172 Pak J Med Sci 2014 Vol. 30 No. 1 www.pjms.com.pk
Staphylococcus aureus is an extracellular Gram
positive pathogen, which causes a number of
infections such as pneumonia, endocarditis, and
septic arthritis.1 In many cases, the infection
originates from hospital derived antibiotic resistant
bacteria, among which the most common is MRSA
whose prevalence varies markedly between
different regions and hospitals.2 Within hospitals,
MRSA accounts for 40–70% of infections in
Intensive care units3 and overall it is responsible for
50% or more of hospital acquired infections in many
countries. Normal nasal carriage of S. aureus is 25-
30% whereas less than 2% of normal individuals are
colonized with MRSA.4
In Pakistan, the prevalence of MRSA has increased
tremendously over the years. It was reported as 5%
in 1989 and since then has increased up to 51%. It is
reported to range from 42 to 51%, increasing from
the 1990 to 20005,6 and from 19.5% in 2001 to 40% in
S. aureus produces many virulence factors
comprising of toxins and enzymes, regulated by
agr and sar systems.8 The accessory gene regulatory
(agr) system down regulates the expression of
surface proteins while up regulates the expression
1-3: Department of Microbiology,
Islamabad 45320, Pakistan.
Sadia Khan, M. Phil,
Faisal Rasheed, M. Phil,
Rabaab Zahra, PhD,
Rabaab Zahra, PhD,
Department of Microbiology,
Islamabad 45320, Pakistan.
* Received for Publication:
* Revision Received:
* Revision Accepted:
July 25, 2013
November 5, 2013
November 6, 2013
Genetic polymorphism of agr Locus and antibiotic resistance
of Staphylococcus aureus at two hospitals in Pakistan
Sadia Khan1, Faisal Rasheed2, Rabaab Zahra3
Objective: The accessory gene regulator (agr) locus in Staphylococcus aureus (S. aureus) is a global
regulator of quorum sensing and controls the production of virulence factors. This study was carried out to
investigate the agr specific groups both in methicillin resistant and sensitive Staphylococcus aureus (MRSA
and MSSA) and their relation with antibiotic resistance.
Methods: A total of 90 clinical S. aureus isolates were studied from two tertiary care hospitals. The
isolates were identified by standard biochemical tests. Methicillin resistance was confirmed by oxacillin
and cefoxitin resistance. Multiplex PCR was used to determine the agr groups.
Results: MRSA prevalence was found to be 53.3%.The agr groups’ distribution in MRSA was as follows:
22 (45.8%) belonged to group I, 14 (29.1%) belonged to group III and 2 (4.1%) belonged to group II. agrIV
was not detected in MRSA. For 17 isolates, the agr group was not detected.agr III isolates showed higher
antibiotic resistance than agrI isolates except in case of oxacillin and linezolid.
Conclusions: Strict infection control policy and antibiotic guidelines should be adopted to control the
problem of MRSA. Higher prevalence of agr I and agr III shows that they are dominant agr groups of our
KEY WORDS: S. aureus, agr, MRSA, MSSA.
How to cite this:
Khan S, Rasheed F, Zahra R. Genetic polymorphism of agr Locus and antibiotic resistance of Staphylococcus aureus at two hospitals
in Pakistan. Pak J Med Sci 2014;30(1):172-176. doi: http://dx.doi.org/10.12669/pjms.301.4124
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Pak J Med Sci 2014 Vol. 30 No. 1 www.pjms.com.pk 173
agr Locus and Antibiotic Resistance of MRSA and MSSA
of expoproteins. It encodes two transcripts, RNAII
and RNAIII where RNAII encodes for agrA, agrB,
agrC and agrD. S. aureus isolates can be divided into
four agr groups on the basis of the specificity of the
auto-inducing peptide (AgrC).9 Further, S. aureus
strains belong to specific agr groups10 implicating
the importance of the knowledge of agr gene groups.
The current study was designed to analyze the
genetic polymorphism of agr locus among S. aureus
isolates and to assess its relationship with antibiotic
Bacterial Isolates: The study includes a total
of 90 S. aureus clinical isolates, out of which
35 were collected from Holy Family Hospital,
Rawalpindi whereas, 55 isolates were collected
from Microbiology Laboratory, Pakistan Institute
of Medical Sciences, Islamabad during the months
of April to Oct 2011. These isolates were taken from
different sources where 18 were from nasal swab,
50 from pus, 3 from peri rectal swab, 10 from blood,
4 from tracheal secretion, and one each from the
following sources: tissue, prostatic secretion, throat
swab, semen and CVP tip.The culture media for
isolation of S. aureus were blood agar, mannitol salt
agar and brain heart infusion (BHI) broth/agar.
Identification of S. aureus Isolates: Identification
of isolates was performed by Gram staining and
routine biochemical tests including catalase,
coagulase, mannitol salt fermentation, and DNase
Antibiotic Susceptibility Testing: Susceptibility
testing was conducted by disk diffusion method
according to the guidelines of Clinical and
Laboratory Standards Institute (CLSI).11 Fusidic
acid (susceptibility and resistance were ≥22 mm and
<22 mm) and tigecycline susceptible breakpoints≥
19 mm zone size were interpreted according
to the European Committee on Antimicrobial
Susceptibility Testing (EUCAST) and US FDA
clinical breakpoints respectively. S. aureus ATCC
25923 was used as quality control strain.
DNA Extraction: Extraction procedure was
followed according to manufacturer’s instructions
using Wizard Genomic DNA Extraction Kit
(Promega Inc., Madison, USA).
PCR Amplification and Detection of agr Groups:
Primers provided by Integrated DNA Technologies
(California, USA) were chosen from published
sequences.10 The PCR assay was performed
using green master mix (Promega Inc., Madison,
USA). Amplified samples were analyzed by
electrophoresis on a 1% agarose gel and stained
with ethidium bromide.
Statistical Analysis: Statistical analysis was
performed using the software SPSS 17.0 (SPSS Inc,
Chicago, USA). Differences among different groups
were analyzed using χ2 test. p value less than 0.05
was considered as significant.
Ninety clinical isolates were confirmed as S. aureus
by Gram staining and standard biochemical tests.
All isolates were mannitol fermenters and positive
for catalase, DNase and coagulase. Resistance to
oxacillin and cefoxitin or both according to CLSI
presented the prevalence of MRSA to be 53.3%.
Prevalence and Association of MRSA and MSSA
with Gender and Age: In our study, MRSA isolates
were comparatively more prevalent in males 55.6%
(35/63) than females 48.1% (13/27) whereas MSSA
isolates were more prevalent in females 51.9%
(14/27) than males 44.4% (28/63) with no significant
statistical difference (p = 0.519). Age was categorized
into three groups i.e., 1-18 years, 19-44 years and
44+ years. Higher prevalence 68.4% (13/19) and
60.9% (14/23) of MRSA was observed in age groups
44+ years and 1-18 years, respectively. Whereas,
comparatively low prevalence (21/48) of MRSA
was found in 19-44 years age group. Prevalence of
MSSA was found to be 56.2% (27/48), 39.1% (9/23)
and 31.6% (6/19) among groups aged 19-44, 1-18
and 44+ years, respectively. Association of MRSA
and MSSA with age was found non-significant (p
Fig.1: Analysis of PCR products for the identification of
agr specific groups. Lane M: 100 bp DNA ladder. Lanes
1-3: PCR product of agr group III (406 bp). Lanes 4-5: PCR
product of agr group I (440 bp). Lanes 6-7: PCR product
of agrgroup II (572 bp). Lane 8: PCR product of agr group
IV (588 bp).
174 Pak J Med Sci 2014 Vol. 30 No. 1 www.pjms.com.pk
Antibiotic Resistance Profile of MRSA and MSSA
Clinical Isolates: Table-I shows the antibiotic
resistance of MRSA and MSSA isolates. Majority
of MRSA isolates exhibited high level of resistance
to penicillin, cefoxitin and fusidic acid and
comparatively low resistance to vancomycin. MSSA
isolates were also highly resistant to penicillin.
Tigecycline against MRSA and vancomycin and
cefoxitin against MSSA clinical isolates were found
to be the most effective antibiotics.
Prevalence of agr Specific Groups in MRSA and
MSSA: Using multiplex PCR, MRSA and MSSA
clinical isolates were grouped in four agr specific
groups (Fig.1). Among all groups, agrI was the most
prevalent followed by agrIII and agrII. agrIV was
absent in MRSA while 4.7% MSSA isolates were
positive for it (Table-II). Non-type able agr group
among MRSA and MSSA were 20.8% and 16.6%,
Association between Antibiotic Resistance and
agr Specific Groups: MRSA and MSSA isolates
showed high resistance against penicillin with
statistically significant difference (p = 0.002) in all
agr groups as shown in Table-III. However, all agr
containing MRSA and MSSA isolates were sensitive
In this study, prevalence of MRSA was found
to be 53.3% which is consistent with the results
reported by other researchers.5,12 Although higher
rate of MRSA was observed in males with non-
significant difference that is similar to the earlier
Higher prevalence of MRSA was observed in case
of patients aged 44+ years (68.4%) and 1-18 years
(60.9%) which is close to the data,3 where it was
61.4% in 41-80 years age group. Similar results were
reported in studies conducted in Malaysia and
Previously, it has been shown that more cases
of MRSA are reported from patients staying in
intensive care units2,3 and the same was observed in
Sadia Khan et al.
Table-I: Antibiotic resistance of
MRSA and MSSA clinical isolates.
8 (16.7 )
31 (64.6 )
Table-II: Distribution of agr groups in
MRSA and MSSA clinical isolates.
agr group MRSA n (%) MSSA n (%) Total n (%)
I 22 (45.8)
II 2 (4.1)
III 14 (29.1)
Non-typeable 10 (20.8)
Total 48 (100)
Table-III: Antibiotic resistance pattern of S. aureus isolates in agr-specific groups.
agrI n (%) agr II n (%) agrIII n (%)
25 (59.5) 3 (50) 13 (56.5)
21 (50) 2 (33.3) 13 (56.2)
41 (97.6) 4 (66.6) 23 (100)
11 (26.1) 1 (16.6) 10 (43.4)
Chloramphenicol 4 (9.5) 0 4 (17.3)
Erythromycin 12 (28.5) 2 (33.3) 7 (30.4)
Tetracycline 6 (14.2) 0 4 (17.3)
Levofloxacin 4 (9.5) 2 (33.3) 5 (21.7)
Vancomycin 0 0 2 (8.9)
Linezolid 9 (21.4) 1 (16.6) 4 (17.3)
Fusidic acid 20 (47.6) 2 (33.3) 11 (47.8)
Tigecycline 0 0 0
* SXT: Trimethoprim-sulfmethoxazole; n: No. of isolates.
agrIV n (%) Non-typeable n (%)
Total n (%)
Pak J Med Sci 2014 Vol. 30 No. 1 www.pjms.com.pk 175
this study. Since the patients in ICUs are acutely ill
and immune-compromised, it generates more risk
for the infections.
A higher percentage (94%) of MRSA was found in
nasal swabs than other specimens in contrast to the
pus and sputum samples reported by others.3,15 This
is due to the reason that nasal swabs constitute the
major portion of samples coming from ICU.
For all the antibiotics tested, MRSA isolates
showed great resistance than MSSA. Most of MRSA
isolates presented multiple drug resistance. All
isolates indicated 21.1% resistance to linezolid while
MRSA presented 20.8% resistance. These results
are in contradiction with other reports3,16 and up to
10% has been reported from Iran.17 This high level
of resistance observed in isolates could be due to
mutations of multiple gene copies, chloramphenicol-
florfenicol resistance (cfr) gene carriage or misuse
of this effective drug. Levofloxacin and tetracycline
presented the resistance in the range of 16%-19% in
MRSA isolates while 14.6% isolates were resistant
to chloramphenicol which is similar to other
This study also identified 3% MRSA isolates
which were resistant to vancomycin. This finding is
in contrast with other studies from the region.3,14,19
Currently the drug of choice for treating life
threatening infection caused by multidrug resistant
MRSA is vancomycin so the emerging resistance
against it would be a serious concern for the
clinicians in Pakistan.
In this study, tigecycline was the only drug with
100% sensitivity showing similarity with the earlier
findings.3 Reports from other studies suggest that
tigecycline is a good choice and has not yet been
influenced by any of the resistance mechanisms
which are involved in other antimicrobials.19,20
By amplification of the hyper-variable domain of
the agr locus, we assigned agr groups to our clinical
isolates. Agr group I was most prevalent group in
both MRSA and MSSA clinical isolates followed
by agr III which is similar with other studies.10,21,22
agr group I and III are closely related having
80% sequence homology that would propose an
exclusive genetic characteristics of our isolates and
selection for the coexistence of S. aureus strains in
No agr group was identified for 17 isolates
which is in accordance with a previous report.21
Only 6 isolates were typed as agr II and 2 isolates
as agr IV which represents reduced prevalence of
these groups in our locality that is opposite to the
earlier findings.22 agr IV was only detected in case of
MSSA while it was absent for MRSA isolates which
is similar to results of few other studies.10,21 Their
absence shows that competition does not favor
Although agr I was dominant in all sources,
hospital wards and age groups, it was higher in nasal
swabs (50%), OPD (50%) and 44+ years age group
(57.8%) respectively. agrIII was more prevalent in
sources other than pus or nasal swabs (31.8%), in
44+ years age group (10.4%) as compared to other
age groups and in outpatients (28.2%) as compared
to hospitalized patients. Resistance profile suggests
that agr III isolates are more resistant than agr I.
Resistance to oxacillin is almost similar in all agr
groups except agr IV and all agr IV isolates were
resistant to penicillin in this study similar to the
report by other study.22
In conclusion, agrI was the most prevalent group
in all the hospital departments, all type of sources
and age groups followed by agrIII. The uniform
fitness of S. aureusagr groups in some cases suggests
that they also have comparable competitive ability
within the host. The allocation of agr groups in this
study perhaps reflects ecological and geographical
structuring or sampling bias.
We thank Dr. Arafat Yameen for providing the
isolates from Holy Family Hospital. This study was
supported by HEC startup research grant to R.Z.
Source of funding: Higher Education Commission,
Conflicts of interest: Nothing to declare.
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SK: Performed the experiments and prepared draft
of paper. FR: Drafting and revising the paper for
intellectual concept. RZ: Conception and design of
research, analysis and interpretation of the data,
approval of final version.