Detection and prevalence of antimicrobial resistance genes in Campylobacter spp. isolated from chickens and humans

Campylobacter spp. are common pathogenic bacteria in both veterinary and human medicine. Infections caused by Campylobacter spp. are usually treated using antibiotics. However, the injudicious use of antibiotics has been proven to spearhead the emergence of antibiotic resistance. The purpose of this study was to detect the prevalence of antibiotic resistance genes in Campylobacter spp. isolated from chickens and human clinical cases in South Africa. One hundred and sixty one isolates of Campylobacter jejuni and Campylobacter coli were collected from chickens and human clinical cases and then screened for the presence of antimicrobial resistance genes. We observed a wide distribution of the tetO gene, which confers resistance to tetracycline. The gyrA genes that are responsible quinolone resistance were also detected. Finally, our study also detected the presence of the blaOXA-61, which is associated with ampicillin resistance. There was a higher (p < 0.05) prevalence of the studied antimicrobial resistance genes in chicken faeces compared with human clinical isolates. The tetO gene was the most prevalent gene detected, which was isolated at 64% and 68% from human and chicken isolates, respectively. The presence of gyrA genes was significantly (p < 0.05) associated with quinolone resistance. In conclusion, this study demonstrated the presence of gyrA (235 bp), gyrA (270 bp), blaOXA-61 and tetO antimicrobial resistance genes in C. jejuni and C. coli isolated from chickens and human clinical cases. This indicates that Campylobacter spp. have the potential of resistance to a number of antibiotic classes.


Introduction
Campylobacter jejuni and Campylobacter coli are considered as being the most common Campylobacter spp. that cause gastroenteritis in humans worldwide. However, other Campylobacter spp. such as Campylobacter lari and Campylobacter upsaliensis have also been implicated in human gastrointestinal infections (Centers for Disease Control and Prevention [CDC] 2013; Obeng et al. 2012). There is a significant concern and paucity of information on the magnitude of antibiotic resistance patterns of Campylobacter spp., particularly in veterinary and human medicine (Hein et al. 2013). When infections are caused by Campylobacter, the symptoms are mild enteritis and are usually selflimiting that rarely requires any antimicrobial treatment. Some severe cases do, however, result in prolonged enteritis and septicaemia wherein antimicrobial treatment is essential (Chatur et al. 2014). Severe cases of Campylobacter infections are commonly treated with macrolides, such as erythromycin and fluoroquinolones like ciprofloxacin are used to treat enteritis. Furthermore, aminoglycosides such as gentamicin are commonly prescribed for the treatment of systemic infections (Noormohamed & Fakhr 2014). Antibiotics prescribed in veterinary medicine vary greatly throughout the world because of different regulations that are specific to countries and regions. Circumstances, where antibiotics are used for growth-promotion purposes as opposed to therapeutic purposes, are of great concern. When low levels of antibiotics are used for nontherapeutic purposes over prolonged periods of time, antimicrobial resistance emerges. Countries such as India, Indonesia, Thailand and parts of Africa have access to veterinary antibiotics without proper prescription or control measures because of the lack of properly regulated veterinary services, mainly because of scarcity of veterinary skills and training. This is of great concern and could accelerate antibiotic overuse in animals (Iovine 2013). Molecular detection of antibiotic resistance genes has demonstrated that identical elements were found in bacteria that colonise both animals and humans. This suggests that bacteria originating from food of animal origin aid in the spread of resistant bacteria and resistance genes from animals to humans via the food chain (Moyane, Jideani & Aiyegoro 2013). Escalating numbers of Campylobacter isolates have developed resistance to fluoroquinolones and other antimicrobial Campylobacter spp. are common pathogenic bacteria in both veterinary and human medicine. Infections caused by Campylobacter spp. are usually treated using antibiotics. However, the injudicious use of antibiotics has been proven to spearhead the emergence of antibiotic resistance. The purpose of this study was to detect the prevalence of antibiotic resistance genes in Campylobacter spp. isolated from chickens and human clinical cases in South Africa. One hundred and sixty one isolates of Campylobacter jejuni and Campylobacter coli were collected from chickens and human clinical cases and then screened for the presence of antimicrobial resistance genes. We observed a wide distribution of the tetO gene, which confers resistance to tetracycline. The gyrA genes that are responsible quinolone resistance were also detected. Finally, our study also detected the presence of the bla OXA-61 , which is associated with ampicillin resistance. There was a higher (p < 0.05) prevalence of the studied antimicrobial resistance genes in chicken faeces compared with human clinical isolates. The tetO gene was the most prevalent gene detected, which was isolated at 64% and 68% from human and chicken isolates, respectively. The presence of gyrA genes was significantly (p < 0.05) associated with quinolone resistance. In conclusion, this study demonstrated the presence of gyrA (235 bp), gyrA (270 bp), bla OXA-61 and tetO antimicrobial resistance genes in C. jejuni and C. coli isolated from chickens and human clinical cases. This indicates that Campylobacter spp. have the potential of resistance to a number of antibiotic classes.

Detection and prevalence of antimicrobial resistance genes in Campylobacter spp. isolated from chickens and humans
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classes such as tetracyclines, betalactams, aminoglycosides and macrolides. The acquisition of resistance mechanisms compromises treatment options against Campylobacter infection in both humans and animals. Although several studies have previously determined the antibiotic resistance patterns exhibited by Campylobacter, they have not investigated the resistance genes which are associated with drug resistant strains (Colles et al. 2016;Maćkiw et al. 2012;Ferro et al. 2015;Fonseca et al. 2014;Ghunaim et al. 2015).
Ciprofloxacin is a fluoroquinolone used for the treatment of human salmonellosis and campylobacteriosis; however, it is also used in the poultry production industry. Fluoroquinolone residues can remain in the animal body post-treatment and this aggravates the evolution fluoroquinolone resistant strains of bacteria. A number of studies have linked the therapeutic and prophylactic use of fluoroquinolone antibiotics in order to select for ciprofloxacin-resistant Campylobacter in poultry products that enter the food chain in an attempt to study the emergence and spread of antimicrobial resistance (Colles et al. 2016;Gallay et al. 2007;Habib et al. 2009;Marinou et al. 2012;Moyane et al. 2013;Zendehbad, Khayatzadeh & Alipour 2015).
Genetic mutations play a major role in the development of Campylobacter resistance. Fluoroquinolone resistance is mediated by amino acid substitutions in the quinolone resistance-determining region (QRDR) (Colles et al. 2016).
The gyrase gene products are large enzymatic quaternary structures consisting of two pairs of subunits gyrA and gyrB.
The gyrA gene, which encodes part of the gyrA subunit of DNA gyrase confers a high-level of resistance to ciprofloxacin because of the point mutation Thr86Ile driven by the C257T change in the gyrA gene. Other mutations of the gyrA gene region in C. jejuni include Thr86Ala which is responsible for high-levels of resistance to nalidixic acid and low-level resistance to ciprofloxacin (Colles et al. 2016).
Natural transformation is a major mechanism for the transfer of chromosomally encoded antibiotic resistance genes, such as for fluoroquinolone and macrolide resistance, while conjugation plays a major role in the transfer of plasmid-mediated resistance, such as for tetracyclines and aminoglycosides (Wieczorek & Osek 2013). Tetracycline resistance in Campylobacter spp. is conferred by the tetO gene, which encodes ribosomal protection proteins (RPPs). This gene is extensively present in both C. jejuni and C. coli and has been demonstrated to confer extremely highlevels of tetracycline resistance (512 mg/L) by displacing tetracycline from its primary binding site on the ribosome and thus eliminating the action of the antibiotic (Abdi-Hachesoo et al. 2014). The high prevalence of conjugative tetO plasmids made it possible to infer that conjugation has contributed significantly to the spread of tetracycline resistance in Campylobacter. Against this background, this study investigated the presence of antimicrobial resistance genes responsible for fluoroquinolone and tetracycline resistance in chicken and human clinical isolates.

Ethical considerations
Human and animal studies were approved by the appropriate ethics committee of the University of KwaZulu-Natal and performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments (REF:BE084/14).

Origin of samples and processing procedures
A total of 100 human Campylobacter isolates were analysed. The samples were cryopreserved in Brucella broth (Oxoid) with 15% glycerol and were part of a collection that was received from a private laboratory in Durban, KwaZulu-Natal during 2014. One hundred faecal samples were randomly collected from commercial broiler chicken farms around the Durban metropolitan area between March and September 2016. Freshly excreted broiler faeces were sampled with sterile swabs and then directly inoculated into charcoal broth (Sigma-Aldrich, St. Louis, MO) and transported back to the laboratory for incubation at 37 °C for 48 h, under microaerophilic conditions (5% O 2 , 10% CO 2 and 85% N 2 ) created by CampyGen (Oxoid, UK) gas generating packs in an anaerobic jar.

Human clinical samples
From cryopreserved samples in Brucella broth (Oxoid) with 15% glycerol, cultures were revived on modified charcoal cefoperazone deoxycholate agar (mCCDA) (Blood-Free Agar) (Oxoid) containing Campylobacter selective supplement SR0155 (Oxoid). A sterile loop was then streaked across the area of inoculation several times to achieve isolated colonies. Thereafter, plates were incubated at 37 °C for 48 h under microaerobic conditions created by CampyGen (Oxoid) gas generating packs in an anaerobic jar. Subsequent to incubation, DNA was extracted for the purposes of species identification using the polymerase chain reaction (PCR) targeting of the hipO gene region (Table 1), that is the hippuricase gene specific for C. jejuni (Marinou et al. 2012) and the asp gene region (Table 1), the aspartokinase gene specific for C. coli (Al Amri et al. 2013).

Chicken faecal samples
Following incubation, the faecal samples in charcoal broth (Sigma-Aldrich, St. Louis, MO) were filtered through a 0.65 µm cellulose nitrate filter (Sartorius Stedim Biotech, Germany) onto mCCDA (Blood-Free Agar) (Oxoid). Approximately 500 µL of the incubated charcoal broth was evenly distributed over the filter aseptically, once the liquid had been filtered through, forceps were used to aseptically remove the filter. The culture plates were then set in an inverted position in an anaerobic jar containing an atmosphere generation system (CampyGen sachet, Oxoid) and then incubated at 37 °C for 48 h. Subsequent to incubation, DNA was isolated for the purposes of species identification using PCR targeting the hipO gene specific for C. jejuni (Marinou et al. 2012) and the asp gene specific for C. coli (Table 1

DNA isolation
Template DNA for PCR was extracted via the conventional boiling method that requires the following: characteristic colonies of Campylobacater spp. were isolated from plates and suspended in 300 µL TE buffer, then vortexed for homogenisation of cells. The suspensions were boiled at 100 °C for 10 min, then immediately cooled on ice. After centrifugation at 14 000 × g for 5 min, the supernatants were transferred to a new tube and stored at -20 °C until use in PCR for detection of antibiotic resistance genes (Datta et al. 2003). A positive Campylobacter spp. control was also prepared by isolating DNA from a reference strain of C. jejuni ATCC 29428, which was incubated under the same conditions and subjected to the same DNA extraction methods. The Thermo Scientific Nanodrop 2000, UV-VIS Spectrophotometer (Wilmington, Delaware, USA) was used to check the concentration and quality of the isolated DNA.

Detection of antibiotic resistance genes using polymerase chain reaction
All samples were subjected to PCR analysis of the hipO and asp gene for confirmation and differentiation of Campylobacter spp. (Table 1). Following identification, 83% of human clinical isolates and 78% of chicken isolates were subjected to detection of the four antimicrobial resistance genes ( Figure 1) as follows: gyrA (235 bp), gyrA (270 bp), bla OXA-61 and tetO. PCR primers were synthesised and sourced from Inqaba Biotechnologies (Pretoria, South Africa); forward and reverse primers specific for the antibiotic resistance genes under investigation were designed based on the gene sequence information in the GenBank database and in previously published studies (Chatur et al. 2014;Gibreel et al. 2004).
PCRs were carried out in the BIO-RAD, T100™ Thermal Cycler (Singapore) for a 25 µL reaction. The amplification conditions for gyrA (235 bp), gyrA (270 bp) and bla  consisted of an initial denaturalisation at 95 °C for 5 min, 35 cycles at 95 °C for 50 s, specific Tm for each primer (Table 1) for 30 s and 72 °C for 1 min, followed by a final extension at 72 °C for 7 min. PCR conditions for the tetO gene, a 559 bp product, were as follows: an initial denaturalisation of 95 °C for 1 min, and then 95 °C for 1 min, 49 °C for 1 min and 72 °C for 1 min, repeated for 35 cycles. PCR products were then electrophoresed on a 1.5% agarose gel run at 60 V for 60 min, stained with ethidium bromide and then visualised using the ChemiDoc™ MP Imaging System (BIO-RAD).

Statistical analysis of virulence genes and antibiotic resistance genes
The four antibiotic resistance genes and two virulence genes detected in C. jejuni and C. coli were analysed using IBM SPSS Statistics (Version 23). Pearson's correlation analysis, Fisher's exact tests, chi-square tests and logistic regression analysis were implemented to evaluate the relationship between the different PCR results obtained and the significance of the presence of virulence genes and antibiotic resistance genes detected in human clinical and chicken samples. Every model included the presence or absence of each antibiotic resistance gene and each virulence gene investigated (0 = absent; 1 = present). Associations were considered significant when p < 0.05.

Results
Antimicrobial resistance genes isolated from Campylobacter spp. from human clinical samples and chicken faeces (Figure 2) demonstrated similar presence in both these hosts.    The 235 bp and 270 bp gyrA genes were present in 49% and 36% of human clinical isolates, respectively; whereas, the Campylobacter from chicken faeces indicated a 51% and 36% presence of these genes, respectively, regardless of differentiation of species. The 270 bp gyrA gene was detected at the same incidence in both hosts. The bla  gene was detected at 53% and 58% in human clinical isolates and chicken faeces, respectively. The tetO gene which confers tetracycline resistance was the most prevalent resistance gene detected compared with the other resistance genes under investigation. The tetO gene was isolated at incidences of 64% and 68% from human clinical isolates and chicken faeces, respectively. Representative PCR products of the antimicrobial resistance genes investigated in this study are depicted in Figure 1. The gyrA genes depicted in lanes 1 and 2 have amplicons of 235 bp and 270 bp. These confer moderate to high-levels of resistance to fluoroquinolones because of mutations occurring in these genes (Chatur et al. 2014). Lane 3 is the bla OXA-61 resistance gene that corresponds to a 372 bp amplicon, which confers ampicillin resistance (Obeng et al. 2012   and tetO) in human clinical and chicken samples investigated in this study (p < 0.05). This was confirmed by both the chi-square and Fisher's exact statistical tests (Table 2).

Target gene Primer sequence (5' -3') Product size (bp) Annealing temperature (°C) References
Logistic regression analyses (Table 3) were conducted to establish the association between the presence of antimicrobial resistance genes (gyrA, bla OXA-61 and tetO), in chicken and human clinical isolates and the source of the isolates. A test of the full model against a constant only model indicated that genes gyrA (235 bp), bla OXA-61 and tetO were not statistically significant (p > 0.05) from the data obtained.
Pearson correlation (Table 4) demonstrated highly significant (p < 0.01) positive correlations between the antibiotic resistance genes investigated in this study. High correlations existed between the ampicillin resistance gene bla OXA-61 and the tetracycline resistance gene tetO (64.3%), the second highest correlation was between the tetO gene and the gyrA gene for fluoroquinolone resistance (56.8%).

Discussion
Our results indicate that C. jejuni is responsible for the majority of infections (83%) in human clinical samples compared with the low incidence of C. coli found in 17% of cases.  B, coefficient for the constant (also called the 'intercept') in the null model; SE, standard error around the coefficient for the constant; p, probability significance value; OR, odds ratio for the independent variable X i and it gives the relative amount by which the odds of the outcome increase (OR greater than 1) or decrease (OR less than 1) when the value of the independent variable is increased by one unit; 95% CI, 95% confidence interval. In chicken samples C. jejuni was detected at 87% compared with C. coli at 13%. This finding correlates with a number of studies that have demonstrated that C. jejuni is more prevalent than C. coli and, therefore, is responsible for most clinical cases related to gastroenteritis (Colles et al. 2016;Marinou et al. 2012;Moyane et al. 2013;Zendehbad et al. 2015). The increase in resistant bacteria has been associated with the injudicious use of antimicrobial agents in feed supplements used in the farming industry. This creates selective pressure, which produces microbial isolates that evolve to become resistant to these antibiotics because of the acquisition of antibiotic resistant genes. This attribute has a high likelihood of easily becoming a human health risk because of crosstransmission via the food chain (Luangtongkum et al. 2010). This study further demonstrated that higher rates of antibiotic resistance genes were detected in chicken samples when compared with the human samples. As a consequence, the injudicious use of antibiotics in chicken production accelerates the emergence of drug resistant strains of pathogens such as Campylobacter spp. This has a long-term effect of substantially reducing the number of antibiotics that can still be used effectively for the treatment of human and animal infections (Ferro et al. 2015). Usually it is recommended that the drug for treatment of human Campylobacter infections is the macrolide erythromycin, followed by ciprofloxacin of the fluoroquinolone family while the third choice would be tetracycline (Ghunaim et al. 2015).
Antibiotic resistance mechanisms for Campylobacter spp. differ between the drugs involved. A mutation in the gyrA gene acts as one of the main mechanisms of resistance for fluoroquinolones. This gene encodes part of the DNA gyrase and in the presence of a single point mutation in QRDR, codon 86 is changed from threonine to isoleucine which results in a high-level of resistance to the antibiotic ciprofloxacin (Wieczorek & Osek 2013 Tetracycline has also been listed as an alternative treatment for Campylobacter gastroenteritis. It is used widely for therapeutic purposes in livestock and poultry, which increases the risk of tetracycline resistant Campylobacter spp. This study further demonstrated that high levels of the tetO gene which could imply that tetracycline may not be a good alternative treatment in cases of Campylobacter infection (Zendehbad et al. 2015). In this study, we analysed two gyrA genes involved in gyrase subunit A. The gyrA (235 bp) and gyrA (270 bp) were detected at 49% and 36% in human samples and 52% and 38% in chicken samples, respectively. Mutations within the gyrA gene lead to phenotypic expression of resistance to fluoroquinolones because of a reduced supercoiling activity of the DNA by the gyrA enzyme. This mechanism is directly linked to gene expression and therefore, the altered function of the enzyme adjusts the 'strength' of the resistant strains, allowing persistence of such strains even in the absence of fluoroquinolone use (Ragimbeau et al. 2014). Unlike the fluoroquinolone resistance in other organisms such as Escherichia coli and Salmonella, acquisition of high-level FQ resistance in Campylobacter does not necessitate stepwise accumulation of point mutations in gyrA. Rather, a single point mutation in the QRDR of gyrA gene is adequate to lead to clinically relevant levels of resistance to fluoroquinolone antimicrobials (Wimalarathna et al. 2013).
The tetO gene was analysed as well because the presence of this gene results in resistance by the organism to the antibiotic tetracycline (Ferro et al. 2015). Pearson correlation coefficients demonstrated significant (p < 0.05) correlations between the tetO gene and all the other antibiotic resistance genes under investigation. This study was successful in elucidating a disperse distribution of a number of antibiotic resistance genes, which play significant roles in resistance to fluoroquinolones, β-lactams and tetracycline antibiotics.

Conclusion
This study in which the antimicrobial resistance for fluoroquinolones in Campylobacter spp. was screened in both human and animal samples from South Africa demonstrated that there is a correlation between the resistance genes in samples from these two species. Restricting the use of certain antibiotics in poultry production systems in South Africa may help to reduce the prevalence of antibiotic resistant Campylobacter strains and hence limit transmission of multiresistant strains to the food chain. This may ultimately contribute to improving the efficacy of antibiotics used to treat human infections by controlling the emergence of resistant strains at the level of the food production industry. Sig. (2-tailed) 0.000 0.000 0.000 -**, Correlation is significant at the 0.01 level (2-tailed).