ABSTRACT A variety of probiotic bacteria have been tested to control animal and foodborne pathogenic bacteria in livestock. The mechanism of inhibition of pathogenic bacteria for several of those probiotic microorganisms is mediated by the production of bacteriocins. Colicins are probably the group of bacteriocins that have been most thoroughly characterized. Colicins are antimicrobial proteins produced by one strains of Escherichia coli to suppress the growth of other strains of E.coli. The present study indicated the preparation of colicin from colicinogenic bacteria. A total of three hundred rectal and rumen swabs isolated from health and diarrheic calves located in Fars province feces. One hundred and fifteen strains were confirmed as E.coli by biochemical test. Polymerase chain reaction was used to determine the following genes encoding colicins. Nearly 100% of isolates were contained at least one gene of colicin. The frequency of several classes of colicin was determined. As a result the most detected gene was Ia Ib and the least detected gene was A.N.S 4 . Colicin should be tested to control animal and foodborne pathogenic bacteria in livestock

A Kargar

M Hyati

M Namvari

Pourbakhsh Z

Vesal Shirazi

Y Shirazi

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Archives of Razi Institute, Vol. 67, No. 1, June (2012) 63-67                                          Copyright ©2012 by 
      Razi Vaccine & Serum Research Institute 
  
                                                
Short Communication  
Detection of Colicin genes by PCR in Escherichia coli 
isolated from cattle in Shiraz-Iran 
 
Tahamtan1, Y., Shirazi2, Z., Pourbakhsh1, A., Kargar2, M., Hyati1, M., Namvari1, 
M.M., Vesal Shirazi3, M.   
 
1. Razi Vaccine and Serum Research Institute-Shiraz, Iran 
2. Department of Microbiology, Jahrom Branch, Islamic Azad University, Jahrom, Iran 
3. Veterinary Organization of Fars Province, Iran 
 
 Received 02 Aug 2011; accepted 18 Oct 2011  
 
 
ABSTRACT 
A variety of probiotic bacteria have been tested to control animal and foodborne pathogenic bacteria in 
livestock. The mechanism of inhibition of pathogenic bacteria for several of those probiotic microorganisms is 
mediated by the production of bacteriocins. Colicins are probably the group of bacteriocins that have been 
most thoroughly characterized. Colicins are antimicrobial proteins produced by one strains of Escherichia coli 
to suppress the growth of other strains of E.coli. The present study indicated the preparation of colicin from 
colicinogenic bacteria.  A total of three hundred rectal and rumen swabs isolated from health and diarrheic 
calves located in Fars province feces. One hundred and fifteen strains were confirmed as E.coli by 
biochemical test. Polymerase chain reaction was used to determine the following genes encoding colicins. 
Nearly 100% of isolates were contained at least one gene of colicin. The frequency of several classes of 
colicin was determined. As a result the most detected gene was Ia Ib and the least detected gene was A.N.S4. 
Colicin should be tested to control animal and foodborne pathogenic bacteria in livestock. 
  
Keywords:  E. coli, Colicin, PCR, Cattle  
 
INTRODUCTION 
The emergence of antibiotic-resistance in many 
bacteria relevant for animal and public health stresses 
the importance of decreasing use of antibiotics in 
animal production. The reduction of antibiotic 
application in livestock can only be achieved if 
 
*Author for correspondence.Email: yahyatahamtan@yahoo.com 
 
alternative antimicrobial strategies are available (Fuller 
et al 1999). A variety of probiotic bacteria have been 
tested to control animal and food borne pathogenic 
bacteria in livestock. The mechanism of inhibition of 
pathogenic bacteria for several of those probiotic 
microorganisms is mediated by the production of 
bacteriocins (Stahl et al 2004). Among the different 
types of bacteriocins, colicins probably have the 
greatest specificity (i.e. E. coli colicins). Colicins are 
antimicrobial proteins produced by one strains of 
Tahamtan et al / Archives of Razi Institute, Vol. 67, No. 1, June (2012) 63-67 
 
64 
Escherichia coli to suppress the growth of other relative 
strains of E. coli (Diez-Gonzalez 2007). Colicins are 
typically produced under stress conditions and more 
than 25 different colicins have been characterized and 
these can be classified according to their mode of 
action and their import pathways (Le Jeune et al 2001). 
Colicin E1 and K inhibit all macromolecular synthesis 
without arrest of respiration(Schamberger & Diez-
Gonzalez 2004), colicins E2 E7 E8 and E9 cleave 
DNA(Schaller & Nomura 1976, (Toba et al 1986, 
Zakharov et al 2006), colicins E3 E4 and E6 hydrolyze 
rRNA(Stahl et al 2004, Boon 1971, Bowman et al 
1971), colicins D and E5 cleave tRNA(Ogawa et al 
1999, Tomita et al 2000), colicin A acts by making tiny 
pores in phospholipid bilayers thus allowing the 
leakage of ions across them(Schein et al 1978, Cavard 
2004). There is no any information of colicin and 
colicinogenic bacteria in Iran. 
The presences of colicin in the gut confirm that 
animal can resist against colibacillosis, when antibiotics 
are not used (Papavassiliou 1961, Russell et al 2002). 
The main objective of the present study was screened 
colicinogenic bacteria from gastrointestinal tract of 
calves. The prevalence of several classes of colicin was 
determined. Polymerase chain reaction was used to 
detect bacteria those posses colicin genes.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
MATERIALS AND METHODS 
Bacterial Strains. A total of 300 rectal and rumen (at 
slaughter house) swabs were collected from feces of 
health and diarrheic calves located in Fars province.  
Biochemical test being Voges-Proskauer negative, 
citrate negative, indole positive and methyl red positive 
were used to confirm the isolates as E. coli. The swab 
sample put into the TSB and the incubated at 37 °C 
over night. The samples were then streaked on Mac 
Conkey agar. Lactose positive colony was chosen for 
biochemical test. 
DNA isolation and PCR amplification. In this 
study we used seven pairs specific primers were 
designed by Gene Bank sequences (Hall 1999, Benson 
et al 2006). Genotypes of all strains were verified by 
PCR. Polymerase chain reaction was used to detect 
bacteria those posses colicin genes (table 1). 
The PCR reactions were in a 25 µL volume and 
comprised 1µL of MgCl2  (15mM), 0.5µL of 2.5 mM 
dNTP, 2.5µL of Tag buffer (10×), 0.5µL of each 
primer (2.5pM each), 0.1µL of Tag DNA polymerase 
and 2 µl of the DNA sample. Sterile filtered water was 
used to bring the final reaction volume to 25µL. The 
thermocycler cycling conditions were 1 cycle of 
denaturation at 94 °C for 2 min, annealing at the primer  
 
 
 
 
 
Table 1. Oligonucleotides (5’-3’) sequences of colicin genes according to farward and  reverse primers. 
 
 
 
 
Target gene 
of colicin 
Primer 
name 
Primer Sequences ( 5’ to 3’ ) 
PCR product 
Size bp 
Annealing 
temperature 
ºC 
A.N.S4 
 
 
 
 
 
 
 
 
NS4: r 
NS4: f 
CGT AGC TAT AAT GAA GCA ATG GCT TCA 
ACC TCC AAC AGG AGA GGT CCC CAG TT 
225 57 
V 
V: f 
V: r 
CAC GCC CTG AAG CAC CAC CA 
CCG TTT TCC AAG CGG ACC CC 
400 68 
Ιa, Ιb 
Iab: f 
Iab: r 
GCA CAA CAG GCC CGT CTG CTC 
CAC CTT CCA CAT CCT CTG TCA CC 
385 68 
E2.E3.E4E5.
E6.E7E8.E9 
Mix: f 
Mix: r 
CGA CAG GCT AAA GCT GTT CAG GT 
TGC AGC AGC ATC AAA TGC AGC CT 
219 60 
U, Y 
Yu: f 
Yu: r 
GTG AAC GGA CAG AAA CCC GCC 
CAA TCT GTC TGA CAG CCT CTC CC 
243 68 
5,10,K 
510K: f 
510K: r 
AAA GCT GAA CTG GCG AAG GC 
CAA CTC ATC ATC CCC TAT GTA AGA AG 
803 60 
E1 
E1: f 
E1: r 
ACG GGA GTG GCT CTG GCG G 
CTC TTT ACG TCG TTG TTC TGC TTC CTG 
389 68 
 
Tahamtan et al / Archives of Razi Institute, Vol. 66, No2, December (2011) 63-67 
 
65
specific Tm for 1min, elongation at 72 °C for 1 min, 
then 35 cycles of denaturation at 94 °C for 1 min. The 
PCR reaction products were stored at ‒20 °C or 
immediately separated on 2% agarose gels in a 
0.5×TBE buffer (per liter: 10×TBE is 108 g of Tris 
base, 55 g of boric acid and 40mL of 0.5 MNa2EDTA) 
(Setia 2009). 
Statistical Analysis. Data were recorded using the 
SPSS version 13. Statistical analysis was performed 
using ANOVA office software (Microsoft).  
 
Table 2. Sources of Escherichia coli and distribution of 
colicinogenic E. coli isolates  
Source of 
isolates 
Number of 
isolates (%) 
E. coli 
isolates (%) 
Colicinogenic 
strains (% 
Rumen 55(18.33) 14(4.66) 12(85.71) 
Feces 245(81.66) 101(33.66) 101(100) 
Total 300(100) 115(38.33) 115(100) 
RESULTS AND DISCUSSION 
Bacterial Strains. One hundred and fifteen samples 
were confirmed as E. coli by biochemical test (Voges-
Proskauer negative, citrate negative, indole positive and 
methyl red positive). According to a previous study 
these strains were not pathogen and they were negative 
for F5, F41, STa and Shiga toxin (Tahamtan et al 2011, 
Shams et al 2010). Of the 115 colicinogenic E. coli 
(CGEC) strains, 100% were found from feces, while 
85.71 % detected in rumen (table 2). 
 
 
 
 
 
 
 
 
 
 
PCR analyses. PCR was used to determine the 
following genes encoding the colicins. Genotypes of 
115 strains were verified by PCR. Approximately 
100% of isolates was contained at least one colicin 
gene (figure 1). The colicins detected in the isolates by 
PCR were E1, V, Ia Ib, E2 E3 E4 E5 E6 E7 E8 E9, UY, 
5.10.K, A.N.S4 (figure 2). The data indicated 39.19% of 
isolates  harbored at least one colicin gene, while 21.73, 
17.42, 13.04 and 3.34 present of isolates posses two, 
three, four and five gene of colicin repectively.                        
E. coli is the major species in the fecal coliform 
group (Daines & Paterson 2008). Coliform bacteria 
normally present in the intestine produce colicins which 
protect intestinal tract from pathogenic bacteria 
(Cascales et al 2007). The normal flora prevents 
colonization of pathogenic microorganisms by 
production of colicins. The colicinogenic bacteria play 
an important role in preventing the colonization of 
gastrointestinal bactrria in livestock. As results of using 
antibiotic, the balance of these normal flora in the colon 
may become disrupted and consequently, the enteric 
pathogen adhere to colon. The use of antibiotic has 
many limitations such as antibiotic resistance.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
0
30
60
A.N.S4 V IaIb E Mix U.Y 5.10.K E1Colicins
N
o 
of
 Is
ol
at
es
Figure 2. Single PCR for colicin gene. M: 100 bp marker, -: 
Negative control, Lan 1 to 7: E Mix, ANS4, UY, IaIb, E1, V, 
and 5 10 K.  
Gastrointestinal tract can also acquire and serve as 
long term reservoirs for resistance genes. Theoretically 
these genes can be transferred to humans. Indeed the 
FDA and CDC are already moving to restrict the use of 
antibiotics in animal agriculture (Majeed et al 2011). 
Because of these limitations, colicinogenic bacteria are 
good alternative and effective replacement.  
Figure1. All colicinogenic E. coli strains harbored at least one 
colicin gene in their genum. IaIb was the dominant among the 
colicins. 
Tahamtan et al / Archives of Razi Institute, Vol. 67, No. 1, June (2012) 63-67 
 
66 
For the first time in Iran we reported colicinogenic 
strain from cattle feces and the prevalence of several 
classes of colicin was determined by PCR method. 
Nearly 100% of the isolates in the present study had at 
least one gene of colicins and some of the isolates had 
more than two colicins. The most detected gene was Ia 
Ib and the least detected gene was A.N.S4. Setia et al 
(2009) indicated no rumen isolates evaluated were 
effective at inhibiting K88, but isolates from cattle 
feces were effective. This is rather interesting because 
it suggests that even though all isolates were 
phenotypically E. coli, there are subtle differences 
between those inhabiting the rumen compared with 
those in the hind-gut (Schamberger and Diez-Gonzalez. 
Schamberger and Diez-Gonzalez in USA screened 
isolates of E. coli from the feces of cats, cattle, 
chickens, deer, dogs, ducks, horses, humans, pigs, and 
sheep. They found colicinogenic strains from all 
sources, but the greatest number was from cats and 
sheep (Schamberger & Diez-Gonzalez 2002). 
However, many factors that might influence the 
differences animal isolates and the type of colicin, 
including colicin diversity, the use of other 
experimental designs, effectiveness of probiotic 
organisms, animal species, diet, and geographical 
region (Murinda et al 1996,  Brashears et al 2003). 
The presence of gene does not indicate whether or 
not gene expression because, under conditions of stress 
a fraction of colicinogenic bacteria are induced to 
produce colicin proteins. The type of stress causing the 
inductive activity needs to be better characterized 
(Diez-Gonzalez 2007, Kuhar et al 2001). Over use of 
antibiotics is the key factor to eliminate the bacteria 
particularly colicinogenic strans from gasterointestinal 
tract in animals and humans (Diez-Gonzalez 2007). 
The colicin will be use to prevent ETEC or VETC 
infection. Further investigation is need to known about 
the inhibitory activity of colicinogenic E. coli against 
pathogen bacteria.  
Aknowledgment 
We thank Dr. MH Hosseini and all of the people who 
collaborated with the authors, particularly the 
Veterinary Organization of Fars Province. This study 
was funded by project No 2-84-18-18-87064 and was 
supported by Razi Vaccine and Serum Research 
Institute.  
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Detection of Colicin genes by PCR in Escherichia coli isolated from cattle in Shiraz-Iran Tahamtan 1

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Detection of Colicin genes by PCR in Escherichia coli isolated from cattle in Shiraz-Iran Tahamtan 1

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