Serotypes, virulence genes, and PFGE profiles of Escherichia coli isolated from pigs with postweaning diarrhoea in Slovakia

Background Postweaning diarrhoea (PWD) in pigs is usually the main infectious problem of large-scale farms and is responsible for significant losses worldwide. The disease is caused mainly by enterotoxigenic E. coli (ETEC) and Shiga-toxin producing E. coli (STEC). In this study a total of 101 E. coli isolated from pigs with PWD in Slovakia were characterized using phenotypic and genotypic methods. Results These 101 isolates belonged to 40 O:H serotypes. However, 57% of the isolates belonged to only six serotypes (O9:H51, O147:H-, O149:H10, O163:H-, ONT:H-, and ONT:H4), including two new serotypes (O163:H- and ONT:H4) not previously found among porcine ETEC and STEC isolated in other countries. Genes for EAST1, STb, STa, LT and Stx2e toxins were identified in 64%, 46%, 26%, 20%, and 5% of isolates, respectively. PCR showed that 35% of isolates carried genes for F18 colonization factor, and further analyzed by restriction endonuclease revealed that all of them were F18ac. Genes for F4 (K88), F6 (P987), F17, F5 (K99), F41, and intimin (eae gene) adhesins were detected in 19 %, 5%, 3%, 0.9%, 0.9%, and 0.9% of the isolates, respectively. The study of genetic diversity, carried out by PFGE of 46 representative ETEC and STEC isolates, revealed 36 distinct restriction profiles clustered in eight groups. Isolates of the same serotype were placed together in the dendrogram, but high degree of polymorphism among certain serotypes was detected. Conclusion Seropathotype O149:H10 LT/STb/EAST1/F4 (14 isolates) was the most commonly detected followed by O163:H- EAST1/F18 (six isolates), and ONT:H4 STa/STb/Stx2e/F18 (five isolates). Interestingly, this study shows that two new serotypes (O163:H- and ONT:H4) have emerged as pig pathogens in Slovakia. Furthermore, our results show that there is a high genetic variation mainly among ETEC of O149:H10 serotype.

Porcine attaching and effacing E. coli (AEEC) induce intestinal lesions similar to those produced by enteropathogenic E. coli (EPEC) in humans. These E. coli carry eae gene encoding a 94 kDa outer membrane protein (intimin) which is responsible for intimate attachment to epithelial cells. However, the pathogenic significance of porcine eaepositive isolates in weaned pigs is unclear [16,17]. A new category of the diarrhoeagenic E. coli family, named enteroaggregative E. coli (EAEC), has been recognized. EAEC elaborate a low-molecular-weight, partially heat-stable, plasmid-encoded enterotoxin named enteroaggregative E. coli heat-stable enterotoxin 1 (EAST1). The gene (astA) encoding the production of EAST1 has been detected in several groups of diarrhoeagenic E. coli (EAEC, EPEC, ETEC, and STEC) isolated from humans and from pigs. The pathogenic significance of EAST1 in diarrhoea in pigs is not known [7,8,[18][19][20].
Although, PWD is frequently observed in Slovakia, there is a lack of information about the prevalence of serogroups, serotypes, and virulence factors of porcine pathogenic E. coli. Thus, the aim of this study was to determine the distribution of serogroups, serotypes, and virulence genes, and to study the genetic relatedness among E. coli isolated from pigs with PWD. This is the first study in Slo-vak Republic of a large collection of pathogenic E. coli isolated from PWD.

Toxin genes
Amplification of the toxin genes by PCR showed that 77% of isolates possessed genes for production of five types of toxins: LT, STa, STb, Stx2e, and EAST1. The gene encoding for EAST1 toxin (65 isolates) was the most prevalent, followed by the STb (47 isolates), STa (27 isolates), and LT (20 isolates) genes. The Stx2e gene was detected in five isolates, which also carried genes for STa and STb (Table  1). Genes encoding Stx1, Stx1c, Stx1d, Stx2, Stx2c, Stx2d, and Stx2g toxins were not detected in any of the 101 porcine isolates studied.

Adhesin genes
The PCR analysis of all 101 isolates of E. coli showed that 61 (60%) carried at least one fimbrial or intimin gene. The most prevalent fimbrial adhesin was F18, detected in 35 isolates. Analysis by restriction endonucleases of PCR F18-positive products revealed that all 35 F18-positive isolates showed the F18ac variant. Three of these 35 F18 isolates were also positive for either F4 or F17 genes. The gene encoding F4 was identified in 19 isolates. F6, F5, F41, and F17 genes were detected in five, one, one, and three isolates, respectively. The eae gene (intimin type β1) was detected in only one isolate (0.9%) of serotype O45:H- (Table 1).

Macrorestriction fragment analysis by pulsed-field gel electrophoresis (PFGE)
A representative group of 46 isolates (45 ETEC and/or STEC) were selected to be analyzed by PFGE: O149:H10 (15 isolates), O163:H-(11 isolates), O147:H-(four isolates), ONT:H-(eight isolates), ONT:H4 (five isolates), O141 (two isolates), and O60:H-(one isolate). The study revealed 36 distinct restriction profiles, considering as significative a difference of a single band (Fig. 1). In the dendrogram produced by the UPGMA algorithm, the isolates were clustered in eight groups (I to VIII; 1 to 13 isolates per group) of 70% similarity according to the Dice similarity index, with 35 isolates clustering in nine subgroups of closely related (similarity > 85%) PFGE profiles. E. coli isolates of the same serotype were placed together in the dendrogram, but high degree of polymorphism among certain serotypes was detected. Thus, the 15 O149:H10 isolates were clustered in three groups (I-III, 70% similarity) with only three small subgroups of closely related profiles (similarity > 85%; five, two and two isolates, each). Genetic distance among O149:H10 isolates was as considerable as similarity < 66%. Group V clustered 13 isolates (all 11 O163:H-and two ONT:H-isolates; similarity > 71%) with three subgroups (two of them clustering five isolates each, similarity > 85%). Curiously, the highest homogeneity (similarity > 92%) was observed among a group of 10 isolates (group VI) belonging to serotypes O147:H-(four isolates) and ONT:H-(six isolates). E. coli isolates of serotypes O141:H-and O141:H34 were clustered in group VII (similarity > 97%). And all five isolates of serotype ONT:H4 clustered in group VIII showing a similarity > 81%.

Discussion
It is widely accepted that specific serotypes and pathotypes of ETEC and STEC are responsible for the major part      LT/STb/EAST1/F4 (14 isolates) was the most prevalent in the present study, and the reason for its predominance is not known. A possible explanation could be that the virulence factor association of these isolates makes them especially adapted to propagation in swine populations and their enviroment [8].
The main discovery of this study was the identification of two new serotypes (O163:H-and ONT:H4) not previously detected among porcine ETEC and STEC isolated in other geographical zones. . In this study, the F17 gene was detected in three E. coli, two of which were also positive for F18, enterotoxins, and EAST1 toxin. Similarly, Osek [6] found that only four (1%) of 372 isolates from PWD in Poland were positive for F17.
The role of EAST1 toxin in swine colibacillosis has not been demonstrated, however, the gene encoding EAST1 toxin is commonly found in isolates associated with PWD [7,8,19,20]. Our results confirm these observations as we found that 65 of 101 isolates harbored astA gene and all F4 isolates were astA positive. The high frequency of the astA gene suggests the necessity of further studies to investigate the significance of this toxin in porcine PWD.
Genotyping methods such as multilocus enzyme electrophoresis (MLEE) and pulsed-field gel electrophoresis (PFGE) have been used for differentiation and epidemiological characterization of E. coli isolated from pigs with PWD and edema disease. PFGE is a powerfull tool to reveal inter-and intra-serotype specific genetic differences among porcine pathogenic E. coli [12,28,29]. However, there are few studies reporting genetic relatedness of E. coli isolated from diarrhoea in pigs. Osek [29] used the PFGE technique to analyze 82 E. coli from pigs with PWD, isolated from geographically separated farms in the western part of Poland. The 82 isolates belonging to four serogroups (O138, O139, O141, and O149) showed 13 different PFGE profiles and although a high degree of polymorphism among different serotypes was observed, isolates belonging to the same serological group showed a close relationship. Thus, the 25 isolates of serotype O149:K91 generated only two PFGE types. In our study, a representative group of 46 ETEC and STEC isolates revealed 36 distinct restriction profiles. Although isolates of the same serotype were placed together in the dendrogram, high degree of polymorphism among certain serotypes was detected.

Conclusion
Our results indicate that in Slovakia, as described in other countries, pathogenic E. coli isolates from PWD belong to a restricted number of serotypes and pathotypes. The ETEC serotype O149:H10 seems to be predominant, but also two new serotypes (O163:H-and ONT:H4) not previously described in porcine ETEC and STEC isolated in other countries are common. The F18ac and F4 fimbriae were the most prevalent colonization factors detected in postweaning E. coli in Slovakia. Macrorestriction analysis showed that, although isolates of the same serotype and virulence markers mainly share the same PFGE group, there is a high genetic variation, especially among ETEC of O149:H10 serotype.

Haemolysin activity
The isolates were inoculated on blood agar base supplemented with 5% sheep blood (Oxoid, UK) and incubated at 37°C for 18h. β-Haemolysis was evident as a zone of lysis surrounding the bacterial growth.

Detection of virulence genes by PCR
The polymerase chain reaction (PCR) for detection of toxins (LT, STa, STb, Stx1, Stx2, Stx2e, and EAST1) and adhesins (F4, F5, F6, F17, F18, F41, and eae) was carried out as described by Vu-Khac et al. [31] and Blanco et al. [32]. Base sequences and predicted sizes of the amplified products for the specific oligonucleotide primers used in this study are shown in Table 2 [31][32][33][34][35]. Typing of eae (intimin) gene detected in one positive isolate identified in this study was carried out by PCR as described elsewhere [32].

Digestion of PCR products with restriction endonuclease
After amplification, the PCR products of F18-positive isolates were digested with restriction enzyme NgoMIV (formerly NgoMI) to distinguish genes encoding F18ab and F18ac [33]. The master mix was prepared with a total of 15µ l volume containing 5µ l of PCR products (after purifying with Wizard PCR Preps [Promega]); 10µ l 1x MULTI-CORE™ buffer (Promega); and 1 unit of enzyme NgoMIV. After incubation at 37°C for one hour, the DNA digestion was analyzed by electrophoresis in 2 % agarose gel.

Pulsed-field gel electrophoresis
PFGE was performed in a CHEF MAPPER system (Bio-Rad, Hemel Hempstead, United Kingdom) at 14°C in 0.5XTBE by the Enternet proposed standard-protocol for PFGE [36]. Cleavage of the agarose-embedded DNA was achieved with 0.2-0.8 U/µ l Xbal (Roche) according to instructions of the manufacturer. Run times and pulse times were 2.20 to 54.0s for 22 h with linear ramping. PFGE was used to establish clonal relatedness and diversity among a representative group of 46 isolates. To perform the comparison of the PFGE pulsotypes, TIFF files were analyzed with BioNumerics software (Applied Maths, Sint-Martns-Latem, Belgium). Cluster analysis of the Dice similarity indices based on the unweighted pair group method using aritmetic averages (UPGMA) was done to generate a dendrogram describing the relationship among EPEC pulsotypes. A difference of at least one restriction fragment in the profiles was considered the criterion for discriminating between clones.