Frequency of Antimicrobial Resistance in Thermophilic Campylobacter Strains from Humans, Poultry and Pigs

Uþestalost antimikrobne rezistencije termofilnih Campylobacter sojeva poreklom od ljudi, živine i svinja


Introduction
Campylobacteriosis is classified as zooanthroponosis.It is an infection caused mainly by thermophilic campylobacters: Campylobacter jejuni, Campylobacter coli, Campylobacter lari, Campylobacter upsaliensis.Campylobacter jejuni and Campylobacter coli are causing the most important bacterial intestinal infections in modern era, with 400 000 000 patients in the world every year.A very important factor in intestinal campylobacteriosis development is a very low infective dosis of only 500 bacteria 1 .
Humans get infected by this bacteria consumming insufficiently thermally processed meat, mostly poultry meat, pork and beef 2,3 , consumming unpasteurised milk and contaminated water 4 , and beeing in contact with domestic pets 5 .Important role of poultry in human infections is demonstrated in Belgium during the Dioxin crisis in 1999, when, due to high levels of poison dioxin detected, domestic poultry and eggs were withdrawn from the market, resulting in lowering number of campylobacteriosis cases by 40% 2 .
Thermophilic Campylobacter spp.mostly produce intestinal disorders, but could also produce extraintestinal disorders.Gut lesions in intestinal campylobacteriosis, similar to infections due to Salmonella and Shigella genera, are manifested as inflammatory infiltrates in lamina propria and abscesses in crypts 6 .The most frequent extraintestinal forms of disease are: meningitis, endocarditis, septic arthritis, os-teomyelitis and neonatal sepsis.Several cases of myocarditis as a complication of Campylobacter jejuni infection were reported.
Secondary diseases reported by various authors 5,7 as a consequence of thermophilic Campylobacter spp.primary infection, are Guillain-Barré syndrom (GBS) and Reiter's syndrom.Arthritis, GBS and Miller-Fisher's syndrom (a form of GBS) are possible complications in campylobacteriosis.Campylobacteriosis is generally a mild and self-limiting disorder.In patients with more severe and prolonged forms, an antibiotic treatment is recommended 8 .
Although a significant percentage of animals is colonized, they rarely develop a disease, but they are reservoirs of infection for humans.Poultry aged 2-3 weeks are in 50%-90% of cases colonized by thermophilic Campylobacter spp. 3 .Swines are less than poultry colonized by the same bacteria.Tambur et al. 9 demonstrated that 80.88% of poultry and 77.27% of swines are contaminated by thermophilic Campylobacter spp.
After inoculation to newborn calves thermophilic Campylobacter spp.produce a mild and self-limiting enteritis and bacteriemia.Campylobacter spp.can produce dysentery in cattle and swine 7 .Campylobacter jejuni produces abortions in sheeps, acute enteritis in calves, dogs and cats, and hepatitis in poultry.Clinical symptoms of hepatitis in poultry are somnolence, weakness, diarrhoea and eggs-laying disorders 10

Treatment of campylobacteriosis and investigation of susceptibility to antibiotics
Drugs, generally used in human campylobacteriosis treatment are: erythromycin, quinolones, tetracyclin, ampicillin, chloramphenicol and gentamycin.Disk-diffusion test, E-test with strips and agar dilution test are used in investigations of susceptibility to antibiotics.
Different results were obtained by the three methods applied in investigation of susceptibility 2 .

Antimicrobial susceptibility testing in Campylobacter spp. and methodology standardization
At present, several methods have been employed for Campylobacter susceptibility testing.Agar dilution is recommended by many authors 8,11 , but it is time consuming and not suitable for routine laboratory work.The E-test, a diffusion method with MIC determination, gives results faster than agar dilution, but its cost and need for standardization can be limitating 12 .Also, some authors recommended broth dilution method 13 (microbroth dilution by Trek, Vet-Mic etc.) as suitable for routine use.Although disc diffusion is the simplest method, absence of available standards limits its application in clinical laboratories.
Disc diffusion and agar dilution are often compared in order to obtain diameter zone for application in routine work.With respect to these methods, Gaudreau and Gilbert 14 reported complete agreement for tetracycline and ciprofloxacin, with only minor differences for erythromycin but poor correlation coefficient for ampicillin.Similarly, Luangtongum et al. 15 , revealed an excellent correlation between the agar dilution and the disk diffusion for aminoglycosidesand, quinolone/fluoroquinolones; a highlevel correlation for erythromycin, clindamycin, and tetracycline, and a weak correlation for ampicillin.They suggested setting the MIC breakpoint for erythromycinsusceptible Campylobacter strains at 2 μg/mL and 8 μg/mL for resistant isolates and the zone diameter breakpoints of the disk diffusion method at 23 mm for susceptible isolates and 18 mm for resistant isolates.Also, they recommended the MIC breakpoints for clindamycin to be 2 μg/ml for susceptible isolates and 8 μg/mL for resistant strains and the zone diameter breakpoints 17 mm for susceptible isolates and 12 mm for resistant ones.Proposed values for the zone diameter breakpoints for tetracycline are 28 mm for susceptible strains and 8 mm for resistant strains.Authors also suggested that the disk diffusion method can be used as a reliable alternative method for susceptibility testing of thermophilic Campylobacter to several classes of antimicrobial agents, particularly to quinolone/fluoroquinolones and aminoglycosides.
Gaudreau et al. 16 recommended zone diameters of 6 mm and 20 mm around the erythromycin disk as resistant and susceptible breakpoints of C. jejuni isolates.Also, for ciprofloxacin susceptibility testing of C. jejuni isolates, zone diameters of 17 mm and 21 mm around the ciprofloxacin disk and the absence or the presence of an inhibition zone around the nalidixic acid disk are suggested as breakpoints for resistance and susceptibility, respectively.
With disk diffusion, the following zone diameters were proposed to be resistant and susceptible breakpoints, respectively, for susceptibility testing of Campylobacter coli: no inhibition zone and 15 mm for erythromycin, and 20 mm and 25 mm for ciprofloxacin, in the absence or presence of an inhibition zone around the nalidixic acid disk.For susceptibility testing of C. coli and C. jejuni, diameter zones 20 mm and 26 mm for tetracycline were recommended 17 .
A recommendation, followed by these findings, is given that disk diffusion could be used to detect C. jejuni and C. coli isolates with reduced susceptibilities to ciprofloxacin and erythromycin in clinical laboratories 18 .
Up to date, The Clinical and Laboratory Standards Institute (CLSI), has established minimal inhibitory concentration (MIC) breakpoints for agar dilution for erythromycin, ciprofloxacin, tetracycline and doxycycline.In addition, for disc diffusion, zone diameter is given only for erythromycin and ciprofloxacin 19 .EUCAST (the European Committee on Antimicrobial Susceptibility Testing) is still working on standards and epidemiological cut off is proposed for C. jejuni and C. coli for erythromycin, ciprofloxacin, tetracycline, streptomycin, gentamicin, chloramphenicol, and nalidixic acid 20 .
Molecular techniques, also, can be applied for resistance determination as the Mismatch Amplification Mutation Assay (MAMA-PCR) 21 , and the Lightcycler mutation assay 22 for the detection of ciprofloxacin-resistant C. jejuni and C. coli isolates.However, these and similar techniques can be applied only if prior knowledge about genetic basis for resistance exist.Usually, they cannot be refered to a routine resistance detection, and may not detect resistance if a new resistance mechanism emerge 22 .Some authors consider that combination of phenotypic and genotypic methods in resistance detection should be more convenient 23 .

Mechanisms of erythromycin resistance in campylobacters
Erythromycin and other macrolide antibiotics bind to the subunit 50S of bacterial ribosome and restrict elongation of polypeptide chain 24 .Sites for macrolide action are parts of subunits 23S rRNA, and ribosomal proteins L4 and L22.Proteins L4 and L22 form parts of exit channel for polypeptide in bacterial ribosome 70S and they are described in several bacterial species 25 .Eyithromycin resistance can be mediated by enzymatic inactivation, can evolve through target modification by mutation or methylation, and by active effluxe 26 .In Campylobacter, resistance to macrolides confer to gene mutation with change of target site for drug binding to bacterial ribosome 27 .Other mechanism that confer resistance is active effluxe 28 .Resistance occurs as synergy between gene modification and efflux pumpe CmeABC activity 29 .Two types of resistance to macrolides are described: resistance to high levels of drug concentration (high level resistance -HLR) 25 and resistance to lower drug concentration (low level resistance -LLR) 28 .In HLR, MICs for erythro- mycin are higher than128 mg/L, and in LLR, MICs are in range from 8-16 mg/L 25,30 .In C. jejuni and C. coli strains, HLR is a consequence of mutation in 23S rRNA V domen in target gene at the positions 2074 and 2075.LLR can be a result of effluxe pumpe activity 31 .Also, it is recognized that modifications of L4 and L22 contribute to low level Ery resistance in C. jejuni 32 .

Mechanisms of fluoroquinolones resistance in campylobacters
Fluoroquinolones inhibit the activity of DNA gyrase due to mutations in the DNA gyrase and DNA topoisomerase IV genes in most bacterial species 8 .Enzyme DNA gyrase is composed of two pairs of subunits, GyrA and GyrB, while topoizomerase IV consists of ParC and ParE 33 .Resistance to fluoroquinolones is a result of aminoacid changes in topoisomerase as well in gyrase.In Campylobacter strains, resistance to fluoroquinolones is a consequence of mutation in gene gyrA which encodes GyrA subunit of DNA gyrase 8 .Up to date, no mutations in DNA gyrase B have been associated with FQ resistance in Campylobacter 34 .The most frequently observed mutation in fluoroquinolones resistant isolates of Campylobacter is the point mutation Thr-86-Ile in gyrA gene 35 which leads to the T86I substitution in the gyrase and confers HLR to fluoroquinolones 33 .Other reported mutations of gyrA in C. jejuni include Thr-86-Ala (HLR to nalidixic acid and LLR to ciprofloxacin), Ala-70-Thr, Thr-86-Lys, Asp-90-Asn, and Pro-104-Ser 35,36 .Double point mutations of gyrA have also been reported 35 .
In C. jejuni and C. coli, a unique modification in the GyrA subunit is sufficient to confer a fluoroquinoloneresistant phenotype.Also, decrease in permeability of outer membrane and activity of effluxe system confer the fluoroquinolones resistance 37 .In Campylobacter jejuni/coli strains, apart of the mutations in GyrA, the multidrug efflux pump, CmeABC, also contributes to fluoroquinolones resistance by reducing the accumulation of the agents in Campylobacter cells 38 .Thus, CmeABC functions synergistically with the gyrA mutations in mediating fluoroquinolones resistance 39 .
To understand the roles of multidrug efflux transporters in the pathobiology of C. jejuni, Jean et al. 40 characterized the function of an MFS transporter (Cj1375) designated CmeG.The results indicated that CmeG functions as a multidrug efflux transporter contributing to antibiotic resistance especially to fluoroquinolones and oxidative defense in Campylobacter.

Mechanisms of tetracyclines resistance in campylobacters
Tetracyclines, (e.g.tetracycline, chlortetracycline, and minocycline) bind to the ribosome and inhibit accommodation of the aminoacyl-tRNA (aa-tRNA) into the ribosomal A site and, therefore, prevent the elongation phase of protein synthesis 41 .Tetracycline resistance can be mediated by different mechanisms: efflux, the enzymatic degradation of drug, protection of the ribosomal binding site and mutations in 16S rDNA 42 .In C. coli and C. jejuni, genes for tetracycline resistance are located on self-transmissible plasmids.They have been identified as a ribosomal protection gene and designated tet(O) 43 .These genes are widely present in Campylobacter isolates recovered from various animal species 23 .They encode ribosomal protection proteins (RPPs) 41 .Tet(O) confers resistance by binding to the ribosome inducing a conformational change with subsequent release of the bound tetracycline molecule and its displacing from its primary binding site, such that the aa-tRNA can bind to the ribosomal A site and protein synthesis can continue 44 .
The presence of tet(O) in different Gram-positive bacteria 45 suggest the origin of the resistance genes and their sharing between species.In C. jejuni, tet(O) was first cloned from a transferable plasmid pUA466 46 .Sequencing of two tetracycline-resistance plasmids, one from C. jejuni strain 81-176 47 , and other from C. coli strain CC31, revealed a high level of sequence identity and genomic organization despite their temporal and spatial distance 48 .
Although, in most strains, the tet(O) gene is plasmidencoded, it can be located on the chromosome, which is reported for 33% of tetracycline-resistant C. jejuni isolates from Alberta, Canada 49 and 76% of tetracycline-resistant isolates from Australia Pratt, Korolik 50 .On tet(O)-carrying plasmids it is described the presence of an insertion element IS607 and therefore it is possible that mobile genetic elements other than transmissible plasmids may be involved in the acquisition and dissemination of tet(O) 51 .
Tetracycline resistance in C. jejuni is also associated with the CmeABC multidrug efflux pump 52 .

Resistance of thermophilic Campylobacter strains isolated from humans, poultry and swines to erythromycin
Alarming is the rise of resistance to erythromycin, the first choice drug for treatment of campylobacteriosis.Detection of the resistant strains started with the use of macrolides, generally thylosine in veterinary practice, mostly in swine farming 8,13,53 .
An investigation 53 detected 12.5% Campylobacter strains isolated from humans resistant to erythromycin.These results are in accordance with the results of other authors [54][55][56] .Lower levels of resistance to erythromycin, ranging from 3.4% to 9.1% are reported by the authors in Brasil, Australia, USA and India 5,[57][58][59] .
A tendency of rising frequency of resistant Campylobacter to erythromycin is evident.For example, in Canada there were 3% Campylobacter jejuni/coli resistant strains in 1998, but the percentage increased to 12% in 2001 60 .
A high percentage of Campylobacter jejuni/coli strains isolated from broilers was found 61 contrary to the fact that erythromycin has not been used in poultry farming.A low level of resistance to erythromycin in thermophilic Campylobacter strains was recorded in Great Britain (0-8%) 62 , USA (3.1%) 63 and Czech Republic (6%) 64 .A high percentage of Campylobacter coli strains resistant to erythromycin isolated from broilers and eggs-laying hens (25% and 40%) was found in Japan 65 .Authors in Italy reported a high level of resistance to erythromycin, up to 45%, in Campylobacter coli strains isolated from poultry faeces 55 .In Africa, high erythromycin resistance levels were observed in human clinical isolates, but low resistance rate to this antibiotic were noticed in C. jejuni and C. coli isolated from husbandry animals 66 .Reports from Asia describe low resistance of C. jejuni to macrolides, but higher resistance of C. coli strains 67 .Also, increased resistance to macrolides was observed among C. coli isolates from pigs in Australia 68 .
Macrolides are widely used in swine farming and, as a consequence of intensive pressure of drugs included in this thylosine group, an increase of Campylobacter strains resistant to erythromycin originating from swines occured.
The investigation detected that even 40% of thermophilic Campylobacter spp.strains isolated from swines were resistant to erythromycin 61 .According to data from Spain, percentage of resistant Campylobacter coli was 81%, in Denmark percentage of resistant Campylobacter jejuni was 33% and of Campylobacter coli 74% 69 .

Resistance of thermophilic Campylobacter strains isolated from humans, poultry and swines to quinolones
A rising frequency of thermophilic Campylobacter spp.originating from humans resistant to quinolones, drugs most frequently used in campylobacteriosis treatment 61,70 is alarming.Emergence of the resistant strains coincided with the beginning of quinolones use in veterinary practice 8,71 .
Thermophilic Campylobacter spp.strains resistant to quinolones were produced diarrhea of mean duration 13.2 days, contrary to susceptible strains with mean duration of diarrhea of 10.3 days 72 .
Investigation of resistance to ciprofloxacin of Campylobacter strains isolated from humans in Serbia, detected 50% resistance 73 .This results are in accordance to the results of others 5,55,58,[74][75][76] .In Chile the resistance of Campylobacter jejuni/coli to ciprofloxacin has not been recorded 77 .Fifty percent of thermophilic Campylobacter spp.originating from humans were characterized as resistant to ciprofloxacin in a controlled investigation of susceptibility to antibiotics 73 .A high level of resistance to ciprofloxacin (71.4%) was demonstrated in Campylobacter jejuni/coli isolated in India from humans, generally children in rural areas 58 .A high level of resistance to ciprofloxacin was registered in Spain, too.Resistance to this antibiotic was found in 75% Campylobacter jejuni and 70.7% Campylobacter coli strains 56 .
A permanent trend of resistance increase to fluoroquinolones is spread worldwide.Enrofloxacin is licenced in Netherlands for use in veterinary medicine in 1987.Resistant Campylobacter jejuni/coli strains isolated from humans represented 8% in 1998, 11% in 1989 and 29% in 1997.A similar trend is registered in Austria, Denmark, Finland, France, Italy, Spain, Thailand, Great Britain and USA 8 .In Canada there were no resistance to ciprofloxacin in 1985/86.In the following period, 1995/97, 12.7% resistant Campylobacter jejuni/coli strains were isolated from humans 60 .A high level of thermophilic Campylobacter spp.resistant to ciprofloxacin has been registered (50% to 60%) 55,62,78 .Cardinale et al. ( 2002) 70 , citing several other authors, reportspercentages of Campylobacter jejuni/coli resistance to ciprofloxacin in several countries: Germany 46%, Japan 46%, USA 23-100%, Kenya 7.7%, Belgium and Spain up to 100%, Taiwan and Thailand 56-84% and Senegal 34%.In Switzerland a very low level of thermophilic Campylobacter spp.isolated from poultry meat resistant to fluoroquinolones is registered: only 0.5%.Resistance to ciprofloxacin in thermophilic Campylobacter spp.isolated from poultry in Norway was also low (2.7%).The reason for this results could be found in the fact that fluoroquinolones were not approved for use in broilers in Norway 78 .
Fluoroquinolones have not been applied in such extent in swine farming as in poultry farming, this being the reason that the percentage of Campylobacter jejuni/coli strains resistant to fluoroquinolones is lower in swines than in poultry.
Results of an investigation 79 demonstrated 26.7% resistant Campylobacter strains isolated from swines.Similar results were reported in Italy and Switzerland 55,80 .A low level of resistance to fluoroquinolones, only 0.5%, was registered in Campylobacter jejuni/coli strains isolated from swines in USA 81 .Hart et al. 82 did not register a resistance to ciprofloxacin in Campylobacter jejuni/coli isolated from swines in Australia, due to the fact that quinolones are not approved for use in veterinary medicine.

Resistance of thermophilic Campylobacter strains isolated from humans, poultry and swines to tetracyclines
It was noted that tetracyclines were used in human medicine without appropriate control 83 .According to numerous authors in the world 30%-40% thermophilic Campylobacter strains isolated from humans are resistant to tetracycline 73,74 .High percentage of resistant thermophilic Campylobacter strains isolated from humans, ranging from 43% to 85%, are reported in Spain, USA and Finland 54,58,75,84 .A lower level of resistance to tetracycline, ranging from 12% to 16%, was reported in Australia, India and Turkey 57,59,76 .Very low level of thermophilic Campylobacter spp.isolated from human, resistant to tetracyclines, only 1.8%, was registered in Chile 77 .The trend of resistance increase to tetracycline in many countries is annoying 2,53 .Many authors report higher percentages of resistance to tetracycline of thermophilic Campylobacter spp.strains isolated from poultry 4,56,61,65 but some reported lower percentages of resistance 83,[85][86][87][88][89] .It was noted that as far as 80% strains of thermophilic Campylobacter spp.originating from swines were resistant to tetracycline 69,82,90 , but some authors registered lower percentages of resistance 80,81 .Aarestrup and Wegener 85 in Denmark, found a low resistance level to tetracyclin in Campylobacter jejuni/coli strains isolated from swines (1%).
Investigation of sensitivity to antibiotics of thermophilic Campylobacter spp.collected from humans, applying disc-diffusion test, detected 47.1% strains resistant to two antibiotics, and 11.8% strains resistant to three antibiotics 91  Hakanen et al. 92 detected 22% Campylobacter jejuni strains resistant to three or more antibiotics.Multiresistance to antibiotics of thermophilic Campylobacter spp.strains in India was 30.6%, most fequently to erythromycin, tetracycline and ciprofloxacin 59 .
It is necessary to emphasize recorded multiresistance of thermophilic Campylobacter isolated from poultry and swines 54,70,81 .

Conclusion
Consuming of food contaminated with thermophilic Campylobacter spp.results in transmission of strains resistance to antibiotics and resistency genes from animals to humans.Humans infected with strains resistant to antibiotics, get illness with more severe symptomatology and with prolonged course.High level of resistance to antibiotics of thermophilic Campylobacter spp.collected from humans and animals, even in high industrialized countries, is a conse-quence of irregular use and misuse of antibiotics, predominantly in veterinary medicine and husbandry, the fact demonstrated in many investigations.It should be emphasized that the level of resistance of 12.5% to erythromycin of Campylobacter strains collected from humans and poultry was detected, contrary to the fact that erythromycin was not being used in poultry farming.Resistance to ciprofloxacin of Campylobacter strains collected from humans and broilers was 50% or more.It was demonstrated that 30% strains originating from humans and 80% strains originating from swines are resistant to tetracycline.A trend of resistance increase to antibiotics of campylobacters collected from humans and animals is extensively evident.