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Isolation and Identification of Methicillin Resistance Staphylococcus aureus

from Clinical Samples and their Drug Resistance Patterns

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 Volume 4, Number 2: 171-175
ISSN: 2313-3937
2016

Research article

Isolation and Identification of Methicillin Resistance

Staphylococcus aureus from Clinical Samples and
their Drug Resistance Patterns
Luma Saeed Mohammed1* and May Talib Flayyih1

ABSTRACT
Methicillin Resistance Staphylococcus aureus (MRSA) is a common multidrug resistant pathogen worldwide, emergence of
community-associated MRSA (CA-MRSA) infections has a fundamentally different epidemiology compare to that of
hospital-associated MRSA (HA-MRSA) infections, which manifests as skin and soft-tissue infections. The study was carried
out to isolate Staphylococcus aureus from different clinical samples and determined the antibiotics susceptibility pattern of
S. aureus, 300 samples were collected from blood, skin, urine, ear and sputum to isolate S. aureus, identify and check the
susceptibility test using Vitek 2 Compact System. Analysis of results revealed that out of 300 clinical samples 103 isolates
(34.33 %) were staphylococci and out of these isolates 40 (38.83 %) were S. aureus. According to antibiotics sensitivity
test MRSA identified through cefoxitin screen test, resistance to oxacillin and presence the modification of mecA gene,
from 40 S. aureus isolates, 38 (95 %) isolates were MRSA. the results revealed that resistance to benzylpenicilline was
100 % and 97.65 % of isolates were produced beta lactamase enzyme. Resistance to gentamycin was 15%, erythromycin
55%, clindamycin 35% and 2.5% of isolates had intermediate resistance to teicoplanin 5%, tertracyclin 47.5% rifampicin
10%, trimethoprim/sulfamethoxazole and tobramycin 5%, while 12.5 % of isolates had intermediate resistance to the last
one. 5% of isolates resisted vancomycin, while 10% had intermediate resistance to it. 92.5 % of isolates were sensitive to
levofloxacin, while 7.5 % had an intermediate resistance to it. 100% of isolates were sensitive to moxifloxacin, linezolid
and tigecyclin and 97.5 % of isolates were sensitive to nitrofurantoin. The results reveal a possibility of potential public
health threat of MRSA. MRSA were highly resistance and multidrug resistance pattern to other antibiotics. The problem of
antibiotic resistance among the bacteria cannot be solved by the production of new and stronger antibiotics. 100% of S.
aureus isolates were sensitive to linezolide, tigecyclin and moxifloxacin.
Keywords: Staphylococcus aureus, Coagulase ,MRSA, SCCmec, Cefoxitin.
Citation: Mohammed LS, Flayyih MT. (2016) Isolation and Identification of Methicillin Resistance Staphylococcus
aureus from Clinical Samples and their Drug Resistance Patterns. World J Exp Biosci 4: 171 – 175.
Received October 19, 2016; Accepted December 8, 2016; Published December 23, 2016.

INTRODUCTION
Received September 06, 2016; Accepted September 30, 2016; Published October 7, 2016.
Staphylococcus aureus is both a commensally organism and our most serious health threats, infections from resistant
also an important opportunistic human pathogen causing a var- bacteria are now too common and some pathogens have even
iety of community and hospital-associated infections, such as become resistant to multiple types or classes of antibiotics, S.
bacteremia,sepsis, endocarditis, pneumonia, osteomyelitis, aureus has become a major public health concern as a result
arthritis and skin diseases [1]. Antimicrobial resistance is one of of the steadily increasing incidence of antimicrobial resistance

*Correspondence: Mohammed LS.Lumasaeed05@gmail.com.

Department of Biology, College Science, University of Baghdad, Baghdad, Iraq.
Full list of author information is available at the end of the article.
Copyright: © 2016, Mohammed LS, Flayyih MT. This is an open-access article distributed under the terms of the Creative
Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any site, provided the original
Unicorn author and source are credited.
171
Mohammed LS, Flayyih MT. (2016).
particularly methicillin-resistant S. aureus (MRSA) [2]. MRSA
is a serious problem in the treatment and control as a result of
multidrug resistant and causes wide variety of human diseases
[3,4]. MRSA had resistance to many commonly used groups of
antibiotics like beta lactams, aminoglycosides, macrolides,
fluoroquinolones, chloramphenicol, and tetracycline [5]. The
present study aims to evaluate the susceptibility of MRSA to
different types of antibiotics.
MATERIALS and METHODS
Sample collection
Three hundred clinical specimens including urine, ear, sputum,
blood and skin swabs, collected from patients attending
Baghdad Hospitals, for the period from January to April 2015.
Isolation of staphylococci
Isolation of staphylococci from different clinical samples by
specific way depending on routine laboratory techniques, all
samples were streaked on mannitol salt agar for detecting
ability of bacterial isolates to grow on this media and
incubated aerobically for 24 h at 37°C [6].
sensitivity card which contain 18-20 types of antibiotics
distributed in 64 wells, 280 microliter of bacterial suspension
were transferred to the antibiotics sensitivity test tubes then
inserted into vitek 2 compact system. There were more than
one concentration for everyone, the turbidity was adjusted to
(0.5-0.63) McFarland measured by using a turbidity meter
DensiChek TM, Results were obtained after 6-12 h of
incubation. Minimum inhibitory concentrations (MIC) were
estimated according to company instruction.
RESULT
Identification of S. aureus
Analysis of results showed that out of total 300 clinical
samples 103 isolates (34.33 %) of staphylococci and out of
these isolates 40 (38.83 %) were S. aureus, which fermented
mannitol salt agar (Fig 1), coagulase test positive (Fig 2),
catalase test positive, oxidase test negative and had B-
hemolysis pettern on blood agar (Fig 1).

Identification of staphylococci
The isolates were identified depending on the morphological
features on culture media and biochemical tests according to
Bergey’s Manual [7] and final detection was done by using
vitek2 compact system.

Microscopic examination
The isolates were stained by Gram stain to detect their
response to stain, cocci shapes and their arrangement.
Fig. 1 Colonies features on mannitol salt agar (a) and blood agar (b).
Colonial morphology on blood agar and
mannitol salt agar
The colonies grown on blood agar plate were tested for their
shape, size, color and blood haemolysis pattern, while those
grown on mannitol salt agar plate, were tested for their ability
to ferment mannitol sugar.

Biochemical tests
Biochemical tests were performed to confirm S. aureus using
and mannitol fermentation test, catalase test, coagulase test
and oxidase test.

Identification of S. aureus by using Biomerieux

vitek 2 compact system
The manufacturer's instructions of VITEK 2 kits were followed
to carry out the test of bacterial identification.
Antibiotics sensitivity test of S. aureus,
determination of MIC values by using
Biomerieux vitek 2 compact system
Susceptibility test was determined for 40 isolates of S. aureus
against 15 different antibiotics; benzylpenicillin, gentamicin
tobramycin, levoflocin, moxifloxacin, erythromycin,
clindamycin, linezolid, teicoplanin, vancomycin, tertracyclin,
tigecyclin, nitrofurantoin, rifampicin and
trimethoprim/sulfamethoxazole), in addition, to detect the
phenotypes like presence of beta lactamase enzyme, inducible
and constitutive clindamycin resistance, resistance to
streptogramine A and B and resistance to vancomycin (VISA,
Hetero VISA, VSSA and VRSA). MIC value for antibiotics
determined by vitek 2 compact system. The antibiotics
172
Fig 2. Coagulase test; a, positive coagulase test; b, negative
coagulase test
Vitek 2 compact system was used for precise and accurate
identification of isolates related to the species of S. aureus
isolates which previously identified by conventional
biochemical. Numbers and presences of S. aureus isolates in
skin was 20 isolates (50 %), 11 isolates isolated from blood
samples (27.5 %), 5 isolates isolated from urine samples
(12.5%) and 2 isolates isolated from sputum samples (5 %)
and ear (Fig 3).
Antibiotic susceptibility test
Detection of MRSA
The detection of MRSA was carried out through cefoxitin
screen test and susceptibility test of S. aureus isolates to
World J Exp Biosci. Vol. 4, No. 2: 171-175.
Mohammed LS, Flayyih MT. (2016).
oxacillin, these two tests confirmed phenotypically by presence
of modification of mecA gene, the results showed that 38 (95
%) of S. aureus isolates gave positive results to cefoxitin
screen test indicated that these isolates were MRSA, 2 (5 %)
isolates (SA34 and SA38) were negative for this test indicated
that these two isolates were MSSA, 95 % of S. aureus isolates
resisted oxacillin (MIC ≥ 4 µg/ml) and 5% of isolates were
sensitive (MIC ≥ 0.5 µg/ml), Oxacillin-resistant S. aureus
(ORSA), more commonly referred as MRSA [8]. The results
showed that all S. aureus isolates which gave positive test of
cefoxitin screen and resistance to oxacilline had modification of
penicillin binding protein (mecA) (95 %).
Fig 3. Distribution of Staphylococcus isolates in different clinical
samples
Susceptibility of S. aureus isolates to different
antibiotics
The results showed that all S. aureus isolates except one
produce beta lactamase enzyme (97.5%). Resistant to different
antibiotics were benzylpenicillin 100%, gentamicin 15%,
erythromycin 55% , clindamycin 35% and 2.5% of isolates had
intermediate resistance, teicoplanin 5%, tertracyclin 47.5%
rifampicin 10%, trimethoprim/sulfamethoxazole and tobramycin
5%, while 12.5 % of isolates had intermediate resistance to the
last one, 5% of isolates resistant to vancomycin, while 10%
had intermediate resistance, 92.5 % of isolates were sensitive
to levofloxacin, while 7.5% had an intermediate resistance,
100% of isolates were sensitive to moxifloxacin, linezolid and
tigecyclin and 97.5% of isolates were sensitive to nitrofurantoin
(Fig 4).
DISCUSION
The skin is the largest organ of the body and with the
underlying soft tissue, which included the fat layers, fascia and
muscle, represented the majority of the tissue in the body, skin
and soft tissue infections (SSTIs) were commonly caused by S.
aureus specifically MRSA (HA-MRSA and CA-MRSA). It
causes mild infections on the skin, like sores or boils but also
caused more serious skin infections like furuncles, impetigo or
infects surgical wounds. S. aureus causes pneumonia, urinary
tract infection and isolated from blood in patients with
endocarditis, osteomyelitis, toxic shock syndrome and scalded
skin syndrome, if bacteria enter blood stream this caused
bactremia in adult and sepsis in infants [9,10]. HA-MRSA
SSTIs in North America found in 45.9% of skin infections [11].
173
Basally, cefoxitin screen test for all MRSA isolates carry the
mecA gene, which caused resistance to all beta lactam
antibiotics, including methicillin and othe beta lactam antibiotics
like cephalosporins and carbapenems [12].
Staphylococcal cassette chromosome mec (SCCmec antibiotic
resistance gene mecA encoded penicillin-binding protein 2a
(PBP2a), which differ from other penicillin-binding proteins as
it's active site did not bind to methicillin or other beta lactam
antibiotics [13]. Resistant of S. aureus isolates to penicillin was
100% (MIC ≥ 0.5 µg/ml) due to beta lactamase a hydrolytic
enzyme secreted by bacteria which cleaved beta lactam ring
the structural skeleton of any β lactam antibiotic, production of
it leads to inactivation of β lactam antibiotics [14]. S. aureus
isolates highly sensitive to gentamycin and tobramycin
antibiotics (MIC for sensitive isolates was (≤ 0.5, 4 and 1
µg/ml) for gentamycin and (≤1 µg/ml) for tobramycin,
enzymatic modifications of aminoglycosides is a common
mechanism of resistance to these antibiotics shown by clinical
bacterial isolates [15]. 92.5% of S. aureus isolates were
sensitive to moxifloxacin and 100% were sensitive to
levofloxacin (MIC ≤ 0.5µg/ml) and an intermediate resistance
to levofloxacin (7.5 %, MIC 4 µg/ml). Fluoroquinolone resist-
ance in S. aureus is widespread and caused by norA, gyrA,
gyrB, grlA and grlB genes, which encoded DNA gyrase and
topoisomerase [16]. S. aureus isolates had 55 % and 32.5 %
resistance to erythromycin and clindamycin, respectively. MIC
≥8 µg/ml for islolates which had constitutive resistance to
clindamycin and (≤ 0.25 µg/ml) for isolates which had inducible
clindamycin ressistance, (0.4%) intermediate resistance to
clindamycin (MIC=2 µg/ml), other study showed that resistance
to these antibiotics were 54% and 44%, respectively which
used in the treatment of Staphylococcal infections [17].
All S. aureus isolates were sensitive to Linezolid (100 %, MIC
1,2 and 4 µg/ml). New oxazolidinone antimicrobial, linezolid,
had been approved for the treatment of infections caused by
various Gram positive bacteria, including MRSA and
vancomycin-resistant enterococci (VRE) [18]. The mechanism
of linezolid action involve the inhibition of protein synthesis,
through binding to the domain V region of the 23S rRNA gene,
resistance had been associated with mutations in the central
loop of the domain V region [19] . Five percentage of S.
aureus isolates (SA13 and SA28) had resistance to
vancomycin (VRSA) (MIC ≥ 32 µg/ml), vancomycin (glycop-
eptide antibiotic) had bactericidal activity against aerobic Gram
positive bacteria, especially staphylococci (including beta
lactamase producing and MRSA), it was associated with
slower clinical response and longer duration of MSSA
bacteremia, and it has been associasted with more frequent
complications in patients with endocarditis [20].
study showed that the resistance to teicoplanin (another
glycopeptide antibiotic) was 2.5% ( MIC=32 µg/ml), VanA-type
resistance was the first to be elucidated and which is the most
common characterized by high levels of resistance to
glycopeptides vancomycin and teicoplanin [21]. 47.5% of
isolates had resistance to tetracycline antibiotic (MIC ≥ 16
µg/ml) and 52.5% of isolates were sensitive to this antibiotic
(MIC 1 and 2 µg/ml), two mechanisms of resistance to
tetracyclanes were identified in Staphylococcus spp. i, active
efflux resulting from acquisition of the plasmid-located genes,
tetK and tetL, and ii, ribosomal protection mediated by
transposon-located or chromosomal tetM or tetO determinants
[22]. bacterial 30S ribosome, blocking the entry of transfer
RNA, this ultimately prevented protein synthesis by halting the
incorporation of amino acids into peptide chains and thus
limited bacterial growth [23].
World J Exp Biosci. Vol. 4, No. 2: 171-175.
Mohammed LS, Flayyih MT. (2016).
Fig 4. Percentage of antibiotic susceptibility for S. aureus isolates.
S. aureus isolates had 97.5% of susceptibility to nitrofurantoin
(MIC≤32 µg/ml), this drug damages bacterial DNA, this make
possibility by the rapid reduction of nitrofurantoin inside the
bacterial cell by flavoproteins (nitrofuran reductase) to multiple
reactive intermediates that attack ribosomal proteins, DNA,
respiration, pyruvate metabolism and other macromolecules
within the cell. S. aureus isolates (10%) resist to rifampicin
(MIC ≥ 32 and 16 µg/ml) and 90% of isolates were sensitive to
it (MIC ≤0.5 and 1 µg/ml), other study suggested that more
than 50% were resistant to rifampicin, rifampicin worked by
stopping the making of RNA in the bacteria by inhibiting
bacterial DNA-dependent RNA synthesis through blocking
bacterial DNA-dependent RNA polymerase [24]. 5% of S.
aureus isolates had resistance to
trimethoprim/sulfamethoxazole (TMP/SMX) also known as co-
trimoxazole (MIC ≥ 320 and 80 µg/ml), TMP/SMX used to
treat variety of bacterial infections, it consist of one part
trimethoprim to five parts of sulfamethoxazole, which used for
urinary tract infections, MRSA skin infections and respiratory
tract infections, MRSA isolates have retained susceptibility to
trimethoprim-sulfamethoxazole in many locations worldwide
despite several decades of exposure to this antibiotic. It
worked by blocking folate metabolism in some bacteria which
results in their death [25].
Acknowledgements
The authors are grateful to Department of Biology College of
Science University of Baghdad.
Conflict of interest
The authors declare that they have no conflict of interests.
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Author affiliation
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of Baghdad, Baghdad, Iraq.
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