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Prevalence of potential virulence markers in Polish Campylobacter jejuni and Campylobacter coli isolates obtained from hospitalized children and from chicken carcasses

¹Department of Clinical Microbiology and Immunology, Children's Memorial Health Institute, 04-730 Warsaw, Aleja Dzieci Polskich 20, Poland ²Department of Safety of Food and Nutrition, National Food and Nutrition Institute, 02-903 Warsaw, Powsinska 61/63, Poland

Correspondence Elzbieta Rozynek fangrat{at}supermedia.pl

Received December 20, 2004
Accepted March 15, 2005

The pathogenicity of thermotolerant Campylobacter species, common food-borne pathogens, depends on certain factors unevenly distributed among strains of different origin. The prevalence of such markers has never been examined in a population of Polish Campylobacter strains of human and poultry origin. Therefore, we analysed the presence of the cadF, cdtA, cdtB and cdtC genes and the iam sequence in Campylobacter jejuni (n = 115) and Campylobacter coli (n = 57) isolates from children with diarrhoea and from chicken carcasses. The cadF gene was present in nearly 100 % of Campylobacter isolates tested, regardless of their origin or species. In contrast, the iam region was found in 83.3 % and 100 % of C. coli isolates from children and chickens, respectively, but in only 1.6 % and 54.7 %, respectively, of C. jejuni isolates. Similarly, the detection rates of cdt genes varied between human and chicken isolates. All three cdt genes were found in nearly all C. jejuni isolates from both children and chickens, but in only 5.6 % of human C. coli isolates as compared to 87.2 % of chicken C. coli isolates. This different distribution of genetic markers between human and chicken isolates indicates that some Campylobacter infections in children may have additional sources other than contaminated chicken meat.

	INTRODUCTION

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Campylobacter jejuni and Campylobacter coli are the most frequent cause of diarrhoeal disease in humans throughout the world (Allos, 2001). However, the results of a 5-year study in Polish children with diarrhoea showed that the mean frequency of C. jejuni and C. coli infections did not exceed 9.1 %, and the majority of cases occurred at an age of 0–5 years (Rozynek & Dzierzanowska, 1994).

Searches for possible sources of Campylobacter infections, carried out in many countries, have revealed a very high level of poultry gut colonization by these micro-organisms. The contamination of poultry carcasses in abattoirs is considered a main risk factor for human infection (Bang et al., 2001; Petersen et al., 2001). A recent study in Poland showed that 88.5 % of chicken carcasses are contaminated with Campylobacter species (Daczkowska-Kozon, 2002). However, according to data from the National Food and Nutrition Institute in Poland, chicken meat is consumed by only 18 % of children under 5 years (Szponar, personal communication), which may suggest an alternative source of Campylobacter infection in this group of patients. Origins of human Campylobacter infections other than poultry meat have been postulated recently by German authors who found a difference in antimicrobial susceptibility patterns between human and poultry strains (Luber et al., 2003).

Some properties of Campylobacter species, such as the ability to adhere and colonize, as well as invasiveness for enterocytes and synthesis of one or more toxins, appear essential in the process of infection (Konkel et al., 2001). Research on the molecular mechanisms of Campylobacter infection pathogenesis has been stimulated by the publication of the C. jejuni NCTC11168 genome sequence (Parkhill et al., 2000). Several potential markers of bacterial virulence have been identified (Bang et al., 2003; Datta et al., 2003; Fouts et al., 2005). One of these is the cadF gene, which encodes a 37 kDa protein belonging to the group of outer-membrane proteins (OMPs) that functions as an adhesin responsible for certain steps of invasion (Konkel et al., 1999). Another interesting region, designated an invasion-associated marker (iam), has been identified in some C. jejuni and C. coli strains (Carvalho et al., 2001). So far, no product attributed to this region has been found. The virulence of Campylobacter species is also associated with the production of a cytotoxin that consists of three subunits of molecular mass 30, 29 and 21 kDa, which are encoded by the cdtA, cdtB and cdtC genes, respectively. All three subunits are necessary for the cytotoxin activity known to be lethal for host enterocytes (Purdy et al., 2000; Lara-Tejero & Galan, 2001).

Since the distribution of potential virulence markers among Polish Campylobacter isolates has never been analysed, our study was aimed at comparing the prevalence of the iam, cadF and cdtABC genes in C. jejuni and C. coli isolates from children and from chicken carcasses.

	METHODS

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Bacterial strains.

Campylobacter isolates were obtained from anal swabs from children with diarrhoea presenting at the Children's Memorial Health Institute, Warsaw, Poland, during 2002–2004, and swabs from chicken carcasses obtained in supermarkets and slaughterhouses in various regions of Poland in the same period. Isolation was performed by direct spreading of the swab-collected specimens on solid selective media (Preston agar and Blazer-Wang agar). Inoculated media were incubated for 48 h at 42 °C in microaerobic conditions (Campy-Pak BBL envelopes). Campylobacter identification and discrimination among thermophilic Campylobacter species were based on colony morphology, Gram's staining and biochemical reactions. Species identification was confirmed by PCR with specific primers (listed in Table 1), as described elsewhere (Linton et al., 1997; On & Jordan, 2003). Reference strains C. jejuni ATCC33560 and C. coli ATCC33559 were used as controls.

Detection of potential virulence markers by PCR.

The presence of the cadF, cdtA, cdtB and cdtC genes and the cdt cluster in all Campylobacter isolates was determined with the primers listed in Table 2, as described elsewhere (Konkel et al., 1999; Eyigor et al., 1999; Bang et al., 2003). Three sets of primers were used for detection of the iam marker: a pair published previously (Carvalho et al., 2001), and two pairs designed in this study (Table 2), selected from the iam locus sequence of C. jejuni strain (GenBank accession no. AF023133). The PCR conditions for all iam-PCRs were the same as in the original study by Carvalho et al. (2001).

	RESULTS AND DISCUSSION

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Among the isolates, 57 were identified as C. coli and 115 as C. jejuni by both classical methods and PCR. Thus, only these 172 well-defined isolates were subjected to further analysis. Eighty isolates were from children (62 C. jejuni and 18 C. coli), and 92 came from chicken carcasses (53 C. jejuni and 39 C. coli).

Analysis of the prevalence of the cadF gene revealed that nearly all of the Campylobacter isolates carried this marker, regardless of their strain origin (Table 3). Similar observations indicating that the cadF gene is present in Campylobacter species isolated from human specimens as well as from chicken carcasses and droppings have recently been reported by other authors (Konkel et al., 1999; Dorrell et al., 2001; Bang et al., 2003; Datta et al., 2003). The product of this gene is an adhesin and fibronectin-binding protein involved in the process of invasion, influencing microfilament organization in host cells (Monteville et al., 2003). It was shown that cadF-negative strains were not able to colonize chicken guts (Ziprin et al., 1999). Thus, the cadF gene, which appears to be essential for chicken gut colonization, may presumably have a similar role in the pathogenesis of human infection.

Another marker potentially associated with the severity of Campylobacter-induced enteritis, called an invasion-associated marker (iam), has been described by Carvalho et al. (2001). Because the iam sequence was detected in 85 % of Campylobacter isolates from children with diarrhoea, as compared to only 20 % of isolates from asymptomatic patients, they suggested that this locus could be used as a marker of invasive Campylobacter strains. In this study, the iam sequence was found in isolates from only 20 % of patients with diarrhoea, and its prevalence varied depending not only on the origin but also on the species of the Campylobacter isolate (Table 3). More than 50 % of C. jejuni and 100 % of C. coli isolates from chicken carcasses were iam-positive. In contrast, only one C. jejuni strain (1.6 %) and 83.3 % of the C. coli isolates from children possessed this sequence. Since three different primer sets were used for iam amplification in all isolates tested, and all except two gave the same results, the lack of the detectable iam locus in some isolates seems to reflect a true absence of this sequence rather than inadequate PCR conditions. The low prevalence (20 %) of the iam sequence in Campylobacter isolates from children with diarrhoea, which was identical to that observed by Carvalho et al. (2001) in asymptomatic patients, indicates that it is not a universal marker of the severity of Campylobacter infection.

Another marker analysed in our study was the cytotoxin-encoding gene cluster. The cytopathic effect of the cytotoxin is associated with damage to nuclear DNA, resulting in the inhibition of the cell cycle in G2 or M phase (Whitehouse et al., 1998). It has also been shown that cytolethal distending toxin (CDT) is involved in inducing the release of pro-inflammatory cytokine IL-8 from intestinal epithelial cells (Hickey et al., 2000). The latter observation suggests that cytolethal distending toxin may also play a part in the development of the inflammatory process in humans. Recent studies analysing the distribution of separate cdtA, cdtB and cdtC genes or the cdt cluster in C. jejuni and C. coli indicate that their prevalence in isolates from poultry and other sources exceeds 90 % (Bang et al., 2001; Datta et al., 2003). Microarray testing of the cdt cluster showed that these genes were present in all C. jejuni isolates from human samples (Dorrell et al., 2001; Volokhov et al., 2003). A similar frequency was also observed in our study of chicken and human C. jejuni isolates (Table 4). However, in C. coli isolates from children with diarrhoea, detection rates of these genes were much lower (Table 4). Because cdt genes have recently been shown to be conserved among different Campylobacter strains (Fouts et al., 2005), and we used separate primer sets for detection of individual cdtA, cdtB and cdtC genes and the cdt cluster, which increased the reliability of PCR results, the low detectability of cdt genes in human C. coli isolates seems to result from a true lack of these markers.

The differences in distribution of cdt genes and the iam sequence between human and chicken isolates observed in our study, together with the low consumption of poultry by young children in Poland, suggests that Campylobacter infections in these patients may have additional sources other than contaminated chicken meat.

	ACKNOWLEDGEMENTS

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
This study was supported in part by research grant KBN PB 371/P05/2002/23.

	REFERENCES

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES