Molecular characterization of Campylobacter spp. isolated from poultry faeces and carcasses in Poland

Campylobacter infection is one of the most common enteric human diseases world-wide but the mechanism of Campylobacter pathogenicity has not been exactly explained yet. One of the main reasons is genotypic, hence phenotypic diversity of the bacterial isolates. The aim of the present study was to perform a molecular characterization of randomly selected C. jejuni and C. coli strains isolated from poultry faeces and carcasses in Poland. Several virulence gene markers were identified by polymerase chain reaction (PCR). Furthermore, genetic typing has also been used by the macrorestriction profiling with pulsed-field gel electrophoresis (PFGE). The results of the present study showed that all analyzed isolates of C. jejuni (n = 24) and C. coli (n = 24) contained the flaA and cadF sequences. On the other hand, the virB11 gene was present only in 6 of 48 (12.5%) of the analyzed isolates, whereas most of the strains contained the cdt genes. Other virulence gene iam linked to Campylobacter invasiveness was present in 34 of 48 (72.9%) strains. The restriction analysis of the whole genome digested with SmaI produced three main clonal groups designed as I, II (with two subgroups IIa and IIb), and III obtained by the comparison of macrorestriction profiling patterns. The results showed a poor correlation between Campylobacter profiles generated by a clonal molecular technique and the presence of virulence markers. Therefore, PCR detection of Campylobacter virulence markers can be utilized as a simple and rapid tool to discriminate stains recovered from different sources, especially when used in conjunction with the PFGE profile analysis as a complex strategy. These kinds of analyses had not been previously carried out in Poland and these results may generate more knowledge regarding the genetic diversity and molecular relationship of Campylobacter. Campylobacter, virulence markers, molecular analysis, PCR, PFGE According to the recent European Food Safety Authority (EFSA) report, the most frequently reported zoonotic disease in humans in the European Union in 2008 was Campylobacter infection, with incidences of 40.7 per 100,000 people (http://www.efsa.europa.eu). The infection with Campylobacter spp. (campylobacteriosis), especially with C. jejuni, and to a lesser extent with C. coli, is one of the leading causes of bacterial diarrhoea worldwide. During last years, laboratory confirmed incidence of campylobacteriosis in Poland. In 2008, according to the EFSA report, there were 257 cases of the disease (0.7/100,000 population), however, it seems that the number of cases is still underestimated due to a lack of proper identification of the infectious agent. Poultry and its meat is considered to be the main vector of C. jejuni; transmission occurs either as a result of cross contamination due to improper handling of raw meat or consumption of undercooked food of animal (mainly poultry) origin. The mechanism of Campylobacter pathogenicity has not been exactly explained yet. One of the main reasons is genotypic, hence phenotypic diversity of the bacterial species belong to the genus of Campylobacter. For the consumers’ safety it is essential to characterize pathogenicity markers in strains that are identified in food. It was found that some C. jejuni strains are not pathogenic at all or induce mild symptoms in humans, whereas other isolates cause a serious illness (Rivera-Amill et al. 2001). It is still not clear which factors of Campylobacter are essential to the disease development. However, it is known that mechanisms of movement, chemotaxy, adhesion, transcytosis and host cell penetration as ACTA VET. BRNO 2011, 80: 019–027; doi:10.2754/avb201180010019 Address for correspondence: Jacek Osek Department of Hygiene of Food of Animal Origin National Veterinary Research Institute, Partyzantow 57 24-100 Pulawy, Poland Phone: +48-81-8893182 Fax: +48-81-8862595 E-mail: [email protected] http://www.vfu.cz/acta-vet/actavet.htm well as toxin production are necessary to induce campylobacteriosis in humans (Snelling et al. 2005). Several putative or defined virulence markers of Campylobacter spp. have been described (Bang et al. 2003; Datta et al. 2003). One of the best characterized Campylobacter virulence markers is the flaA gene which determines the flagella formation, hence bacteria motility and enterocyte colonization (Nuijten et al. 2000). Molecular identification and differentiation of Campylobacter strains isolated from the same or different samples is very important in order to trace the sources of human infection. The methods used for genetic characterization differ in their taxonomic range, discriminatory power, reproducibility, easiness of interpretation, and standardization. Macrorestriction profiling (MRP) by pulsed-field gel electrophoresis (PFGE) has been proved to be useful for this purpose, and its discriminatory power can be enhanced by increasing the number of restriction enzymes used (On et al. 1998). This method is currently a golden standard for the typing of Campylobacter spp. (Schouls et al. 2003). The aim of the present study was to perform a molecular characterization of randomly selected C. jejuni and C. coli strains isolated from poultry faeces and carcasses in Poland. For this purpose, 7 virulence genes important in pathogenesis of campylobacteriosis were chosen and identified by the PCR method. These molecular markers participate in adhesion and colonization (flaA, cadF), invasion (virB11) and toxin production (cdtA, cdtB, cdtC). Moreover, the iam sequence connected with diarrhoeal form of the disease was also identified. Furthermore, molecular characterization of the investigated Campylobacter strains were further performed by macrorestriction profiling with PFGE. Materials and Methods Bacterial strains The following positive and negative reference strains were included: C. jejuni ATCC 33291, C. coli ATCC 43478, Escherichia coli EDL 933, and Salmonella Typhimurium ATCC 14028. All Campylobacter isolates were isolated from randomly chosen poultry faeces and carcasses in Poland during the period of September 2004 and July 2005. The samples were obtained from different regions of the country (administrative division – voivodship), including the following areas: northern (Pomorskie – P; WarminskoMazurskie – WM, Zachodniopomorskie – ZP voivodships), western (Dolnoslaskie – D, Lubuskie – L voivodships), southern (Opolskie – O, Slaskie – S voivodships), and eastern (Lubelskie – LU, Ludzkie – LD, Mazowieckie – M voivodships). All faecal samples were taken using swab method at farm level. From each farm one pooled sample (taken from at least ten fresh droppings) were examined. The carcass samples were collected at slaughterhouse at the end of processing level, from final products stored chilled (< 4 °C). For isolation of thermophilic Campylobacter spp., swabs inoculated with faeces or carcass samples were plated onto Campylobacter bloodfree selective medium such as mCCDA (Oxoid, UK) or Karmali Agar (Oxoid, UK) followed by incubation at 41.5 °C for 40-48 h in microaerofilic conditions generated by the Campy Gen gas-generating kit (Oxoid, UK). Bacteria from individual colonies were stored (at -80 °C in nutrient broth, with glycerol added to 15% for genotypic analyses. Suspected bacterial colonies were tested by multiplex PCR (m-PCR) for the simultaneous detection of the C. jejuni and C. coli in a single reaction based on 16S rRNA (specific for theromphilic Campylobacter), ceuE (specific for C. coli), and mapA genes (typical for C. jejuni), respectively (Wieczorek and Osek 2005). Total of 48 isolates (24 from faeces and 24 from poultry carcasses) were used in this study. Detection of putative virulence genes by PCR Campylobacter strains were grown at 41.5 oC in Karmali agar for 24 h under microaerophilic condition. A bacterial colony was suspended in 1 ml of sterile water and centrifuged at 13 000 g for 1 min. Afterwards, DNA was extracted using the Genomic – Mini kit (A&A Biotechnology, Poland) according to the manufacturer’s instruction. The purity and concentration of the DNA preparations were estimated using spectrophotometry at 260 and 280 nm. Characteristics of all primers used in the study are shown in Table 1. The PCR primers were commercially synthesised (Symbiosis, Poland). All PCRs were carried out in a thermal cycler (PTC-100, MJ Research, USA) under the following conditions: initial DNA denaturation at 94 oC for 5 min followed by 30 cycles of 94 oC for 1 min, 55 oC for 1 min (with the exception for the flaA gene – 48 oC for 1 min) and 72 oC for 1 min. The final extension step was run at 72 oC for 5 min. The analysis of the amplified products was performed in 2% agarose (Sigma, USA) in Tris-Acetate–EDTA (TAE) buffer at 100 V. The DNA bands were visualised by staining with ethidium bromide, analysed under UV light (300 nm) and photographed using the Gel Doc 2000 documentation system (Bio-Rad, USA). The size of the PCR amplicons was compared to the 100 bp DNA marker (Fermentas, EU). 20