Serotyping and Identification of Campylobacter Jejuni and Campylobacter Coli Strains of Human and Animal Origin Using the Pcr Method

Steinhauserová I . , K. Foj t íková: Serotyping and Identification of Campylobacter jejuni and Campylobacter coli Strains of Human and Animal Origin Using the PCR Method. Acta Vet. Brno 1999, 68: 149–154. A total number of 178 strains of Campylobacter spp. from human patients suffering from diarrhea and from the intestinal content of slaughtered pigs and poultry was isolated during years 1996 to 1997. Campylobacter strains were isolated and identified by standard tests and the PCR method using fla A and fla B, as well. Isolates originating from human patients were identified as Campylobacter jejuni (98% = 108 strains) and C. coli (2% = 2 strains). Campylobacter spp. (20) strains obtained from slaughtered pigs were identified using standard tests in 45% (9 strains) and 40% (8 strains) as C. coli and C. jejuni, respectively. Strains of Campylobacter spp. (48) isolated from slaughtered poultry were identified as C. jejuni (89% = 42 strains). Standard tests failed to identify 9 strains; 6 strains were identified as Campylobacter jejuni and C. coli using the PCR method. Serotyping using 34 antisera of the Penner’s serotyping scheme was performed in 130 strains of Campylobacter jejuni isolated from poultry and human patients. This method was suitable for identification of almost 80% of C. jejuni strains. We found 10 different serotypes of human C. jejuni strains. There were serotypes (HS) 4, 1 and 9 (32%, 23% and 15%, respectively) predominating in the human population; they were followed by serotypes (HS) 10, 2 and 23 (8%, 5% and 3%, respectively). Animal strains included 7 (HS) serotypes. Like in the human strains, the serotype 4, found in 37% of examined isolates, was predominating. Serotypes 23 and 1 (17% and 15%, respectively) were the next most frequent ones. Comparing the obtained serotypes of human and animal strains we see that there are only 4 identical ones (i.e., 4, 1, 10, 23), which are, however, most frequently occurring in both groups of strains. The PCR methods and serotyping may facilitate and make more accurate the results of standard cultivation and in epidemiologically serious cases help in explaining the route of transmission. Campylobacter jejuni/coli, PCR, HS antigen, food-borne diseases Diseases caused by Campylobacter spp. belong to the most prevalent infections in many countries. From all the known Campylobacter species, diseases are caused most frequently by Campylobacter jejuni and to a lesser extent by Campylobacter coli. Diseases caused by other species, such as C. upsaliensis and C. hyointestinalis, have been, however, described (Saleha et al. 1998). Campylobacter spp. are very common in domestic and wild animals from which they are rather often and easily isolated. On the other hand, isolations of Campylobacter spp. from risk foodstuffs succeed with difficulty. There may be many causes for this. One of them is the low number of Campylobacter spp. cells which are not detected by standard methods. Another reason is the high sensitivity of Campylobacter spp. to the external environmental factors (such as pH, humidity, NaCl, presence of oxygen and others) leading to the damage of cells (Steinhauserová 1998). The existence of viable but nonculturable cells not only of Campylobacter spp. but also of other microorganisms has been proven recently. These forms of microorganisms cannot be found using standard methods but it is probable that they may cause the disease (McDougald et al. 1998). ACTA VET. BRNO 1999, 68: 149–154 Address for correspondence: Doc. MVDr. Iva Steinhauserová, CSc. University of Veterinary and Pharmaceuticcal Sciences Dept. of Meat Hygiene and Technology Palackého 1-3, 612 42 Brno, Czech Republic Phone: +420 5 4156 2660 Fax: + 420 5 4132 1230 E-mail: [email protected] http://www.vfu.cz/acta-vet/actavet.htm Transmission of Campylobacter jejuni to humans may take the route of direct contact with animals or a sick person but most frequently indirectly by consuming contaminated food and water (Steinhauserová 1998). With the increasing number of campylobacter enteritis there are growing efforts to explain routes of Campylobacter jejuni/coli transmission. Means and routes of Campylobacter spp. transmission have not been fully clarified yet. Serotyping is one of possibilities to identify and evaluate the epidemiological relation of strains isolated from different sources and patients. Numerous methods using different surface structures such as polysaccharides, lipopolysaccharides or proteins have been described in the past. Two systems began to predominate in the course of time. One of them is based on the use of thermostable polysaccharides (the Penner’s serotyping scheme) and the other on the identification of thermolabile flagellar proteins (the Lior’s scheme). The use of serotyping methods is limited, and, therefore, methods of genotyping have been employed more and more recently. It is possible to combine phenotypic methods and genotypic identification using molecular methods which may reliably identify strains isolated from animals and human patients, their characteristics and probable routes of transmission (Aarts et al. 1995; Madden et al. 1998). There is no laboratory engaged in routine serotyping of Campylobacter jejuni and Campylobacter coli in the Czech Republic, so the frequency of occurrence of individual serotypes is not known. It is the aim of our work to perform serotyping and identification by the PCR method using the collection of isolated strains of human and animal origin. Materials and Methods Isolat ion of Campylobacter spp. s t ra ins Campylobacter strains from human patients and animals were collected during the years 1996 to 1997. Human strains were obtained owing to the cooperation with the laboratory of microbiology Bioplus and originated from rectal swabs from the south Moravian region patients suffering from gastrointestinal diseases. Animal strains were obtained by sampling the intestinal content of the large intestine and caecum of pigs and poultry immediately after slaughter. Strains from poultry were isolated from slaughtered animals from 5 different Moravian herds; the slaughtered pigs originated from the whole south-Moravian region. The isolation and identification of Campylobacter jejuni/coli was performed using the modified âSN ISO Norm 10272 “The microbiology of food and foodstuffs the horizontal method of identification of thermotolerant species of the genus Campylobacter.“ After taking of about 1 g of the intestinal content, the sample was directly plated on a solid selective medium (Campylobacter Agar Base (Karmali) Oxoid) and simultaneously enrichment in 10 ml of the Preston broth. The solid culture media were incubated at 37 °C for 48 h. The propagation lasted for 16 to 18 h under microaerophiolus conditions at 42 °C. For the purpose of achieving microaerophilous conditions we used the Campylobacter Gas Generating Kit (Oxoid). Propagated samples were plated on the Blood Free Campylobacter Selective Agar Base (Oxoid) and Campylobacter Agar Base (Karmali) Oxoid and cultivated at 37 or 42 °C under microaerophilous conditions. Suspect colonies were re-plated on the Blood Agar Base No. 2 (Oxoid) with the aim to obtain pure Campylobacter cultures. Standard biochemical examinations were performed according to the recommendation of the âSN ISO Norm (i.e., catalase, oxidase, hippurate hydrolysis, hydrogen sulphide production in TSI, growth at 37 °C (aerobically, anaerobically and microaerobically), 30 °C aerobically, 43 °C microaerobically; susceptibility to discs containing cephalothin (30 μg) and nalidixic acid (30 μg). Isolated strains were kept at -60 °C in the Meat Cooked Broth (Oxoid). Serotyping Selected strains were serotyped using the Penner’s serotyping scheme described by Penner and Hennessy (1980). For the purpose of typing the strains using the thermostable (HS) lipopolysaccharide antigens, the method of passive hemagglutination by commercial sera (PHL Manchester) was employed. Serotyping was performed using 34 antisera: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 23, 24, 26, 27, 28, 30, 34, 37, 39, 46, 48, 49, 50 and 51. Reference strains CCM 6214, serotype HS 23, and CCM 6212, serotype HS9, were used in order to check the results of agglutination. PCR method Results of standard cultivation and typing were verified using the PCR method. This method was also employed when the results of standard typing were ambiguous. A total number of 50 strains of various serotypes was examined by the PCR method. 150