resistance to fl uoroquinolones has become a serious issue on pig farms across the world, since this drug is used for treating several diseases such as neonatal diarrhea, post weaning diarrhea, the edema disease and others.In order to reduce the economical losses caused by these diseases, fl uoroquinolones are used in veterinary medicine, which has resulted in the acquisition of the resistance of pathogenic strains of E coli and of commensals (Huang et al., 2014).Table 1 shows the licensed quinolones and fl uoroquinolones used in livestock production in various regions in the world.Th e most recent research has shown that in China, the E coli resistance to fl uoroquinolones in farm animals, especially pigs, prevails in comparison to other countries.High level of resistance to these microbial agents arose as a result of the application of prophylactic antimicrobial therapy during the period of seven days.Because of this established situation, Huan et al. (2014) investigated the eff ect of ciprofl oxacin administered in feed on the dynamics of acquiring the resistance of E coli strains isolated from pig feces of the Landrace, Duroc and Yorkshire breed.According to these results, the minimal inhibitory concentration (MIC) for ciprofl oxacin in the experimental group, aft er three days of application, increased from ≥0.5mg/L to ≥8mg/L, while it was stable in the control group ≥1mg/L as well as in the experimental group before the application.Aft er the sixteenth day of application, the MIC for the experimental group was ≥128mg/L.Using the PCR method, point mutations were detected on the gyrA and parC genes.
According to the research results obtained by the Japanese Veterinary Antimicrobial Resistance Monitoring System (JVARM), in E. coli (n=358) strains derived from pigs, the resistance to fl uoroquinolones (enrofl oxacin and ofl oxacin) was not established.In most of the isolates (n=267), the MIC ≥0.05mg/L to enrofl oxacin was noticed, whereas MIC value for ofl oxacin was ≥0.1mg/L (n=230) (Kijima-Tanaka et al., 2003).
A study that examined antimicrobial resistance of zoonotic enteric bacteria Campylobacter spp., E. coli and Enterococci spp.isolated from pig meat and carcasses was conducted in two regions in Australia (New South Wales and South Australia).Th e resistance to ciprofl oxacin has not been established in isolated E. coli strains (Hart et al., 2004).
During the year 2004, resistance to ciprofl oxacin was not established in E. coli strains isolated from feces on 20 pig farms in Canada (in the Alberta state).In the total number of the E. coli isolates (n=1439), which were included in this study, the MIC to ciprofl oxacin was ≥0.0015mg/L in 1426 of the isolates.Th e highest MIC of ≥0.5mg/L was observed in one of the examined E. coli isolates (Rosengren at al., 2008).

THE SITUATION IN EUROPE
Th e resistance to fl uoroquinolones is mostly observed in multidrug resistant bacteria.Consequently, the therapy for animals and humans is compromised, so caution when using antimicrobial drugs is strongly advised.In some European countries, the use of fl uoroquinolones is banned in the animal husbandry.
Th e legislation in Denmark from 2002 restricts the use of fl uoroquinolones in animal husbandry, unless for treating infections, where pathogens are resistant to all the other antimicrobials.According to data from Hammerum et al. (2007), the consumption of fl uoroquinolones in 2001 was around 114 kg.Aft er implementation of this law, the use of fl uoroquinolones was reduced to 18 kg in 2005.With the 2005 legislation, the use of fl uoroquinolones and cephalosporin's was completely prohibited in livestock production, with special emphasis on pigs, because pig production shares 82% of the total animal production in Denmark.Th e implementation of this law makes Denmark the only country in Europe where the use of fl uoroquinolones (ciprofl oxacin, enrofl oxacin, difl oxacin, and marbofl oxacin) is banned in the livestock industry, which was reported by Hendriksen et al. (2008).
In the period 2002-2004, Hendriksen et al. ( 2008) conducted a study aimed at continuous monitoring of antimicrobial resistance of pathogenic bacteria and commensals, where 12 EU Member States were involved.Th is was the fi rst report on bacterial resistance to antimicrobial agents in pigs, which also confi rmed the resistance of E. coli (isolated from pig feces) towards fl uoroquniolones (ciprofl oxacin and enrofl oxacin).While during these three years there was no evidence of E. coli resistance to fl uoroquinolones in Denmark, Portugal had the highest average prevalence of resistance being 48.0% for the period 2002-2004.Spain is just behind Portugal with an average prevalence of resistance of 14.7%, while in other countries the prevalence of resistance was in the range from 1.3% to 6.5%.Data for the Netherlands was not available, while Lithuania, Norway and Switzerland are lacking evidence for all three years (Table 2).According to data from the European Food Safety Authority (EFSA) from 2013, signifi cant decrease of the prevalence of resistance to ciprofl oxacin was established in E. coli strains isolated from pigs in Holland.While in 2009 the prevalence of the resistance was around 10%, it was not found in any of the 289 E. coli strains examined in 2013.
Th e results obtained by Guerra et al. (2003) revealed that 61% of the analyzed E. coli isolates (n = 42) originating from cattle, pigs and poultry in Germany demonstrated no resistance to ciprofl oxacin, i.e., the MIC was in the range from ≥0.012mg/L to ≥2mg/L.In 39% of the isolates, the resistance to ciprofl oxacin (MIC ≥4mg / L) and the presence of a double mutant on the gyrA and parC was determined.
Th e use of antibiotics in the UK on annual basis amounts to 440-480 tons.Th e half of this amount belongs to tetracyclines, while the other half is shared between sulfonamides, aminoglycosides, β-lactams, macrolides and fl uoroquinolones.Th e prevalence of antimicrobial resistance is diff erent for various domestic animals.Th e highest resistance is established for tetracyclines used in pig production.Th ere is still no offi cial data on the prevalence of resistance to ciprofl oxacin for E. coli, isolated from pigs.However from total 2480 of the isolates 0.6% was resistant to nalidixic acid (Enne et al., 2008).
Testing of the resistance to ciprofl oxacin and enrofl oxacin in Iceland was a part of the research of Th orsteinsdottir et al. ( 2008) conducted from October 2005 to May 2007.Th e research objectives was to evaluate the frequency of resistance occurrence and the genetic similarity of E. coli strains isolated from healthy pigs and broilers, pig and broiler meat from slaughterhouses, and human isolates.Th e resistance prevalence is shown in table 3.According to the data available from the literature, the resistance to fl uoroquinolones of E. coli isolated from the feces of healthy and infected pigs in Europe is higher than in other countries worldwide.

THE SITUATION IN THE REGION
Enrofl oxacin and fl umequin are the most frequently used quinolones in the livestock industry in Serbia and countries in the region.Th ere is no precise data about the prevalence of the E. coli resistance towards quinolones and fl uoroquinolones in the surrounding region, except for Croatia and Hungary.Th e research conducted by Habrun et al. (2011) in the Republic of Croatia demonstrated an increase in the prevalence of the E. coli resistance to fl uoroquinolones.In 1990, the resistance was not established, while in 1996 it increased to 7.5%.During the years 1997 and 1998, the prevalence of enrofl oxacin-resistance reached 29.7% .Th e testing conducted on eight pig farms (2005)(2006)(2007) on 256 E. coli isolates isolated from organs of dead piglets with clinical signs of diarrhea revealed an increase of 39% in the resistance prevalence, also towards enrofl oxacin, while 11% of the isolates showed intermediate sensitivity.Th e authors concluded that the increase in the prevalence of the resistance resulted from an excessive use of fl uoroquinolones (Habrun et al. 2010).
According to EFSA data from 2013, in Hungary, the prevalence of resistance to ciprofl oxacin of E. coli isolated from pigs (n = 152) was 9.2%.Hungary is the only country in our region, which uses ciprofl oxacin in the livestock industry.

CONCLUSION
Fluoroquinolones are one of the most important classes of antimicrobial agents used for treatment of various infections in swine production.Th eir uncontrolled use in veterinary medicine may lead to the development of antimicrobial resistance of zoonotic pathogens.Meat and meat products from such animals may present a major reservoir for resistant strains and the source of infection for humans.Th e transmission of resistant bacterial strains through food and between humans and animals presents a major threat for public health.Th e use of fl uoroquinolones should be limited when treating less dangerous infections or completely prohibited in the livestock industry.Despite numerous studies, the eff ects of diff erent amino acid substitutions in topoisomerase genes are still not fully understood.In order to overcome the problem of resistance, all antimicrobial agents must be used with caution, especially fl uoroquinolones, and the code of practice for issuing prescription for antibiotics through must be respected.

3 B
Th e prevalence of resistance of E. coli in some European countries Year B DK E FIN F LV NL N Belgium DK Denmark E England and Wales, FIN Finland, F-France, LV-Lithuania, NL-Netherlands, Norway N-P-Portugal, EC-Spain, Sweden S-CH-Switzerland; *enrofl oxacin

Table 1 .
Quinolones and fl uoroquinolones most commonly used in the livestock industry in various regions around the world (adapted from Webber and Piddock 2001).

Table 3 .
Th e prevalence of ciprofl oxacin and enrofl oxacin resistance of E. coli isolated from pigs and pig meat in Iceland expressed in % (adapted from Th orsteinsdottir et al., 2008).