International Journal of Agriculture: Research and Review. Vol.

, 2 (6), 862-866, 2012

Available online at http://www.ecisi.com
ISSN 2228-7973 2012 ECISI Journals

A STUDY ON TRANSFER OF ANTIBIOTIC RESISTANCE PLASMIDS BETWEEN

SALMONELLA ENTERITIDIS AND ESCHERICHIA COLI K12
JAFAR AKBARMEHR
Department of Microbiology, Islamic Azad University Sarab Branch, Sarab, Iran
*Corresponding Author Email: ja_mehr@yahoo.com
ABSTRACT: In recent years multidrug-resistant Salmonella strains, have emerged due to
extensive use of antibiotics in both animals and humans.The aim of this study was to
investigate if conjugative transfer of antimicrobial resistance plasmids could occur between
Salmonella enteritidis (donor) and Escherichia coli K12 (recipient). 22 S. enteritidis isolates,
from poultry sources, were tested for their susceptibility to ten common antibiotics by disc
diffusion method on Muller Hinton agar and the interpretation of results was carried out
according to the recommendation of National Committee for Clinical Laboratory
Standards(NCCLS). Transfer of antibiotic resistance plasmids between donor and recipient
was carried out by in vitro conjugation experiment following the protocol of Hart et al,
(1996). Based on antibiotic sensitivity test results, resistance to Tetracycline was the highest
(40.90%) followed by Streptomycin (31.81%) and Nalidixic acid (22.72%). Cephalotin with
100% sensitivity was the most effective antibiotic against S. enteritidis followed by
Enrofloxacin (95.45% sensitivity) Ciprofloxacin (90.90% sensitivity) and Gentamicin
(86.36% sensitivity ). 9 out of 22 Salmonella isolates (40.9%) were resistant to more than
one antibiotic and identified as multidrug-resistant isolates. In conjugation experiment, 7, out
of 15 Salmonella isolates (46.66%) transferred their resistance patterns (1-3 patterns) to E.
coli K 12. This research demonstrated that the horizontal transfer of antibiotic resistance
plasmids can occur among Salmonella isolates via conjugation and also revealed the
emergence of multidrug-resistant Salmonella isolates as a significant health problem in Iran.
Key words: Resistance, Plasmid, Salmonella, Conjugation, Antibiotic
INTRODUCTION
Enteric Salmonella infection is a global
problem and has been a major challenge
worldwide.
Salmonellosis
is
common
throughout the world (Zhao et al., 2001).
Outbreaks are usually associated with ingestion
of contaminated food of animal origin. Poultry
meat and egg have been implicated as important
sources of human Salmonella infections (Doyle
et al., 2007). Salmonella enterica serovar
Typhimurium and Salmonella enterica serovar
Enteritidis are the most frequently isolated
serovars worldwide (Zhao et al., 2001; Zahraei
et al., 2005; Akbarmehr et al., 2010) and
considered the main cause of Salmonella
poisoning in humans (Doyle et al., 2007).
Although some Salmonella serotypes such as S.
Pullorum(in poultry) and S. Typhi (in man) are
host specific, most serotypes can be isolated

from a wide variety of animal species and

humans. Many kinds of antibiotics are used in
animals and humans for both prevention and
treatment of salmonellosis. In animal husbandry
they are also used as growth-promoting agents.
The widespread use of antibiotic in poultry
farms has lead to an increase in the number of
resistant Salmonella strains (Zahraei et al.,
2005; Peighambari et al., 2010; Enabulele et
al., 2010). Bacterial resistance to antibiotics
may be natural or acquired. Natural resistance is
a characteristic of bacterial species which are
resistant to a particular antibiotic. It may be due
to the bacterial cell wall which is impermeable
to the antibiotic. Acquired resistance can
originate from chromosomal mutation or from
acquisition of transferable genetic materials
(Meervenne, et al., 2012). Plasmids,
transposons and integrons are the most
important vehicle for transfer of resistance

Intl. J. Agric: Res & Rev. Vol., 2 (6), 862-866, 2012

genes in bacteria (Prescott et al., 2005).

Plasmids are extrachromosomal, replicable
DNA molecule that may contain resistance
gene. They have been a major factor in the
spreading antibiotic resistance between bacteria
(Hart et al., 1996; Mandal et al., 2003;
Peighambari et al., 2010). In addition plasmids
are important in bacterial evolution because
they affect in bacterial fertility,replication,
metabolism as well as resistance to antibiotics.
Bacterial conjugation is an important
mechanism whereby plasmid or other genetic
material is transferred from the donor bacteria to
the recipient via cytoplasmic bridge (Poole et
al., 2009). Recently drug resistant strains of
bacteria have emerged presumably due to
extensive use of antimicrobial agents both in
humans and animals. Misuse of antibiotics has
been associated with high prevalence of
antibiotic resistance among isolates and
promotes the spread of multiple-drug resistance
(MDR) ( Rahman et al., 2001; Halawani et al.,
2008 ; Lan-Ho Chiu et al., 2010; Meervenne et
al., 2012). In Iran as well as other developing
countries, factors such as insufficient control of
drug prescribing and widespread use of
antibiotics, promote and favour drug resistance.
Knowledge of the epidemiology of Salmonella
antibiotic resistance and information about their
resistance mechanisms are necessary for control
of drug resistant Salmonella isolates in both
animals and humans. This study was undertaken
to characterize the antibiotic resistance patterns
of the Salmonella enteritidis isolates and also to
investigate the horizontal transfer of Rplasmids between S. enteritidis and E.coli k12.
MATERIALS AND METHODS
Drug sensitivity test of Salmonella isolates
In this study 22 S. enteritidis isolates
which obtained from poultry sources, were
examined for conjugative transfer of
antimicrobial resistance plasmids to E.coli K12.
Salmonella isolates were tested fo their
susceptibility to ten common antibiotics by disc
diffusion method on Muller Hinton agar
(Merck) and the interpretation of results was
carried out according to the recommendation of
National Committee for Clinical Laboratory
Standards (NCCLS). The following commercial
antibiotic discs were used: Amoxicillin (10 g),

863

Streptomycin (10 g), Gentamicin (30 g),

Nalidixic acid (30 g), Trimethoprim (30 g),
Tetracycline (30 g), Ciprofloxacin (5 g),
Cephalotin (30 g), Ampicillin (10 g),
Enrofloxacin (5 g).The antibiotic discs were
provided by Padtan Teb Co (Tehran,Iran).
Conjugation experiment
In this experiment those Salmonella
isolates which were sensitive to Nalidixic acid
and resistant to one or more other tested
antibiotics were choosed as donor bacteria. The
transfer of R-plasmid was determined by
conjugation between donor (S. enteritidis) and
recipient (E.coli K12) according to the method
of Hart et al., (1996). The recipient E.coli K12
was susceptible to all drugs except Nalidixic
acid. Transconjucants were selected on Mac
Conkey agar (Merck) containing Nalidixic acid
(32mg/l). All transconjucants were tested for
antimicrobial susceptibility.
RESULTS
Antibiogram results of 22 Salmonella
isolates are shown in Table 1. According to the
table
Cephalotin was the most effective
antibiotic (100% sensitivity) followed by
Enrofloxacin (95.45% sensitivity) Ciprofloxacin
(90.90% sensitivity) and Gentamicin (86.36%
sensitivity ). The highest resistance (40.90%)
was observed to Tetracycline followed by
Streptomycin
(31.81%)
and
Nalidixic
acid(22.72%). Also 18.18% of isolates were
equally resistance to Amoxicillin, Trimethoprim
and Ampicillin. Of 22 Salmonella isolates 15
(68.18%) were sensitive to Nalidixic acid. The
frequency of drug resistance patterns of 22
Salmonella enteritis isolates is shown in Table
2. As it shows 9 Salmonella (40.9%) isolates
were resistance to more than one antimicrobial
agents (MDR). The MDR patterns were varied
from 2 to 5 antibiotics. In conjugation
experiment 15 Salmonella isolates which were
sensitive to Nalidixic acid and resistant to one
or more other tested antibiotics were examined
for transfer of R-plasmids to E.coli K12.
According to Table 3, seven (46.66%)
Salmonella isolates transferred their resistance
patterns to E.coli K12. The number of resistance
patterns was varied from 1 to 3.

Intl. J. Agric: Res & Rev. Vol., 2 (6), 862-866, 2012

Table 1.The results of Antibiotic sensitivity test of 22 S .enteritidis isolates

--------------------------------------------------------------------------------------------------------------------------Antibiotic
Sensitive(S)
Intermediate(I)
Resistant(R)
No. of isolates(%)
No. of isolates(%)
No of isolates(%)
Amoxicillin
16(72.72)
2(9.09)
4(18.18)
Streptomycin
12(54.54)
3(13.63)
7(31.81)
Gentamicin
19(86.36)
3(13.63)
0(0)
Trimethoprim
14(63.63)
4(18.18)
4(18.18)
Nalidixic acid
15(68.18)
2(9.09)
5(22.72)
Enrofloxacin
21(95.45)
1(4.54)
0(0)
Tetracycline
12(54.54)
1(4.54)
9(40.90)
Cephalotin
22(100)
0(0)
0(0)
Ampicillin
11(50)
7(31.81)
4(18.18)
Ciprofloxacin
20(90.90)
2(9.09)
0(0)
-----------------------------------------------------------------------------------------------------------------------------Table 2.The frequency of multidrug- resistance patterns among 22 Salmonella enteritidis isolates
-----------------------------------------------------------------------------------------------------------------------Resistance patterns
No. of isolates
------------------------------------------------------------------------------------------------------------------------TC-TR-NA-S-AM
3
TC-TR- S-AM
1
NA-S
2
TC-S
1
AP-TC
2
Total
9
----------------------------------------------------------------------------------------------------------------------------Table 3. The frequency of different resistance patterns transferred from resistant Salmonella isolates to
E.coli k12
--------------------------------------------------------------------------------------------------------------------------Transferred resistance pattern to E.coli K12
No. of Salmonella isolates
TC-TR- -AM
2
TC-AP
2
AP
2
TC
1
Total
7
--------------------------------------------------------------------------------------------------------------------------DISCUSSION
Antibiotic resistant Non-typhoidal
Salmonella (NTS) appear to arise with different
resistant patterns in different animal species
(Linton, 1986). In recent year's antibiotic
resistance in Salmonella has assumed alarming
proportion worldwide (Murugkar et al., 2005).
Increased MDR has been reported in Salmonella
isolates in many countries including Iran
(Rahman et al., 2001; Zahraei et al., 2005;
Halawani et al., 2008; Peighambari et al.,
2010). In the present study the highest number
of Salmonella isolates, were resistant against
Tetracycline
(40.90%)
followed
by
Streptomycin (31.81%) and Nalidixic acid
(22.72%). Cephalotin (100% sensitivity),
Enrofloxacin
(95.45%
sensitivity),

Ciprofloxacin
(90.90%sensitivity)
and
Gentamicin (86.36% sensitivity) were the most
effective antibiotics in vitro against all
Salmonella isolates respectively. Tetracycline
and Streptomycin are two common antibiotics
which are used in veterinary medicine in Iran.
The high ratio of resistance against these
antimicrobial agents may be due to their
uncontrolled and wide use in poultry farms.
Although many other factors such as
pharmacokinetic characteristics, insufficient
dose, too short duration of treatment, long term
use, and active concentration of the drug may
influence antibiotic resistance of bacteria(Davis
1994). In particular the long term use of
insufficient doses, is regarded as one of the
major factor responsible for development of
antibiotic resistance (Corpet et al., 1989).

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Intl. J. Agric: Res & Rev. Vol., 2 (6), 862-866, 2012

According to Table 2, nine Salmonella isolates

(40.9%) were resistance to more than one
antimicrobial agents and the MDR patterns were
varied from 2 to 5 antibiotics. This may be due
to the intensive use of antibiotics in Iranian
poultry industry which lead to collection of
resistance genes in Salmonella with poultry
origin. Peighambari et al., (2010) in a study
which carried out in Iran examined 49
S.enteritidis for drug resistance. They observed
the highest antibiotic resistance (38.8%) to
flumequine. They also showed that 61.2% of the
isolates were multidrug- resistant which is
higher compared to our results (40.90%).
Zahraei et al.,(2005) in another study which
conducted in Fars province,Iran reported that
20.6%of Salmonella strains in the studied area
were MDR which shows lower rate compared
to our results. Halawani et al.,(2008) who
investigated on 22 Salmonella strains in Saudi
Arabia have found MDR Salmonella in 68% of
Salmonella isolates. Multidrug resistance in
bacteria is generally attributed to the acquisition
of transposons, integrons, or plasmids via
different mechanisms (Perscott et al., 2005).
The emergence of multidrug resistant foodborne pathogens such as Salmonella and E.coli
has become a global public health concern (Pool
et al., 2009). The first well documented
bacterial outbreak involving MDR bacteria was
an epidemic of typhoid fever caused by
Salmonella typhi in Mexico, with more than
10000 confirmed case in 1972 (Amobile et al.,
1995).
Zoonotic
infections
such
as
salmonellosis are examples of the development
and spread of MDR bacteria from animal to
man via food chain. The use of antibiotics in
food animal production may be one of the
factors contributing to the rapid spread of MDR
(Meervenne et al., 2012). Many scientists are
concerned that the use of antibiotics to prevent
and treat disease in animals may apply selective
pressures , leading to the emergence of
antibiotic resistant bacteria that can cause
infection in humans (Rahman et al., 2001).
Plasmids are the most important vehicles for
resistance genes which transfer between bacteria
via conjugation(Poole et al., 2009). Bassed on
our results in conjugation experiments, in 7 of
15 (46.66%) MDR S. entertidis, 1-3 R-plasmid
resistance patterns transferred in vitro to E.coli
k12. Resistance to four antibiotics (Tetracycline,
Amoxicillin, Ampicillin, Trimethoprim) appears
to be transferable (Table 3). In recent years
several authors have studied on multidrugresistance and transfer of Salmonella Rplasmids via conjugation. Hart, et al, (1996)
who studied on 90 NTS resistant isolates,

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demonstrated that 61/90 (67.7%) of them

transferred their resistance to one or more
antimicrobials to E.coli k12. Mandal et al.,
(2003) reported that in S. enterica serovar
Typhi, the R-plasmid encoded resistance to
Ampicillin, Chloramphenicol, Cotrimoxazole,
and Tetracycline was transferable. Many other
studies have demonstrated that R-plasmid
transfer via conjugation between Salmonella
isolates and other gram negative bacteria has an
important role in antimicrobial resistance of
enteric bacteria (Formal et al., 1970; Carattoli,
2003; Poole et al., 2009). Finally the present
study has demonstrated that horizontal transfer
of antibiotic resistance plasmids via conjugation
can occur among Salmonella isolates with
poultry origin. The results of this research also
revealed that the antibiotic resistance in
Salmonella is a significant problem in poultry
industry which should be considered by
veterinary organization in Iran.
ACKNOWLEDGMENTS
This research was supported by the Islamic
Azad University of Sarab Branch. We are
grateful to the Dr. Farmani and Mr. Rezanavaz
for their valuable collaboration.
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