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African Journal of Pig Farming ISSN 2375-0731 Vol. 3 (2), pp. 001-008, February, 2015. Available online at 
www.internationalscholarsjournals.org © International Scholars Journals 

 

Author(s) retain the copyright of this article. 

 

 

Full Length Research Paper 

 

Genetic diversity of Escherichia coli isolated from 

commercial swine farms revealed by enterobacterial 

repetitive intergenic consensus  
 

Li Wan1, Zhi-Biao Wang1, Qi-gui Yan1,2*, Xu Wang1, Yan Lei1, Lan Zuo1, Yu 
Cheng1, Yu-Peng Ren1 and Wan-zhu GUO1

  
1
College of Veterinary Medicine, Sichuan Agricultural University, Ya’an, 625014, P. R. China. 

2
Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Ya’an, 

625014, P. R. China. 
 

Accepted 09 November, 2014 
 

The objective of this study was to use enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) 
and repetitive extragenic palindrome PCR (REP-PCR) for the analysis of genetic diversity among 
Escherichia coli strains isolated from commercial swine farms in Sichuan province of China. Thirty four 
strains of E. coli were selected by selective medium and conventional biochemical test from fresh stool 
samples of swines in five farms in Sichuan province. The isolates were identified by 160 kinds of E. coli 
O serums. The results show that 30 strains were determined among 34 E. coli isolates, 12 kinds of O 
serogroups were obtained on the basis of the agglutination test. The predominant types are O23, O 113 
and O120, representing 35.4%. Furthermore, the genotypes and phylogenetic relationship of all isolates 
were analysed by Enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) and repetitive 
extragenic palindrome PCR (REP-PCR), 34 E. coli isolates were clustered to 19 ERIC-PCR genotypes 
and 13 REP-PCR genotypes. The isolates from the same farm or sharing the same serotyping showed 
different genotype. And the isolates which could not be serotyped were genotyped by ERIC-PCR and 
REP-PCR. The analysis of genetic type and original source revealed that isolates from different farms 
had different genetic types. The subtypes of E. coli are also different within a single farm. Genetic 
variability with E. coli strains isolated from swine farms in China has been demonstrated. The presence 
of ERIC-PCR and REP-PCR sequences in the genome of E. coli was confirmed. ERIC-PCR and REP-PCR 
techniques are more rapid methods for molecular typing of E. coli strain. They are also useful methods 
for diversity survey of E. coli and the two methods analyzes genetic diversity of E. coli isolated in 
Sichuan of China. 

 
Key words: Escherichia coli, serotype, enterobacterial repetitive intergenic consensus PCR (ERIC-PCR), 

repetitive extragenic palindrome PCR (REP-PCR). 
 
 
INTRODUCTION 

 
Escherichia coli is one of the strain widely distributed in 

nature and intestinal tract of animal. Genetic diversity 

exists widely among strains, so that using traditional  
 
 
 
*Corresponding author. E-mail: yanqigui@126.com. 

 
 
 
 
 
bacterial taxonomy proves problematic when 
differentiating between strains that share a close genetic 
relationship. The traditional method for subspecies typing 
among the E. coli has been serotyping. The serotype is 

based on the antigenic properties of the O-antigen 
(surface polysaccharide) and H antigen(s) (flagellar). 
Typing the O-antigen itself denotes the serogroup, and 



 
 
 

 
Table 1. The results of E. coli isolation.  

 
 Swine house Serial number Original King of sample Number 

 A A1 Farrow Stool 1 

 A A2 Stag Stool 1 

 A A3, A4, A5, A6 Weaner Stool 4 

 B B1, B3, B4 Pregnancy sow Stool 3 

 B B2, B5 Farrow Stool 2 

 B B6 Stag Stool 1 

 C C1, C2 Pregnancy sow Stool 2 

 C C3, C6, C7 Weaner Stool 3 

 C C4, C5 Farrow Stool 2 

 D D1, D6 Pregnancy sow Stool 2 

 D D2, D4, D7 Weaner Stool 3 

 D D3, D5 Stag Stool 2 

 E E1, E2, E6, E7, E8 Pregnancy sow Stool 5 

 E E3, E4, E5 Weaner Stool 3 

 Total    34 
 

 

the serotype is obtained by characterizing the flagella, 
which is often biphasic (Popoff and Le Minor, 2001). 
Although, all the methods are reliable, they present some 
disadvantages either in the time needed to perform the 
test or in the cost of sera and reagents. Moreover, 
readings are not always easy to interpret. With the 
development of molecular biology, some classification 
methods based PCR technique had been recognized as 
having great ability to differentiate separate strains by 
producing DNA fingerprints that are specific for individual 
strains. These methods were proved to be fast, sensitive 
and reliable for differentiation of microorganism. Several 
of them had been used widely for studying genetic 
relationship of different bacteria such as the random 
amplified polymorphic DNA PCR (RAPD-PCR) (Welsh 
and McClelland, 1990), enterobacterial repetitive 
intergenic consensus sequence PCR (ERIC- PCR) (Wei 
et al., 2004; Wenz et al., 2006; Chen et al., 2010) and 
repetitive extragenic palindromic PCR (REP-PCR) 
(Trombert et al., 2007; Bonacorsi et al., 2009), other 
analysis methods such as pulsed-field gel electrophoresis 
(PFGE) (McLellan et al., 2003; Casarez et al., 2007), 
amplified fragment length polymorphism (AFLP) 
(Engstrom et al., 2003) and ribotyping also had been 
applied widely. REP consensus sequence consist of 38 
bp inverted repeat sequence, ERIC sequences are 126 
bp long and appear to be restricted to transcribed regions 
of the genome (Versalovic et al., 1991). REP-PCR and 
ERIC-PCR were used for studying bacterial source 
tracking in the field and analysis of genetic diversity. 
Identification of the spread of airborne Escherichia coli 
using ERIC- PCR and REP-PCR (Dombek et al., 2000; 
Duan et al., 2009; Yuan et al., 2010) has been reported 
using ERIC-PCR fingerprinting to assess E. coli strains 
diversity isolated from natural water (Casarez et al., 
2007). 

 

 

Sichuan is one of the biggest pig raising provinces in 
China. The infectious diseases caused by E. coli are very 
serious. Analysis of E. coli strains genetic diversity is 
important for epidemiology. The objective of this study 
was to use REP- PCR and ERIC-PCR for the analysis of 
genetic diversity among E. coli strains isolated from 

commercial swine farms in Sichuan province of China. 
 

 
MATERIALS AND METHODS 
 
Five commercial swine farms in Sichuan were chosen, fresh stool 
samples were aseptically collected from sick pig that have being alo 
laxata from the five farms, respectively, a total of 40 stool samples 
were collected, they are listed in Table 1. Samples were diluted and 
spread onto the surface of MacConkey agar plates which were 
incubated at 37°C for 18 to 20 h. Colonies with typical pink on 
MacConkey agar plates were picked and streaked onto eosin 
methylene blue (EMB) agar plates. After overnight incubation at 
37°C, one or two colonies with a metallic sheen on EMB agar were 
selected and cultured in Luria-Bertani broth. All cultures were stored 
at -70°C in Luria-Berintani broth with 30% glycerol. Then, the 
strains species identifications were made using conventional 
biochemical tests (not shown in the paper). A total of 34 E. coli 
strains were chosen due to their varying sources. 

 

Serotyping procedures 
 
All the 160 standard E. coli O factor serums (O17, O23, O24 O198, etc) 
were kindly provided by China Institute of Veterinary Drug Control, 
and the serotyping procedures used including the production of 

heat-stable antigen extracts by heating at 121°C for 2 h have been 

described in detail elsewhere (Biao et al., 2009). 

 

DNA preparation 
 
Genome DNA of all E. coli was isolated by the CTAB/NaCI mini-

prep protocol (Barney et al., 2001) used for all PCR- based 

methods. All E. coli isolates strains were grown in 5 ml of Luria- 



  
 
 

 
Table 2. The serological result of E. coli O antigen by slide agglutination.  

 
Serotype Strain Numbe (%) 

O198 A1, A2, C1 3 (8.8%) 

O150 A3, B2, E4 3 (8.8%) 

O46 A5, D6, E7 3 (8.8%) 

O113 A6, C3, D3, E8 4 (11.8%) 

O26 B4 1 (2.9%) 

O120 B5, D2, E1, E2 4 (11.8%) 

O23 B6, C2, D4, E3 4 (11.8%) 

O17 C4, C5 2 (5.9%) 

O68 C6, C7 2 (5.9%) 

O24 D5 1 (2.9%) 

O78 D7 1 (2.9%) 

O129 E5, E6 2 (5.9%) 
 

 
Bertani (LB) broth (Oxoid) with moderate shaking for 18 h at 37°C. 
1.5 ml LB broth was removed and centrifuged at 10,000 g for 2 min. 
The cell pellet was resuspended in 567 l TE and mixed with 30 10% 
SDS and 3 l 20 mg/ml protein K; and the mixture reacted for 1 h at 
37°C; followed by 100 l ABTC and 100 l 5mol/L NaCl were added in 
the mixture, 65°C for 10 min, and DNA templates were extracted by 
phenol/chloroform method, and DNA preparation were stored at -
20°C until used by PCR amplifications. Three microlitres of DNA 
preparation was used as the source of DNA template in the PCR 
reaction. 

 

ERIC-PCR 
 
The primers 5’-ATG TAA GCT CCT GGG GAT TCA C-3’ (ERIC1); 
5’-AAG TAA GTG ACT GGG GTG AGC G-3’ (ERIC2) (Versalovic el 
al., 1991) were synthesized by Invitrogen. The PCR reaction (25 l) 
contained 10× buffer 2.5 l, dNTPs (2.5mmol/L) 2.5 l, TaqDNA 
polymerase (5 U/ l) 0.5 l, MgCl2 (25 mmol/L) 1.5 l, primers (10 
mol/L) each 1 l, template DNA 1 | and addition ddH2O to 25 l. 
Amplification was performed in a Thermal Cycler (Bio-BRI) with 
different temperatures: denatured for 7 min at 94°C, followed by 40 
cycles of 1 min at 94°C, 1 min at 48°C and 5 min at 72°C; and then 
extension at 72°C for 7 min.  

The reaction products were stored at 4°C until they were 
electrophoresed on a 2% (w/v) agarose gel containing 1×TAE and 

0.5 g ml
-1

 ethidium bromide together with the DL2000 DNA markers 
(TaKaRa) for 2 to 3 h. 

 

REP-PCR 
 
The primers REP1R (5 -III ICG ICG ICA TCI GGC-3 ) and REP2I (5 
-ICG ICT TAT CIG GCC TAC-3 ) (Versalovic et al., 1991) were 
synthesized by Invitrogen. The PCR reaction (25 l) contained 10× 
buffer 2.5 l, dNTPs (2.5mmol/L) 2.5 l, TaqDNA polymerase (5 U/ l) 
0.5 l, MgCl2 (25 mmol/L) 1.5 l, primers (10 mol/L) each 1 l, template 
DNA 1 l and addition of ddH2O (25 l) . The amplification was carried 
out by incubation of the mixture for 5 min at 95°C for pre-
denaturation, followed by 35 cycles of denaturation at 94°C for 1 
min, annealing at 45°C for 1 min and extension at 65°C for 8 min. A 
final extension was performed at 65°C for 16 min.  

The reaction products were stored at 4°C until they were 
electrophoresed on a 1.5% (w/v) agarose gel containing 1×TAE and 

0.5 gml
-1

 ethidium bromide together with the DNA marker III 
(TaKaRa) for 2 to 3 h. 

 

 
ERIC-PCR and REP-PCR fingerprints analyses 
 
ERIC-PCR and REP-PCR fingerprints of amplified DNA fragments 
obtained from the agarose gel electrophoreses were recorded. The 
observed bands in the gels were evaluated based on the presence 
(coded 1) or absence (coded 0) of polymorphic fragments for the 
ERIC and REP-PCR primers. Cluster analysis was performed with 
NTSYS- pc (Version 2.10), a numerical taxonomy and multivariate 
analysis software package (Rohlf, 2000), based on Dice’s similarity 
coefficient (SD) with a 1% position tolerance and the unweighted 
pair group method using arithmetic averages (UPGMA). In addition, 
each isolate was considered as an operational taxonomic unit 
(OTU). In order to reduce the number of OTUs in the dendrogram, 
to facilitate interpretation, isolates of 70% similarity were treated as 
a single isolate. 
 
 
RESULTS 
 
Serotyping 
 
As noted earlier, 30 strains are determined among 34 E. 
coli isolates, 12 kinds of O serogroups were obtained on 
the basis of slide agglutination. The results are listed in  
Table 2. The predominant types are O23, O113 and O120, 
representing 35.4%, but 3 strains are nontypeable by the  
test. The nontypeable isolates may either exist due to the 
fact that some isolates do not likely contain a sufficient 
level of serovar-specific antigens or that there exist novel 
serovars that have not been identified yet. The remaining 
serovars were detected at low frequencies, ranging from 
2.9 to 8.8% (Table 3). 

As indicated in Table 3, 5 serovars were identified 
among 6 strains isolates from A farm, 4 serovars present 
in B farm, 7 serovars present in C farm, 6 serovars 
present in D farm, 8 serovars present in E farm, and O-
type 23 was reported to be the most prevalent serotype in  
B, C, D and E, followed by O113 (A, C, D and E). 

Comparing the type strains, many field isolates with  
mixed serotypes showed that there are many kinds of 

serogroups in the same pig farm and they have 

predominant types in most pig farms. Some predominant 



 
 
 

 
Table 3. The distribution of O serogroups of E. coli in different pig farms.  

 

 
Pig farm 

Number of E. coli Numberof identified Serotype 
 

 
isolate isolate (Number of isolate)  

  
 

 A 6 5 O198(2) O150(1) O46(1) O113(1) 
 

 B 6 4 O150(1) O26(1) O120(1) O23(1) 
 

 C 7 7 O198 (1) O23(1) O113(1) O17(2) O68(2) 
 

 D 7 6 O120(1) O113(1) O23(1) O24(1) O46(1) O78(1) 
 

 E 8 8 O120(2) O23(1) O150(1) O129(2) O46(1) O113(1) 
  

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Figure 1. ERIC-PCR fingerprints obtained for E. coli isolate from A to E farms with primers ERIC1 and ERIC2. Lane: M, DNA marker DL2000; lane: A1 to A6, E. coli strains isolated  
from A pig house of Sichuan; lane: B1 to B6, E. coli strains isolated from B pig house of Sichuan; lane: C1 to C7, E. coli strains isolated from C pig house of Sichuan; lane: D1 to 
D7, E. coli strains isolated from D pig house of Sichuan; lane: E1 to E8, E. coli strains isolated from E pig house of Sichuan. 

 

 

serogroups in different districts are identical, 

others are different. Serogroups of different pig 

farms in the same district are generally different. 

 

ERIC-PCR 
 
The ERIC-PCR fingerprints analysis showed that 
there were 6 to 19 bands with molecular weight 
ranging from 100 bp to more than 3 kb generated 
by ERIC primers (Figure 1), and a common 
intensive band of about 270 bp in most E. coli 

(Figure 1). The data matrices based on the DNA 

 
 

 

fragments and the dendrogram using the NTSYS-
pc software were constructed, all E. coli isolates 
obtained in this study were grouped into different 
clusters or branches based on the ERIC-PCR 
pattern similarity. 

According to the UPGMA dendrogram (Figure 

2), all generated DNA patterns are relatively 
complex. Each subjects exhibited a unique 
banding pattern with similarity ranging from 0.17 
to 0.93. These 34 isolates were grouped into 19 
different groups with the similarity coefficients 
0.78, designated as I-XIX. The cluster X includes 

 
 

 

5 strains isolated from D farm with similarity 
coefficients 0.83. This second level of clustering 
suggested that a remarkable polymorphism exists 
among all strains in these groups. The two 
samples collected at different farms showed much 
higher similarity, 0.89 between C6 and E5 
and0.81 between A1 and B5. 
 

 

REP-PCR 

 

REP-PCR fingerprinting profiles and cluster 



   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Figure 2. Dendrogram established by the biostatistical analysis program NTSYS-pc using the dice similarity coefficient and UPGMA on the basis of the ERIC-PCR profile of E.  
coli strains obtained with primers ERIC1 and ERIC2. 

 

 

analysis of 34 E. coli strains are listed in Figure 3. 

With primers REP1R and REP2I, REP-PCR 

fingerprinting profiles were obtained. Analysis of 

the strains with REP-PCR yielded one to fourteen 

 
 

 

bands depending on the strains. The size of these 

bands ranged from 200 bp to more than 4500 bp. 
REP-PCR fingerprinting profiles of E. coli strains 

were different from each other. According to 

 
 

 

UPGMA dendrogram, 34 E. coli strains were 

separated into thirteen types with 0.81 similarity 
named 1 to 13 (Figure 4); however, E. coli strains 

of every pig house were not in the same type. 



    
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 

Figure 3. REP- PCR fingerprints for 34 E. coli strains A to E farms with primers REP1R and REP2I. Lane: M, DNA marker III; A1 to A6, E. 
coli strains isolated from A pig house of Sichuan; B1 to B6, E. coli strains isolated from B pig house of Sichuan; C1 to C7, E. coli strains 
isolated from C pig house of Sichuan; D1 to D7, E. coli strains isolated from D pig house of Sichuan; E1 to E8, E. coli strains isolated from E 
pig house of Sichuan. 
 

 

Thirteen E. coli strains (A1, A4, A5, B1, B4, B6, C1, C3, 
C4, D7, E1, E4 and E5) sharing common band of about 
700 bp were clustered together and belonged to type 1; 
among them A4, D7, E5 and B1, E4, E5 shared 1.0 
similarity, however, they were not from simple pig house. 
Type 2 only had one strain E7. Four E. coli isolates D2, 
D4, D5 and D6 isolated from D pig house were clustered 
to type 3. Other E. coli isolates sharing high similarity 

were separated into same type such as B2 and B3, E3 
and E8, C5 and C6, A6 and C2.The remaining E. coli 

strains were unique. 
 

 

DISCUSSION 

 

REP-PCR and ERIC-PCR techniques that prove to be 
fast, sensitive and reliable for the differentiation of 
microorganism were applied for analysis of genetic 
diversity and epidemiology. REP-PCR and ERIC-PCR 
technique were established from highly conserved 
repetitive DNA sequence distributed among the gene of 
microorganism. Combined with PCR and gel 
electrophoresis, highly complex and specificity genome 
fingerprintings profile produced could discriminate spices 
and strains. REP-PCR and ERIC-PCR technique are 
widely used method for DNA typing analysis and has 
been shown to successfully differentiate strains of 
Bradyrhizobium japonicum (Judd et al., 1993), 
Lactobacillus johnsoni (Ventura and Zink, 2002) and 
Clostridium perfringens (XiaoLi et al., 2009). Many 
genotyping studies were done on clinical isolates of E. 
coli, as well as environmental and veterinary isolates 
identified via biochemical and/or culture based approach 
(Carvalho de Moura et al., 2001; Gordon and Cowling, 
2003; Higgins et al., 2007).  

In this study, two molecular methods, REP-PCR and 

ERIC-PCR, were applied and their power to discriminate 

 
 

 

34 E. coli strains was compared with serogrouping 
method. Difference in PCR fingerprinting profile existed 
among identical swine farms. It was indicated that the 
diversity of E. coli strains of swine farms in Sichuan 
province were high.  

Genetic diversity of E. coli isolated from different five 
swine farms demonstrated in dendrogram based on 
ERIC-PCR fingerprinting profiles, namely, E. coli strains 
from different sources showed certain difference at the 
level of gene and were separated into different sub-
genetypes. E. coli isolated from the same swine farms 
were clustered into several groups, and had low cross 
with other farms. According to analysis of the dendrogram 
of ERIC-PCR, it can be seen that farm A was mainly 
concentrated in I, II and XIV, farm B was mainly 
concentrated in III, IV and V, farm C was mainly 
concentrated in IX, XII and XVI, farm D was mainly 
concentrated in X, XV and XIII, farm D was mainly 
concentrated in VI, VII, VIII, X, XI, XVI, XVII and XIX.  

When compared with ERIC-PCR, REP-PCR has lower 
discrimination power. Although, genetic differences of E. 
coli of farms were demonstrated on dendrogram by REP-
PCR, the sub-genetypes of identical farm has higher 
cross with others. Especially, genetypes of E. coli from 
farm A were crossed with other farms, and it was difficult 
to analyze the major subgroup of E. coli of some farms. It 
was determined that the major groups of farm D were 3 
and 6, farm E were 2, 5 and 8.  

Each E. coli strain has only one O serovars, so it is 
usually chosen for serotype identification. The results 
show that E. coli O serovars diversity was presented in 
five swine house. The method of analysis of E. coli 
diversity by O serovars diversity is a practical method 
indeed. But the quantity of E. coli O serovars is much and 

it is difficult to collect all identified O serovars. 
Accordingly, O serovars of 4 strains were not identified 
because only 160 kinds of O serovars were provided. 



   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Figure 4. Dendrogram established by the biostatistical analysis program NTSYS-pc using the Dice similarity coefficient and UPGMA on the basis of the REP-PCR profile of E. coli 

strains obtained with primers REP1R and REP2I. 
 

 

Predicting  Salmonella  enteric  serotypes  by When  compared  with  both  sides,  O  antigen study were quite complex, which made it difficult 
repetitive   sequence-based   PCR   has   been serotypes  of  Salmonella  strains  studied  by for ERIC-PCR and REP-PCR to identify the O 

reported   recently  successfully  and   it   was Albufera were relatively single (Albufera et al., antigen serotypes of E. coli. For example, C6 and 

discovered  that  REP-PCR  and  PADF  analysis 2009; Wise et al., 2009). E5 are closer that E6 and E5 on the dendrogram, 

have good correlation with O antigen serotyping. However, O antigen serotypes of E. coli in this but E5 and E6 belonged to the same serogroup 



 
 
 

 

and C6 to another. It was consistent with previous study. 
Hence from this study, the results of PCR fingerprinting  

profiles with the primers REP and ERIC revealed that 
genetic variability of E. coli strains isolated from the five 

swine farms was obvious. It was indicated that the 
diversity of E. coli strains of swine farms in Sichuan 

province were high. 
 

 

ACKNOWLEDGEMENTS 

 

This study was supported by “Program for Changjiang 

Scholars and Innovative Research Team” in University 

with grant on IRTO848. 
 

 
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