Introduction

Infection with Campylobacterspp. is the leading cause of bacterial gastroenteritis in Europe and North America. It is a typical food infection with a source of domestic and wild birds, mainly broilers, the human disease most often caused by the socalled crossbreeding in the preparation of various foods being in contact with poultry meat. Less often the source may be unpasteurized milk, contaminated drinking water, pets or a sick person. Campylobacteriosis is also one of the causes of diarrhea in travellers [1-5].

Leading agents of diarrheal diseases in humans are Campylobacter jejuni and Campylobactercoli. Although rarely C. jejuni is described as a cause of some autoimmune diseases such as Guillain-Barre syndrome, post-infectious arthritis, neuropathies, etc. [1].

In practice, macrolides (Azithromycin, Clarithromycin), especially fluoroquinolones (Ciprofloxacin) are most commonly used in antimicrobial therapy of children and adults. A number of authors describe tetracyclines as a possible choice, but in practice they are seldom applied. Due to the frequent use of fluoroquinolones in broiler breeding and the abuse of antibacterial therapy, numerous studies have shown a sharp increase in resistance to fluoroquinolones, tetracyclines and even some macrolides [5].

The purpose of this study is to detect the prevalence and antimicrobial resistance of C. jejuni and C. coli isolated from hospitalized patients with diarrheal syndrome.

Materials and Methods

Collection of samples and reference strains

The bacterial strains used as positive controls in this work are ATCC 33560 for C. jejuni and C-14.2 for C. coli.

In 2017, 260 faecal samples were collected from patients hospitalized with diarrheal syndrome aged 0-80.

Rapid test and sample processing

Each faeces was tested by an immunochromatographic assay of CerTestBiotec, S.L. according to the manufacturer’s instructions. Of all positive samples, plain blood agar with 10% defibrillating sheep blood (BulBio, Bulgaria) was performed on a membrane with nitrocellulose membranes (SartoriusStedimBiotech) with a pore size of 0.45 μm. The seeds were cultured in a microaerophilic atmosphere (5% O₂+10% CO₂+ 85% N₂), which was generated from gas-again “Helyco-Campy Pac” in anaerobic jar (Oxoid, USA) at 42-43°C for 24-72 hours.

Phenotypic identification of Campylobacterspp

For the validation tests according to ISO 10272-1: 2006 and ISO/ TS 10272-2: 2006 pure culture was used. We used a catalase and oxidase test, hydrolysis of sodium hippurate and indoxil acetate .

DNA extraction and PCR identification

For isolation of DNA from fresh culture, lysis buffer PrepMan^™ Ultra (Applied Biosystems, Foster City, CA) was used.

Three pairs of primers, identifying and differentiating strains of C. jejuni/C. coli were used for Multiplex-PCR analysis. The primers amplifying the genes have the following sequences: for cadF-F-TTGAAGGTAATTTAGATATG, for cadF-R-CTAATACCTAAAGTTGAAAC; for hipO-F-GAAGAGGGTTTGGGTGGTG, for hipO-R-AGCTAGCTTCGCARAATAACTTG; for asp-F-GGTATGATTTCTACAAAGCGAG, for asp-R-ATAAAAGACTATCGTCGCGTG. The final reaction volume was 25 μl and in this volume were included 1x TaqDNAPolymerase buffer; 4 mM MgCl₂; 0.2 mM dNTP; 0.03 U/μl of SuperHotTaq DNA Polymerase (set Applichem GmbH, Germany); 0.6 μMcadF-F/R; 0.2 μMasp-F/R; 0.2 μMhipO-F/R and 5 μl DNA. The temperature regime was 94°C for 5 min, initial denaturation, followed by 35 cycles: 94°C, 45 s, denaturation, 52°C, 45 s, hybridization, 72°C, 60 s, elongation, 72°C, 2 min, final elongation step. All PCR products in the assay methods were split into standard agarose electrophoresis in 1x TBE buffer at 6 V/cm, 100 Ams in 2% agarose gels. Visualization of the amplified products and molecular markers was performed by illuminating by ultraviolet a light transilluminator.

Antibiotic sensitivity test

We used the Kirby-Bauer standard disk-diffusion method as the test was performed according to the recommendations and standards of the CLSA Guidelines. For the interpretation of the size of the inhibition zones, we used the CLSA standards related to the Enterobacteriaceae family.

Results

Of the 260 probes tested, 66 (25.38%) were positive for Campylobacterspp., of which 60 (91%) were identified as C. jejuni and 6 (9%) as C. coli.

Isolates were tested for susceptibility to 5 antimicrobials used in clinical practice to treat Campylobacterspp. infection. The data interpretation revealed that all isolates are susceptible to at least one of the antimicrobial agents. All C. jejuni/coli isolates are Azitromycin (AZI) sensitive, and all C. coli isolates are susceptible to Clarithromycin (CLA) and Erythromycin (ER). Three isolates (5%) of C. jejuni are resistant to CLA and 7 (11.6%) to ER. 35 (58.3%) of C. jejuni isolates are resistant to Ciprofloxacin (CIP) and 2 (33.3%) of C. coli. For Tetracycline (TE), the resistance is 23 (38.3%) for C. jejuni and 1 (16.6%) for C. coli. In C. jejuni there were 3 (5%) isolates multi-resistant to CIP-CL-ER-TE simultaneously; also 3 (5%) to CL-ER-CIP, and 7 (11.6%) to TEE-ER. In C. coli there was 1 (16.6%) TE-ER resistant isolate (Table 1).

Table 1The frequency of antibiotic drug resistance in Campylobacter spp.

Discussion

Although campylobacterios is often manifests itself as a mild self-limiting diarrheal disease, it is common for children and immunocompromised patients to develop as severe gastroenteritis or severe extra-intestinal complications. In the case of severe circuit and underlying diseases, the antimicrobial treatment of the infection is mandatory, with macrolides and fluoroquinolones being used most frequently in clinical practice [1]. In Bulgaria, the very commonly used agents are Clarithromycin, Erythromycin and Ciprofloxacin [6]. The antimicrobial resistance of some Campylobacterstrains is becoming increasingly common in the world. Campylobacterjejuni has a relatively low resistance to ER - about 5%, and is one of the most widely used drugs in the European Union (EU). The worrying data on the increase in CIP-resistant strains have been known since the end of the 20th century. Currently, in the EU, it has reached an average of 47.4%. Due to the fact that resistance to fluoroquinolones is due to two synergistic mechanisms, fluoroquinolone resistant mutants develop rapidly during antibiotic treatment, and the strains persist even after removal of the selection [1,5]

As reported by other EU countries, our data also show an alarmingly high resistance of C. jejuni strains to CIP. Although significantly lower, C. jejuni’s resistance to ER also occurs in our country approximately as in other European countries. According to many authors, the resistance of C. coli to macrolides is higher than that of C. jejuni, but in our study we do not find such regularity. It is very possible that this is due to the small number of C. coli strains we have worked with. None of the strains of Campylobacterspp. in our work did show resistance to AZI, which is a prerequisite for it to be most commonly seen as a means of choice when using macrolides. Tetracyclines are rarely the first choice in the treatment of campylobacteriosis, and the high resistance of our strains to it is comparable to that in other EU countries [3].

Very soon, in 2017, the World Health Organization announced the list of the world’s most problematic bacteria for which it is urgently needed to develop new antibiotic molecules, such as fluoroquinolone-resistant Campylobacterspp. which are ranked fourth in the “high priority” category [5]. In recent years, multiresistant strains of Campylobacterspp. have also been reported more frequently, and our isolates have also been reported in our work.

References

1. Blaser B, Engberg J (2008) Clinical aspects of Campylobacter jejuni and Campylobacter coli infections. In: Nachamkin I, Szymanski CM, Blaser MJ, editors. Campylobacter. Washington DC: American Society Microbiol 99-121.
2. Kaakoush NO, Castaño-Rodríguez N, Mitchell HM, Man SM (2015) Global epidemiology of Campylobacter infection. Clin Microbiol Rev 28: 687-720.
3. Rozynek E, Dzierzanowska-Fangrat K, Korsak D, Konieczny P, Wardak S, et al. (2008) Comparison of antimicrobial resistance of Campylobacter jejuni and Campylobacter coli isolated from humans and chicken carcasses in Poland. J Food Prot 71: 602-607.
4. Gaudreau C, Boucher F, Gilbert H, Bekal S (2014) Antimicrobial susceptibility of Campylobacter jejuni and Campylobacter coli isolates obtained in Montreal, Quebec, Canada, from 2002 to 2013. J Clin Microbiol 52: 2644-2646.
5. Szczepanska B, Andrzejewska M, Spica D, Klawe JJ (2017) Prevalence and antimicrobial resistance of Campylobacter jejuni and Campylobacter coli isolated from children and environmental sources in urban and suburban areas. BMC Microbiol 4: 17-80.
6. Maramski A, Daskalov H (2009) Campylobacter resistance isolated from broiler carcasses. Collection of scientific papers of National Institute of Veterinary Medicine, Bulgaria 40-44.