a study on transfer of antibiotic resistance plasmids between salmonella enteritidis and escherichia...

7/26/2019 A Study on Transfer of Antibiotic Resistance Plasmids Between Salmonella Enteritidis and Escherichia Coli k12

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International Journal of Agriculture: Research and Review. Vol., 2 (6), 862-866, 2012

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

ASTUDY ON TRANSFER OF ANTIBIOTIC RESISTANCE PLASMIDS BETWEEN

SALMONELLA ENTERITIDIS ANDESCHERICHIA COLIK12

JAFAR AKBARMEHR

Department of Microbiology, Islamic Azad University Sarab Branch, Sarab, Iran

*Corresponding Author Email: [email protected]

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 betweenSalmonella enteritidis(donor) and Escherichia coliK12 (recipient). 22S. enteritidisisolates,

from poultry sources, were tested for their susceptibility to ten common antibiotics by discdiffusion 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 15Salmonella isolates (46.66%) transferred their resistance patterns (1-3 patterns) to E.coli K 12. This research demonstrated that the horizontal transfer of antibiotic resistanceplasmids can occur among Salmonella isolates via conjugation and also revealed the

emergence of multidrug-resistantSalmonellaisolates as a significant health problem in Iran.

Key words: Resistance, Plasmid,Salmonella, Conjugation, Antibiotic

INTRODUCTION

EntericSalmonellainfection 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 ingestionof contaminated food of animal origin. Poultry

meat and egg have been implicated as important

sources of humanSalmonella infections (Doyle

et al., 2007). Salmonella enterica serovar

Typhimurium and Salmonella enterica serovar

Enteritidis are the most frequently isolated

serovars worldwide (Zhaoet 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 someSalmonella serotypes such asS.

Pullorum(in poultry) andS. 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 ofresistant Salmonella strains (Zahraei et al.,

2005; Peighambari et al., 2010; Enabuleleet

al., 2010). Bacterial resistance to antibioticsmay 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


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

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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;Peighambariet 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) ( Rahmanet al., 2001; Halawaniet al.,2008 ; Lan-Ho Chiuet al., 2010; Meervenneet

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 ofSalmonella

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 patternsof theSalmonellaenteritidis isolates and also to

investigate the horizontal transfer of R-

plasmids betweenS. enteritidisandE.colik12.

MATERIALS AND METHODS

Drug sensitivity test of Salmonella isolatesIn this study 22 S. enteritidis isolates

which obtained from poultry sources, were

examined for conjugative transfer of

antimicrobial resistance plasmids toE.coliK12.

Salmonella isolates were tested fo theirsusceptibility 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 LaboratoryStandards (NCCLS). The following commercial

antibiotic discs were used: Amoxicillin (10 g),

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 experimentIn 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.coliK12) according to the method

of Hartet al., (1996). The recipientE.coliK12

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 Nalidixicacid(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 Table2. 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 oneor 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 toE.coliK12. The number of resistancepatterns was varied from 1 to 3.


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Table 1.The results of Antibiotic sensitivity test of 22 S.enteritidisisolates

---------------------------------------------------------------------------------------------------------------------------

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 22Salmonella enteritidisisolates

------------------------------------------------------------------------------------------------------------------------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 resistantSalmonellaisolatesto

E.colik12

---------------------------------------------------------------------------------------------------------------------------Transferred resistance pattern toE.coli K12 No. ofSalmonellaisolates

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 inSalmonellahas assumed alarming

proportion worldwide (Murugkar et al., 2005).

Increased MDR has been reported inSalmonella

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 antibioticswhich 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 aspharmacokinetic 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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According to Table 2, nineSalmonella 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 ofresistance 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 MDRSalmonella in 68% of

Salmonella isolates. Multidrug resistance inbacteria is generally attributed to the acquisition

of transposons, integrons, or plasmids via

different mechanisms (Perscott et al., 2005).

The emergence of multidrug resistant food-

borne pathogens such as SalmonellaandE.colihas 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 (Amobileet 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%) MDRS. entertidis,1-3 R-plasmidresistance patterns transferred in vitro toE.coli

k12. Resistance to four antibiotics (Tetracycline,

Amoxicillin, Ampicillin, Trimethoprim) appears

to be transferable (Table 3). In recent years

several authors have studied on multidrug-

resistance and transfer of Salmonella R-

plasmids via conjugation. Hart, et al, (1996)who studied on 90 NTS resistant isolates,

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 Salmonellaisolates and other gram negative bacteria has an

important role in antimicrobial resistance of

enteric bacteria (Formalet 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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a study on transfer of antibiotic resistance plasmids between salmonella enteritidis and escherichia...

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