|Year : 2020 | Volume
| Issue : 2 | Page : 157-161
Investigation of antimicrobial resistance and virulence genes of Campylobacter isolates from patients in a tertiary hospital in Edirne, Turkey
Canan Eryildiz1, Kiymet Tabakcioglu2, Gulcan Kuyucuklu1, Nermin Sakru1
1 Department of Medical Microbiology, Faculty of Medicine, Trakya University, Edirne, Turkey
2 Department of Medical Biology, Faculty of Medicine, Trakya University, Edirne, Turkey
|Date of Submission||24-Feb-2020|
|Date of Decision||10-Jun-2020|
|Date of Acceptance||08-Jul-2020|
|Date of Web Publication||29-Aug-2020|
Dr. Canan Eryildiz
Department of Medical Microbiology, Faculty of Medicine, Trakya University, Balkan Campus, Edirne 22030
Source of Support: None, Conflict of Interest: None
Purpose: Campylobacter is one of the most common pathogens that cause food-borne infections worldwide. The aim of this study was to determine the antimicrobial resistance rates and the presence of multiple virulence genes in Campylobacter isolates obtained from humans. Materials and Methods: In this study, 71 Campylobacter isolates obtained from human faecal samples were used. Antimicrobial susceptibility tests were performed through the gradient strip method. The presence of virulence genes was investigated by monoplex and multiplex polymerase chain reaction. Results: The rate of resistance of the 66 Campylobacter jejuni isolates was 12.1% for erythromycin, 40.9% for tetracycline and 68.2% for ciprofloxacin. Only one of five Campylobacter coli isolates was resistant to these three antimicrobial agents. The flaB, pldA, cdtA, cadF, cdtC and ceuE genes were found in all 66 of the C. jejuni isolates. In the C. jejuni isolates, positivity rates of 92.4% for flaA, 96.7% for cdtB, 98.5% for ciaB, 90.9% for dnaJ and 96.7% for racR were observed. The flaA, flaB, ciaB, cdtA and cdtC genes were present in all C. coli isolates. Conclusions: It was detected that there is an increase in antimicrobial resistance of Campylobacter strains in our region, and most of the isolates harbour virulence genes.
Keywords: Antimicrobial resistance, Campylobacter, human, virulence genes
|How to cite this article:|
Eryildiz C, Tabakcioglu K, Kuyucuklu G, Sakru N. Investigation of antimicrobial resistance and virulence genes of Campylobacter isolates from patients in a tertiary hospital in Edirne, Turkey. Indian J Med Microbiol 2020;38:157-61
|How to cite this URL:|
Eryildiz C, Tabakcioglu K, Kuyucuklu G, Sakru N. Investigation of antimicrobial resistance and virulence genes of Campylobacter isolates from patients in a tertiary hospital in Edirne, Turkey. Indian J Med Microbiol [serial online] 2020 [cited 2020 Sep 26];38:157-61. Available from: http://www.ijmm.org/text.asp?2020/38/2/157/293914
| ~ Introduction|| |
Campylobacter is one of the major causes of food-borne acute gastroenteritis worldwide. C. jejuni is the most common species (approximately 90%) responsible for Campylobacter infections, and the second one is Campylobacter coli. Campylobacter species are also associated with some gastrointestinal and extra-gastrointestinal manifestations and various post-infectious complications., The primary reservoirs of Campylobacter are poultry, cattle and swine; however, these bacteria can also be isolated from other animals and the environment. Campylobacter gastroenteritis is a self-limited infection in most of the cases and does not require any antimicrobial therapy. However, antimicrobial drugs can be used in some situations, such as fever, bloody diarrhoea, prolonged illness and immunocompromised patients. Macrolides and fluoroquinolones are commonly used in antimicrobial therapy. In addition, tetracycline, doxycycline and chloramphenicol are among the alternative drugs used to treat infections. There have been significant increases in the resistance rates to the antimicrobials used in the treatment, and this increased resistance is an important public health problem. Antibiotic-resistant Campylobacter strains have spread worldwide, mainly due to the continuous use of antibiotics in animal husbandry. However, insufficiency in wastewater treatment, pollution of water resources with human and animal wastes and improper preparation of animal-derived foods are other factors that have affected the increase in antibiotic resistance. Antimicrobial susceptibility testing is important to manage treatment and monitor epidemiological data.
Although the pathogenesis of Campylobacter infections is not completely understood, some genes are considered to be responsible for different stages of infection. Flagellum, which is involved in bacterial motility and colonisation, is important in the pathogenesis of campylobacteriosis. Major flagellin protein FlaA and the minor flagellin protein FlaB are components of the extracellular filament of flagellum. FlaA plays a role in flagellation and motility, however, the necessity of FlaB varies depending on the species., CadF, an outer membrane protein, contributes to the binding of Campylobacter to intestinal epithelial cells. RacR, a response regulator protein, plays a role in response to specific environmental signals of bacteria. It has been demonstrated that inactivation of the racR gene reduces the colonisation ability of bacteria. Furthermore, it has been demonstrated that the ceuE gene encodes the periplasmic enterochelin-binding lipoprotein., The ceuE gene was characterised to identify C. jejuni and C. coli. Mutation of the C. jejuni ciaB gene, which encodes Campylobacter invasion antigen B, reduces the invasion potential of bacteria. Cytolethal distending toxin consists of three subunits encoded by the cdtA, cdtB and cdtC genes. The CdtA and CdtC subunits are involved in binding to cell, whereas CdtB subunit shows toxin activity, which blocks cell division. The dnaJ gene encodes a heat shock protein, which is associated with colonisation. It is thought that the difference among Campylobacter isolates in terms of the presence of virulence genes may explain differences in virulence potential.
The aim of our study was to determine the antimicrobial resistance rates and the presence of several virulence genes in Campylobacter isolates obtained from humans.
| ~ Materials and Methods|| |
This study was approved by the Ethical Committee of Trakya University School of Medicine.
Campylobacter isolates obtained from the faecal samples of patients with acute gastroenteritis at the Trakya University Health Research and Application Center Department of Medical Microbiology in Edirne, Turkey, between June 2016 and December 2016 were included in this study. Faecal samples were streaked on Campylobacter agar (Becton Dickinson, USA) containing 7% horse blood and incubated at 42°C in a microaerophilic atmosphere for 48 h. Gram staining and catalase and oxidase tests were performed on suspected colonies to identify Campylobacter species. Campylobacter isolates were stored in Mueller-Hinton broth containing 20% glycerol at −80°C for further use.
Antimicrobial susceptibility testing
Campylobacter isolates were subcultured on 5% sheep blood Campylobacter agar (Liofilchem, Italy) and incubated at 42°C in a microaerophilic atmosphere for 48 h. A bacterial suspension of 0.5 McFarland turbidity was inoculated onto Mueller-Hinton agar with 5% sheep blood agar. Strips of erythromycin (0.016–256 mg/L), tetracycline (0.016–256 mg/L) and ciprofloxacin (0.002–32 mg/L) (Liofilchem, Italy) were placed on the agar media, and the plates were incubated at 42°C in a microaerophilic atmosphere for 24 h. The results were interpreted according to the Clinical and Laboratory Standards Institute guidelines.
Genomic DNA was extracted with a commercial kit (PureLink Genomic DNA Mini Kit, Invitrogen, by Thermo Fisher Scientific, USA) according to the manufacturer's instructions and stored at −20°C until used.
Genus and species identification
A multiplex polymerase chain reaction (mPCR) was performed using the previously described primers and protocol with minor modifications. C. jejuni NCTC 13367 and C. coli NCTC 11350 were used as positive controls, and Escherichia coli ACTC 25922 was used as a negative control in all PCR experiments.
Detection of virulence genes
PCRs (monoplex and multiplex) were carried out with the previously described primers and PCR conditions with slight modifications to investigate the presence of the flaA, flaB, cdtA, cdtB, cdtC, cadF, ciaB, dnaJ, racR, pldA and ceuE virulence genes. The primers are shown in [Table 1]. The C. jejuni NCTC 13367 strain was used as a positive control for the flaA, flaB, cdtA, cdtB, cdtC, ciaB, dnaJ, racR and pldA genes, and the C. jejuni NCTC 11351 strain was used as a positive control for cadF gene. The C. jejuni NCTC 10983 strain and C. coli NCTC 11350 strain were used as positive controls for the ceuE gene.
| ~ Results|| |
A total of 71 Campylobacter isolates were obtained from humans; 66 isolates were identified as C. jejuni and 5 isolates were identified as C. coli by mPCR. Resistance to erythromycin, tetracycline and ciprofloxacin was detected in 12.1%, 40.9% and 68.2% of the 66 C. jejuni isolates, respectively [Table 2]. One C. coli isolate was resistant to all three of these antimicrobials. In addition, two C. coli isolates demonstrated intermediate resistance to ciprofloxacin.
|Table 2: Interpretative criteria for Campylobacter susceptibility testing and antimicrobial resistance percentages of Campylobacter jejuni isolates|
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The flaB, pldA, cadF, cdtA, cdtC and ceuE genes were present in all C. jejuni isolates. The flaA, cdtB, ciaB, dnaJ and racR genes were present in 92.4%, 96.7%, 98.5%, 90.9% and 96.7% of C. jejuni isolates, respectively. All of the C. coli isolates were positive for the flaA, flaB, ciaB, cdtA and cdtC genes. Four C. coli isolates had the cdtB, pldA, racR, cadF and ceuE genes, and one isolate had the dnaJ gene [Table 3]. There were 23 monodrug-resistant isolates (erythromycin, 2 isolates; tetracycline, 1 isolate; and ciprofloxacin, 20 isolates), whereas there were 24 isolates resistant to two drugs (erythromycin and tetracycline, 2 isolates; erythromycin and ciprofloxacin, 1 isolate; and tetracycline and ciprofloxacin, 21 isolates). Three isolates were resistant to all three drugs studied.
|Table 3: Percentage of virulence genes detected in Campylobacter isolates|
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| ~ Discussion|| |
Macrolides and fluoroquinolones are commonly used to treat Campylobacter infections that require antimicrobial treatment. Tetracycline is one of the alternative drugs for treatment. In the strains isolated from gastroenteritis cases, increasing resistance to several antibiotics, including macrolides and fluoroquinolones, has been observed., It is thought that the use of fluoroquinolones in the agricultural industry and veterinary medicine plays a role in high fluoroquinolone resistance rates in Campylobacter spp.
In a study performed in Qatar, the antibiotic resistance rates of C. jejuni isolates from patients with diarrhoea were investigated, and resistance rates of 63.2% to ciprofloxacin and 8.6% to erythromycin were found. In a study performed in Switzerland, resistance to ciprofloxacin, tetracycline and erythromycin was found in 37.5%, 33.1% and 0.7% of C. jejuni isolates obtained from humans, respectively. In Italy, ciprofloxacin, tetracycline and erythromycin resistance rates were 76%, 61% and 7% in C. jejuni isolates and 70%, 76% and 32% in C. coli isolates, respectively. Erythromycin resistance was found in 7% of Campylobacter isolates obtained from faecal samples in Turkey. In another study performed in Turkey, 152 Campylobacter isolates were obtained from patients, and 74.3%, 25% and 5.9% were resistant to ciprofloxacin, tetracycline and erythromycin, respectively. In a study evaluating 4-year data in a university hospital in Ankara, 81.8% of Campylobacter isolates were resistant to quinolones, and 18.9% were resistant to tetracycline, whereas no resistance to erythromycin was detected. Multidrug resistance was observed in 15.9% of the isolates.
The resistance rates among the isolates we tested were highest for ciprofloxacin. The lowest resistance rate was to erythromycin. Our results are consistent with those of other studies in Turkey and in other parts of the world. In a study performed in Edirne in 2005, an 8% resistance rate to erythromycin and ciprofloxacin was detected in C. jejuni strains, whereas no resistance to tetracycline was found. When compared with the data of this study, there was a significant increase in the rate of antimicrobial resistance in C. jejuni in our region.
Although the molecular basis of Campylobacter pathogenesis has not been completely elucidated, multiple factors, such as adherence, cytotoxin production, motility and invasion, are thought to play a role. Several genes that are thought to be associated with the infection process have been identified.,
In a study that included 174 C. jejuni isolates from different geographical regions, the ciaB, cadF and cdtB genes were investigated and were positive in 71.8%, 59.8% and 87.4% of isolates, respectively. A total of 155 C. jejuni isolates from humans with diarrhoea were investigated, and all isolates had the flaA, cadF, racR, cdtA, cdtB, cdtC and ciaB genes. In a study that investigated virulence genes in 83 Campylobacter clinical isolates, the cadF, ciaB, dnaJ, pldA, cdtA, cdtB and cdtC genes were present in 100%, 45%, 46%, 49%, 33%, 20% and 30% of C. jejuni isolates and 100%, 43%, 50%, 71%, 29%, 14% and 36% of C. coli isolates, respectively. In 30 C. jejuni isolates from humans in Turkey, the cdtA, cdtB and cdtC genes were found in 86.6%, 96% and 90% of isolates, respectively. In another study from Turkey, of 126 C. jejuni isolates obtained from patients with acute gastroenteritis, some isolates had the flaA (88.1%), flaB (96.8%), cadF (95.2%), dnaJ (92.1%), cdtA (87.3%), cdtB (44.4%), cdtC (81.7%), pIdA (69.0%) and ciaB (57.1%) genes.
In our study, the presence of virulence genes ranged from 90.9% to 100% in C. jejuni isolates and from 20% to 100% in C. coli isolates. Our results appear to be similar to those of other studies involving human isolates in Turkey and worldwide. However, the low number of C. coli isolates seems to be a limitation of the assessment. It is thought that the high virulence gene rates may have been due to the fact that our study was performed on isolates that caused gastroenteritis and were obtained from clinical samples.
| ~ Conclusions|| |
In Campylobacter strains that caused gastroenteritis, there was an increase in resistance to antimicrobials used to treat infections in our region, and most of the isolates had virulence genes. Monitoring resistance in Campylobacter spp. has an important role in the determination of the antimicrobials used in empirical therapy. The detection of virulence genes in bacteria is important for providing data to explain the pathogenesis of infections and to develop different approaches in advanced genetic studies to prevent infections.
Financial support and sponsorship
This study was financially supported by the Trakya University Scientific Research Fund (TUBAP-2017/54).
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3]