ABSTRACT

BACKGROUND/AIMS:

Campylobacter is a major cause of foodborne diarrheal disease, and the incidence of campylobacteriosis has significantly increased in both developed and developing countries. The purpose of the present study was to identify the species of Campylobacter isolates and to evaluate the distribution of Campylobacter infections according to various characteristics in our region.

MATERIALS AND METHODS:

Campylobacter isolates obtained from patients at a tertiary hospital in Edirne, Turkey were included in this study. The distribution of Campylobacter infections was evaluated according to age, season, and gender. Species identification was performed by multiplex polymerase chain reaction (PCR). The RNA polymerase beta-subunit gene (rpoB) of selected samples was amplified, and DNA sequencing was performed.

RESULTS:

Campylobacter species were isolated from 226 (4.3%) of the 5,241 samples. One hundred and seventy-six (89.3%) of 197 samples were identified as C. jejuni and 19 (9.6%) as C. coli by multiplex PCR. Two isolates showed a band profile compatible with both C. jejuni and C. coli. DNA sequencing was performed for 21 isolates. Sixteen isolates were compatible with C. jejuni and 5 isolates were consistent with C. coli. There was no statistically significant difference in Campylobacter isolation rates according to gender and season (p>0.05). Campylobacter species were most frequently isolated from children in the age group of 0-14 years (p<0.01).

CONCLUSION:

Campylobacter is one of the main causes of diarrhea in Turkey, and this infection is more common in children. This study contributes to information about the situation of Campylobacter infection and the genetic features of isolates in Turkey.

Keywords:

Campylobacter, multiplex PCR, DNA sequencing, Turkey

INTRODUCTION

Campylobacter species are Gram-negative bacteria that are a major cause of gastroenteritis around the world.¹ Although the primary reservoirs of Campylobacter species are poultry, cattle, and pigs, these bacteria can be colonized in the gastrointestinal tract of many warm-blooded animals.²Transmission to humans is often caused by ingestion of contaminated food or water or by contact with fecal material from infected animals or people.³To date, 32 Campylobacter species and 9 subspecies have been isolated from various sources. However, the majority of human infections are caused by C. jejuni and C. coli.⁴ In recent years, infections caused by Campylobacter species have increased in both developed and developing countries.¹ The infection occurs especially in children younger than 5. However, high incidence can also be seen in other age groups.^3,5 According to “The European Union One Health 2019 Zoonoses Report”, there was a seasonality in campylobacteriosis cases, with peaks in the summer months.⁶

The aim of this study was to determine the distribution of Campylobacter species and to evaluate the effect of age, gender and seasonality on Campylobacter infections.

MATERIALS AND METHODS

Ethics Statement

This study was approved by the Ethical Committee of Trakya University Faculty of Medicine (approval number: TUTF-BAEK 2016/196).

Bacterial Isolates

Campylobacter isolates obtained from 5,241 fecal samples by conventional methods at the Trakya University Health Center for Medical Research and Practice, Microbiology Laboratory between October, 2013 and May, 2016 were included in this study. Fecal samples were streaked on Campylobacter agar (Becton Dickinson, USA) containing 7% horse blood and incubated at 42 °C in a microaerophilic atmosphere for 48 hours. Gram staining, and catalase and oxidase tests were performed on suspected colonies to identify the Campylobacter species. Gram-negative, curved rods with positive catalase and oxidase tests were considered to be Campylobacter. Laboratory records were examined retrospectively, and the data were evaluated in terms of age, gender, and seasonal distribution.

DNA Extraction

DNA isolation of Campylobacter strains that were subcultured and stored at -20 °C was performed using the Invitrogen PureLink Genomic DNA Mini Kit (ThermoFisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol.

Identification of Campylobacter Species

A multiplex polymerase chain reaction (PCR) was performed according to the protocol established by Wang et al.⁷ with minor modifications. Each multiplex PCR tube contained 2.5 µL of 10X Taq buffer [100 mM Tris-HCl (pH 8.8), 500 mM KCl, 0.8% (v/v) Nonidet P40], 0.2 mM dNTP, 2 mM MgCl₂, 0.5 µM C. jejuni and C. lari primers, 1 µM C. coli primers, 2 µM C. upsaliensis primers, 0.2 µM 23S rRNA primers, 1.25 U of Taq DNA polymerase (ThermoFisher Scientific, Waltham, MA, USA), and 2.5 µL of whole-cell template DNA in total 25 μL reaction volume. The primers used for the identification of Campylobacter species are shown in Table 1. In all PCR reactions, C. jejuni NCTC 13367 and C. coli NCTC 11350 were used as positive controls, and E. coli ATCC 25922 was used as a negative control. PCR products were subjected to electrophoresis on 1.5% agarose gel, and the bands were evaluated under ultraviolet light.

Table 1

DNA Sequencing

DNA sequencing was performed for 21 isolates. According to the multiplex PCR results, 13 C. jejuni and 6 C. coli isolates were randomly selected for DNA sequencing. Sequence analysis was also performed on two samples exhibiting double bands. The RNA polymerase beta-subunit gene (rpoB) was amplified using the previously described primers.⁸ PCR products were purified using the Invitrogen PureLink Quick PCR Purification Kit (ThermoFisher Scientific, Waltham, MA, USA). DNA sequencing was performed by the Trakya University Technology Research Development Application and Research Center. The rpoB gene sequence analysis was carried out on the Applied Biosystems 3500 Series Genetic Analyzer (ThermoFisher Scientific, Waltham, MA, USA) using the Applied Biosystems BigDye Terminator v3.1 Cycle Sequencing Kit (ThermoFisher Scientific, Waltham, MA, USA). The sequencing chromatograms were analyzed using the ProSeq v2 and BioEdit programs. The nucleotide sequences of the isolates were compared with sequences in GenBank using the Basic Local Alignment Search Tool.

Statistical Analysis

NCSS (Number Cruncher Statistical System) 2007 software (Kaysville, Utah, USA) was used for statistical analysis. Data were analyzed by descriptive statistical methods. Qualitative data were compared by Pearson chi-square and the Fisher-freeman-halton exact test with a significance level of p<0.05.

RESULTS

Bacterial Isolates

A total of 5,241 stool samples were sent to the microbiology laboratory for Campylobacter culture between October, 2013 and May, 2016. Campylobacter spp. were detected in 226 (4.3%) of these samples, which were obtained from 215 individual patients. Of the patients with Campylobacter infection, 44.2% were female and 55.8% were male. There was no statistically significant difference in Campylobacter isolation rate by gender (p=0.584), and the difference in seasonal rates of Campylobacter isolation was not statistically significant (p=0.141) (Table 2). The highest culture positivity rate was detected in May (p=0.001), while the lowest rate was seen in September (p=0.014) (Figure 1). When the 0-5 and 6-14 age groups were evaluated together, Campylobacter spp. were isolated more frequently in the 0-14 age group compared to the other age groups (p<0.01) (Figure 2).

Table 2

Figure 1

Figure 2

Identification of Campylobacter Species

Bacterial DNA was obtained from 197 isolates of 187 patients. By multiplex PCR, 176 (89.3%) isolates were identified as C. jejuni and 19 (9.6%) isolates as C. coli. Two samples showed a band profile compatible with both C. jejuni and C. coli.

DNA Sequencing

The results of the DNA sequencing of 21 isolates were evaluated. The DNA sequences of 13 isolates were found to be compatible with the C. jejuni sequences in the GenBank. The DNA sequences of the five isolates matched C. coli. One isolate was identified as C. coli by multiplex PCR and C. jejuni by sequence analysis. Additionally, both of the isolates showing double bands in multiplex PCR were found to be compatible with C. jejuni. One of the C. coli isolates was submitted to the DNA Data Bank of Japan database (accession no: LC511784). The results of multiplex PCR and sequencing analysis of the isolates included in DNA sequencing are shown the Table 3.

Table 3

DISCUSSION

Campylobacter is one of the most common causes of diarrheal diseases worldwide.⁹ There has been a significant increase in the incidence of campylobacteriosis in North America, Europe, and Australia. Although epidemiological data are insufficient, it is known that campylobacteriosis is endemic in Africa, Asia, and the Middle East.¹ In European Union countries, 220,682 cases (59.7 per 100,000 population) were reported in 2019.⁶ In a study performed in Denmark, it was found that the incidence of Campylobacter infection was higher in males. For domestic cases, age groups 20-29 and 0-4 years had a higher incidence compared to other age groups. For travel-related cases, the incidence was highest in young adults aged 20-29 years. Also, it was revealed that cases not related to travel increased from May to October and peaked in August.⁵ In a study conducted in the United States, the highest incidence of campylobacteriosis was observed in males and children under five years of age in most regions, while in three states, the highest incidence was found in people over 60 years of age. The incidence was highest from June to August and lowest from December to February for all regions and age groups.¹⁰ In Ireland, the highest incidence of Campylobacter infection was reported in children aged 0-4 years. Cases reported in males were more than female cases in the surveillance system.¹¹ In our study, 44.2% of the patients with Campylobacter infection were female, and 55.8% of the patients were male; but no statistically significant difference was found by gender (p>0.05). Infection most commonly occurred in May (p=0.001), and least commonly in September (p=0.014); nonetheless, we observed no seasonal patterns. The majority of cases were detected in children, and these data are consistent with other studies in Turkey.^12-14

CONCLUSION

Campylobacter is a significant bacterial agent in gastroenteritis; therefore, it is important to investigate Campylobacter in routine laboratory diagnosis. In the present study, it was determined that there was no statistically significant difference in Campylobacter isolation rates according to gender and season in our region. However, it was observed that Campylobacter infection is more common in children. This study contributes to information about the epidemiological features of Campylobacter in Turkey. To obtain more comprehensive epidemiological data about Campylobacter infection and examine the the genetic features of Campylobacter, there is a need for further research conducted over an extended period and which uses multiple molecular methods.

MAIN POINTS

- Campylobacter species were isolated from 4.3% of the fecal samples.

- The most common Campylobacter species isolated from fecal samples was C. jejuni (89.3%) followed by C. coli (9.6%).

- No statistically significant difference was found in the Campylobacter isolation rate according to gender and season (p>0.05).

- Campylobacter species were most commonly isolated from children aged 0-14 years (p<0.01).

Acknowledgment: A part of the this study was presented as a poster at the 10^th Balkan Congress of Microbiology; November 16-18^th, 2017, Sofia, Bulgaria.

ETHICS

Ethics Committee Approval: This study was approved by the Ethical Committee of Trakya University Faculty of Medicine (approval number: TUTF-BAEK 2016/196).

Informed Consent: Informed consent is not necessary due to the retrospective nature of this study.

Peer-review: Externally peer-reviewed.

Authorship Contributions

Concept: C.E., N.Ş., K.T., M.C.U., Ş.B., Design: C.E., N.Ş., K.T., M.C.U., Ş.B., Supervision: C.E., N.Ş., K.T., Materials: C.E., N.Ş., Ş.B., Data Collection and/or Processing: C.E., N.Ş., M.C.U., Analysis and/or Interpretation: C.E., N.Ş., K.T., M.C.U., Literature Search: C.E., Ş.B., Writing: C.E., Critical Review: N.Ş., K.T.

DISCLOSURES

Conflict of Interest: No conflict of interest was declared by the authors.

Financial Disclosure: This study was financially supported by the Trakya University Scientific Research Fund [TUBAP-2017/53].

References

Kaakoush NO, Castaño-Rodríguez N, Mitchell HM, Man SM. Global epidemiology of Campylobacter infection. Clin Microbiol Rev. 2015; 28(3): 687-720.

Wagenaar JA, Newell DG, Kalupahana RS, Mughini-Gras L. Campylobacter: Animal reservoirs, human infections, and options for control. Sing A, (editor). Zoonoses - Infections affecting humans and animals: Focus on public health aspects. Dordrecht: Springer; 2015.p.159-77.

Fitzgerald C. Campylobacter. Clin Lab Med. 2015; 35(2): 289-98.

Costa D, Iraola G. Pathogenomics of emerging Campylobacter species. Clin Microbiol Rev. 2019; 32(4): e00072-18.

Kuhn KG, Nielsen EM, Mølbak K, Ethelberg S. Epidemiology of campylobacteriosis in Denmark 2000-2015. Zoonoses Public Health. 2018; 65(1): 59-66.

European Food Safety Authority and European Centre for Disease Prevention and Control (EFSA and ECDC). The European Union One Health 2019 Zoonoses Report. EFSA J 2021; 19(2): 6406. DOI: 10.2903/j.efsa.2021.6406.

Wang G, Clark CG, Taylor TM, Pucknell C, Barton C, Price L, et al. Colony multiplex PCR assay for identification and differentiation of Campylobacter jejuni, C. coli, C. lari, C. upsaliensis, and C. fetus subsp. fetus. J Clin Microbiol. 2002; 40(12): 4744-7.

Korczak BM, Stieber R, Emler S, Burnens AP, Frey J, Kuhnert P. Genetic relatedness within the genus Campylobacter inferred from rpoB sequences. Int J Syst Evol Microbiol. 2006; 56(5): 937-45.

World Health Organization; [last accessed on 8 June 2021]. Available from: https://www.who.int/news-room/fact-sheets/detail/campylobacter.c2020

Geissler AL, Bustos Carrillo F, Swanson K, Patrick ME, Fullerton KE, Bennett C. Increasing Campylobacter infections, outbreaks, and antimicrobial resistance in the United States, 2004-2012. Clin Infect Dis. 2017; 65(10): 1624-31.

O’Connor L, McKeown P, Barrasa A, Garvey P. Epidemiology of Campylobacter infections in Ireland 2004-2016: What has changed? Zoonoses Public Health. 2020; 67(4): 362-9.

Gülmez D, Gür D, Hascelik G, Guleşen R, Levent B. Experiences of a university hospital participating in the National Enteric Pathogens Surveillance Network (UEPLA): Four- year data of Salmonella, Shigella and Campylobacter. Türk Mikrobiyol Cem Derg. 2012; 42(3): 85-92.

Kayman T, Abay S, Hızlısoy H. Identification of Campylobacter spp. isolates with phenotypic methods and multiplex polymerase chain reaction and their antibiotic susceptibilities. Mikrobiyol Bul. 2013; 47(2): 230-9.

Ciftci N, Hatice Turk-Dagi H, Tuncer I. Prevalence and antimicrobial susceptibility of Campylobacter and Salmonella species in patients with acute gastroenteritis. Klimik Derg. 2019; 32(2): 127-31.

Patrick ME, Henao OL, Robinson T, Geissler AL, Cronquist A, Hanna S, et al. Features of illnesses caused by five species of Campylobacter, Foodborne Diseases Active Surveillance Network (FoodNet) - 2010-2015. Epidemiol Infect. 2018; 146(1): 1-10.

Hamidian M, Sanaei M, Bolfion M, Dabiri H, Zali MR, Walther-Rasmussen J. Prevalence of putative virulence markers in Campylobacter jejuni and Campylobacter coli isolated from hospitalized children, raw chicken, and raw beef in Tehran, Iran. Can J Microbiol. 2011; 57(2): 143-8.

Ateş Yılmaz A, Tuğrul HM. Investigation on Campylobacter species as diarrheogenic agents in Edirne, Turkey, and their antimicrobial susceptibility patterns. Turkish Journal of Infection. 2005; 19(1): 53-9.

Wirz SE, Overesch G, Kuhnert P, Korczak BM. Genotype and antibiotic resistance analyses of Campylobacter isolates from ceca and carcasses of slaughtered broiler flocks. Appl Environ Microbiol. 2010; 76(19): 6377-86.

Molecular Identification of Campylobacter Species Isolated from Patients with Gastroenteritis in Edirne, Turkey