The Prevalence of Virulence Genes and Multidrug Resistance in Thermophilic Campylobacter Spp. Isolated from Dogs

Abstract The aim of the study was to determine the role of dogs as a potential reservoir of Campylobacter spp. At the next stage of the research the frequency of occurrence of selected virulence genes, i.e. cadF, flaA and iam as well as genes responsible for the formation the cytolethal distending toxin (CDT), i.e. cdtA, cdtB and cdtC was determined. The isolates obtained in the research were tested for their resistance to selected antibiotics: ciprofloxacin (CIP), enrofloxacin (EF), erythromycin (E) and tetracycline (TE). Campylobacter spp. was found in 63 (12.6%) out of a total number of 500 isolates. 61 (12.2%) isolates were identified as C. jejuni. The number of C. jejuni isolates found in the younger animals was smaller (p <0.05) than in the older ones. The frequency of occurrence of virulence genes and the genes responsible for the formation of CDT was significantly (p <0.05) higher in the older dogs. A comparison of the effect of antibiotics showed that the isolates obtained from both age groups exhibited low resistance to erythrosine (13.5% in the group aged under 1 year and 8.6% in the group aged over 1 year). Both groups exhibited the highest resistance to ciprofloxacin and enrofloxacin.


Introduction
Thermotolerant bacteria of the Campylobacter genus (mainly Campylobacter jejuni and Campylobacter coli) are part of the natural intestinal flora in mammals and birds. They can also be found in water and soil contaminated with animal faeces [1]. As these bacteria are so common, animal products, especially red meat and poultry, are at risk of contamination. Close contact with a sick animal and lack of hygiene may cause campylobacteriosis in humans and become a serious epidemiological problem in the 21 st century [2,3]. The incidence of this disease in humans has been increased in Europe since 2005 [4]. In 2015 the overall incidence of infections in the EU was 65.5 cases per 100.000 inhabitants [5]. 20-40% of human infections were caused by contact with poultry or poultry meat [6]. The most common clinical symptoms are diarrhea, fever and abdominal pain. The infection usually disappears spontaneously, although there may be complications such as arthritis [7]. According to the latest EFSA report [8], each year about 200.000 cases of campylobacteriosis infections diagnosed in humans were caused by the consumption of contaminated food. The actual number of infections is estimated at 9 million per annum. According to the EFSA estimates, the annual costs generated by the incidence of campylobacteriosis in public healthcare in the EU amount to 2.4 billion Euros (€). Due to the fact that Campylobacter spp. isolates from humans are increasingly often resistant to quinolone chemotherapeutics [9], aminoglycosides and macrolide antibiotics, they pose a threat to public health [10]. The development of resistance is often associated with spontaneous point mutations of the genes encoding enzymes synthesised by Campylobacter spp. [11]. The bacteria were proved to have various pathogenic properties resulting from high genetic diversity of their strains. The pathogenicity of bacteria is caused by the genes that determine their motility, adhesion, invasiveness and synthesis of a cytolethal distending toxin (CDT), which is encoded by three genes -cdtA, cdtB and cdtC. This cytotoxin causes cell cycle arrest at the G2/M phase [12]. Other virulence genes are: fla, cad, rac, vir, cia, pld, iam. The fla genes (flaA and flaB) are responsible for bacterial motility. They encode flagellin -the ciliary protein which enables Campylobacter spp. cells to move. The cadF gene encodes the fibronectin binding protein of enterocytes, which participate in adherence. Many researchers believe that this gene, which is necessary to induce symptoms of campylobacteriosis, is a conservative gene in C. jejuni and C. coli. The vir gene, which can be found in the Campylobacter spp. plasmid (the plasmid is not always present), also encodes proteins responsible for pathogenicity. The iam sequence, which is responsible for adhesion and invasiveness, is found in C. coli more often than in C. jejuni [13]. In consequence, it may disorder intestinal absorption.
The aim of the study was to determine the frequency of occurrence of Campylobacter bacteria in dogs, to identify species within the Campylobacter genus, selected virulence genes in isolated strains and genes determining the occurrence of CDT, and to determine resistance to selected antibiotics.

Sampling
Swab samples (swabs with transport medium, Stuart swabs. Sterile, product No. 300295, Deltalab, Spain) collected from the rectum of 500 dogs (n = 250 dogs younger than 1 year and n = 250 dogs older than 1 year) from shelters located in the Wielkopolska region were used as material for the tests. No dog showed gastrointestinal symptoms. The samples were collected on three consecutive days and transported to a laboratory within 6 hours while stored at 4°C.

Ethical approval:
The research related to animals use has been complied with all the relevant national regulations and institutional policies for the care and use of animals.

Isolation and identification of Campylobacter spp. from swab samples (PN EN ISO10272 under modification)
Swab samples were initially placed in 3 cm 3

Identification of genes determining CDT occurrence by means of multiplex PCR
The primers shown in Table 1 were used to identify the cdtA, cdtB, and cdtC genes. A 25μL reaction mixture was used. It was composed of: 2.5 μL 10 x PCR buffer, 6 x 1 μL primers (5 μM), 1 μL dNTP (2.5 mM), 0.2μL Taq polymerase, 1.8 μL DNA, 13.5 μL water. The thermal conditions of the reaction were as follows: initial denaturation at 94 °C/2 min. It was followed by 30 cycles, each of which consisted of: denaturation (94 °C/0.5 min), primer attachment (50 °C/0.5 min) and extension (72 °C/1 min and 72 °C/5 min). The resulting products were analysed by means of electrophoresis in 1.5% agarose gel.

Assessment of sensitivity to antibiotics
The isolated Campylobacter bacterial strains were tested for sensitivity to antibiotics by means of the Kirby-Bauer disc diffusion test and E-test. The method was compatible with the EUCAST [18]

Statistical analysis
The counts of microorganisms measured in the investigations were analysed statistically using the GLM procedure of the SAS program. The significance of differences was verified with Duncan's test (α=0.05).

Isolation and identification of Campylobacter spp.
Campylobacter spp. was found in 63 (12.6%) out of the total number of 500 isolates. 61 (12.2%) isolates were identified as C. jejuni (Table 3). There were 2 isolates (0.4%) that should not pose a threat to human health, so they were not further differentiated. There were no C. coli or C. lari strains detected. The total number of positive isolates found in the dogs aged under 1 year was lower (n = 27; 10.8%) than the number of positive isolates found in the dogs aged over 1 year (n = 36; 14.4%). The analysis of the species composition of the isolates (n = 61; 100%) showed that all of them belonged to C. jejuni. There were 26 (10.4%) C. jejuni isolates found in the dogs aged under 1 year. This number was lower than in the group of dogs aged over 1 year (n = 35, 14.0%). The differences were statistically significant (p <0.05).

Identification of genes determining CDT occurrence
There were 3 dogs aged under 1 year (11.5%) and 15 dogs aged over 1 year (42.8%) with the cdtA gene. The cdtB gene was found in 2 dogs aged under 1 year (7.7%) and 17 dogs aged over 1 year (48.6%). The cdtC gene was found in 4 dogs aged under 1 year (15.3%) and 20 dogs aged over 1 year (57.1%). The differences were statistically significant (p <0.05). The frequency of occurrence of the genes determining CDT occurrence was higher in the animals aged over 1 year (Table 4).

Sensitivity to antibiotics (the percentage of resistant isolates is the average of two tests)
Tables 5 -6 show the results of Campylobacter jejuni sensitivity (S) and resistance (R) to the antibiotics applied in the experiment. The E Test and disc diffusion test were two methods compared in the study. Enrofloxacin was the only antibiotic which resulted in statistically significant differences (p <0.05) between the methods, both in the group of dogs aged under 1 year and in the group of older animals. The isolates from the younger animals exhibited the highest resistance to ciprofloxacin (46%) and enrofloxacin (30.7%). The analysis of resistance of the isolates obtained from the older animals showed that they also exhibited high sensitivity to ciprofloxacin (50%) and enrofloxacin (22.9%) (this was observed only in the disc diffusion test). Both the isolates from the younger and older dogs exhibited low resistance to erythromycin (13.5% and 8.6%) and tetracycline (7.7% and 8.6%).

Discussion
Initially, biochemical tests were used to detect species such as C. coli and C. jejuni in research on Campylobacter spp. Particular attention was paid to the ability of C. jejuni to hydrolyse sodium hippurate. Since the description of hippurate-negative C. jejuni strains it has seemed more reliable to use the PCR method and the Real-Time PCR system with complementary primers [19]. Our study revealed a low incidence of Campylobacter spp. in the group of dogs (n = 500), i.e. 12.6%. This result stood in opposition to our earlier studies [1,20] and research conducted by other authors [5,21]. Chaban et al. [22] analysed a group (n = 70) of healthy dogs and found bacteria of the Campylobacter genus in 58% of the samples. Likewise, the authors did not find C. coli or C. lari, whereas C. jejuni was detected in 7% (n = 5) of the positive samples. Differences in the results of studies conducted by various authors may have been caused by many factors, e.g. the sampling method, the culturing conditions in a laboratory (in vitro) or high species diversity within the Campylobacter genus [23]. Other authors have suggested that the age of animals may affect the population of Campylobacter spp. [24]. Hald et al. [25] noticed a relation between the age of animals and the incidence of thermotolerant Campylobacter spp. The authors observed that the incidence of these bacteria increased from 60% in young animals (3 months old) to 100% in animals aged 12 months, but it then decreased to 67% in dogs aged 24 months. The C. jejuni genome WCTC 11168, sequenced in 2000, revealed the presence of genes encoding a protein with potential toxic properties [19]. The strain contained cdt genes encoding the CDT protein (cytolethal distending toxin). These were two genes encoding proteins with haemolytic domains and one phospholipase gene. CDT was the first described genotoxin that damages the genetic material of sensitive eukaryotic cells by stopping the cell cycle and causing the death of cells as a consequence [26]. A CDT locus consists of three jointly constitutively transcribed open reading frames (ORFs): cdtA, cdtB and cdtC, which are usually located on the chromosome [27]. All three genes were detected in our study. Hyun-Ho et al. [28] recorded similar results in their study on dogs. It is noteworthy that the frequency of occurrence of these genes in the dogs aged over 1 year was higher than in the younger animals. The cdtB subunit is responsible for the nucleolytic effect. In our study the occurrence of this gene in older dogs was higher than in the younger animals, i.e. 48.6% vs. 7.7%. Other authors have observed higher values. Andrzejewska et al. [29] researched a group of dogs and found that the percentage of animals with cdtB was as high as 72.7% or even 100% [30], whereas Findik et al. [31] noted 97.1%. The role of the cdtA and cdtC subunits should not be neglected either, because they may transport the cdtB subunit into the target cell (Krutkiewicz et al.) [19].
The pathogenesis of campylobacteriosis may be directly related with factors such as: motility, chemotaxis (mutants without these features do not colonise intestinal epithelial cells) as well as adhesion and invasiveness. Many genes and the effects of their expression are thought to be responsible for pathogenicity, e.g. fla -the motility gene, cadF -the adhesion gene and iam -the invasiveness gene [30,32]. Our study showed a high percentage of the cadF, flaA genes and iam sequence in both groups of dogs. Rodrigues et al. [33] observed a high incidence of the cadF and flaA genes in dogs and noted that these genes may act as reporters in research on the mechanisms of C. jejuni pathogenicity.
The progressive increase in bacterial resistance is closely correlated with the misuse of chemotherapeutics. Apart from that, as a result of the widespread application of antibiotics to animals, often without medical indications, bacteria become selected towards antibiotic resistance. In consequence, they develop multi-drug resistance [34]. Campylobacter spp. strains isolated from animals and food exhibited the highest resistance to ciprofloxacin and tetracycline [8]. The main problem with Campylobacter spp. is the resistance of these bacteria to aminoglycosides (gentamicin), macrolides (erythromycin) and quinolones (ciprofloxacin), which are commonly used to treat campylobacteriosis [4]. Research conducted in Poland between 2003 and 2006 showed that all strains isolated from humans were sensitive to erythromycin. On the other hand, the percentage of clinical strains of Campylobacter jejuni which were resistant to ciprofloxacin was very high, i.e. 58% [35]. Research conducted in the US showed that 99.5% of Campylobacter spp. strains isolated from poultry were resistant to one or more antibiotics. 99.5% of C. jejuni strains and 96.3% of C. coli strains were resistant to tetracycline. Research conducted in Slovenia also showed a high percentage of C. jejuni isolates resistant to enrofloxacin (58.2%). The strains were less resistant to erythromycin (14.5%) and tetracycline (12.7%) [36]. These results are in line with our research findings. Maćkiw et al. [37] observed the highest resistance to ciprofloxacin (97.9%), and lower resistance to tetracycline (64.3%) and erythromycin (9.1%). The authors noted that 7% of 143 resistant isolates exhibited resistance to 3 unrelated antibiotics. Research conducted on dogs in India [36] revealed that all the isolates were resistant to tetracycline (100%), and most of them were resistant to ciprofloxacin and enrofloxacin (97.3%). The values recorded by these authors were higher than the values noted in our study.

Conclusions
To sum up, the dogs were identified as asymptomatic carriers of Campylobacter spp. and were found to be vectors carrying and transmitting Campylobacter infections to humans. It is necessary to conduct further research on these bacteria as risk factors to better understand the epidemiology of Campylobacter spp. and to emphasise the dangers of the unreasonable use of antibiotics in both human and animal therapy.

Conflict of interest:
Authors state no conflict of interest.