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mec A AND mec C GENES PROFILE OF CLINICAL ISOLATES OF
Staphylococcus aureus
Zainab Dakhil Degaim*, Abbas D. Mater*, Haydar Khamis Al- Malky**
* Department of Microbiology,-College of Medicin,-University of Thi-Qar, Iraq.
** Department of Microbiology ,College of Veterinary Medicine, University of Thi-Qar, Iraq.
Key word: mecA, mec C, S. aureus, PCR
Corresponding author E.mail:zainabdegaim@yahoo.com
ABSTRACT
Methicillin resistant Staph. aureus (MRSA) was a substantial bacteria that caused diverse
hospital and systemic infections. The detection of mec gene of this pathogen must be used as a
rapid screening technique. The current study was aimed to characterize the frequency of mecA
and mecC genes in Staph. aureus were isolates which phenotypically were resistance to
methicillin which were recovered from patients with tonsillitis that was happened at Al-
Habboby teaching hospital during the period from February to November, 2016 in Thi-Qar
province/Iraq by using PCR technique. From a complete of 109 (63%) Staph. aureus isolates,
only 71 isolates were identified phenotypically as MRSA. The molecular results were
documented that (62% and 31%) of isolates expressed mecA and mecC, respectively. Sixty nine
percentage of all Staph. aureus isolates showed negative results of mecC gene. The current
results of were established the significance of mecC gene in MRSA recognition than mecA gene
and highlighted the increasing manner of its frequency in south of Iraq.
INTRODUCTION
Methicillin resistant Staphylococcus aureus (MRSA) is one of the greatest vital multiresistant
human pathogens universal, causing the infections in both hospitals and community
and in the livestock [1]. The gaining of the mec A gene by S. aureus isolates, therefore those
isolates become resistant to methicillin antibiotic and recorded as MRSA, and the mec A gene
situated on the staphylococcal cassette chromosome mec (SCCmec) [2].
The SCCmec elements were characterized through the presence of two indispensable loci: the
mec gene complex comprising the methicillin resistance determinant with intact copies of the
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mec regulatory genes (mecI,mecR, and mecR2) and the ccr gene complex, which encodes siteand
orientation-specific recombines responsible for SCCmec mobilization [3]. The incidence of
hospital associated and similarly community acquired infections triggered by S. aureus strains,
especially MRSA which have gained worldwide notoriety as hospital 'superbugs' and that are
resistant to numerous antibiotics [4].
The MRSA poses a serious problem for infection prevention, control and antibiotic treatment
globally. In MRSA, resistance against almost all beta-lactam compounds in clinical use is
caused by the expression of an alternate penicillin binding protein (PBP2a) that is encoded by
the mecA gene and those genes can be found in different staphylococci [5].
A novel mec gene type was discovered in 2011, which located on a novel SCCmec element
designated as type XI [6,7]. Because of its highly divergent sequence, it cannot be detected by
routinely used molecular assays designed to identify mecA (formerly mecALGA251) [6,8]. This
gene renamed mec [5]. mecC had been isolated from various animals including cattle, sheep,
dogs, cats, a guinea pig, rabbits, rats, and a chaffinch as well as from humans from Ireland,
England, Scotland, Germany, Denmark, Sweden, Norway, France, Switzerland, Belgium and
The Netherlands [9,10,11]. The aim of the this study was to characterize a sensitivity of S.aureus
isolates to methicillin antibiotic, to detect the attendance of mecC and mecA genes that found in
isolates of S. aureus recovered from patients with tonsillitis.
MATERIAL AND METHODS
Ethical approval
This research was approved by the Medicine College Ethics Committee, Thi-Qar University,
Thi-Qar Province, Iraq.
Laboratory methods
All S. aureus were isolated from 173 swabs which collected from tonsillitis patients whom
admitted to ENT unit in AL-Habbuby Teaching Hospital of Thi-Qar province through the period
from February to November, 2016 and identified depending on cultural properties (LAB/ United
Kingdom), followed by biochemical tests [12,13]. The confirmed diagnosis was performed by
using API system (BioMerieux/France).
Antibiotic sensitivity test
To detect the S. aureus sensitivity to methicillin antibiotic (5μg /disc) (Bioanalyse, Turkey)
by using the disc diffusion method described by Kirby, 1966 [14]. The diameters of inhibition
zone were measured and interpreted according to CLSI [15].
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Preparation of bacterial DNA
The S. aureus chromosomal DNA extraction was carried out on entirely S. aureus isolates
using Genomic DNA Extraction kit (Geneaid/Korea).
PCR diagnosis of mecA and mecC genes
The specific primer pairs of mecA as following: forward: 5’-GTA GAA ATG ACT GAA
CGT CCG ATA A-3’ and reverse: 5’-CCA ATT CCA CAT TGT TTC GGT CTA A-3’16.
While for mecC gene: forward: 5’-GAA AAA AAG GCT TAG AAC GCC TC -3’ and reverse:
5’-GAA GAT CTT TTC CGT TTT CAG C-3’[17].
The PCR cycling conditions of mecA gene: initial denaturation at 94°C for 4 min, followed by
30 cycles of denaturation at 94°C for 45 sec, annealing at 50°C for 45 sec, extension at 72°C for
1 min and final extension for 2 min [18]. Whereas for mecC gene: initial denaturation at 94°C
for 15 min, followed by 30 cycles of denaturation at 94°C for 30 sec, annealing at 59°C for 1
min, extension at 72°C for 1 min and final extension for 10 min after the last cycle2.
Electrophoresis of PCR product was carried out in 1.4% agarose gel and the presence of a 310
bp and 138 band indicate a positive result for mecA and mecC genes, respectively.
RESULTS AND DISCUSSION
The results of the current study presented that the prevalence of S. aureus was 109 isolates
(63%) from completely collected swabs. The S. aureus was an important causer of tonsillitis
infection [19,20].
From 109 isolates of S. aureus, only 71 isolates (65%) exhibited resistant to methicillin disc and
which recorded phenotypically as MRSA.
In similar study conducted in Thi-qar province, Hamim, [21] recorded an approach percentage
of MRSA infection outbreak (53%) in comparison with the results of the present study. On other
hand, the recent results dissimilar with local studies that recorded a low percentages of MRSA
among S. aureus isolates such as Taha et al. [22] in Erbil, Abdullah,[23] in Baghdad, reported
that the rates of MRSA were 30.24% and 41.54%, respectively. Also the present results
disagreed with results of study piloted in Nigeria by Nwokah et al. [24] exhibited that 25
(12.2%) out of 205 isolates of S. aureus were resistant to oxacillin.
The molecular detection of mecA gene revealed that 44/71 (62)% of isolates contained this
gene with the molecular weight of approximately 310bp (Fig1). The genetic profile of mecA is
commonly used as a reference standard for MRSA identification, and used as a main test or for
validation [28]. The percentage of mecA gene in current study approached with other studies
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like Hamim,21 and Nwokah et al. [24] showed that 88 (73.3%) and 17 (68%) of isolates had the
targeted gene.
Sixty eight isolates from total S. aureus harbored mecA gene, the current results disagreed with
studies performed by Lepainteur et al.[25]; Becker et al.[26] showed that all 98 methicillin
sensitive S. aureus (MSSA) strains exhibited negative results for mecA.
Not all S. aureus isolates which recorded phenotypically as MRSA had mecA gene, because of
the resistancy to methicillin may be due to not only to the existence of the mecA gene alone;
nevertheless by a cluster of ica gene with this gene [27], furthermore must be due to the present
of mecC gene that encoded to the same resistancy among human and bovine MRSA isolates [5,
6].
Among the examined MRSA, 22 amplified of the goal gene (mecC); the percentage was 30%
with the molecular weight of approximately 138bp (Fig.2).
The recent percentage of mecC gene differenced from the results of Stegger et al.[17] showed
that 12 (6%) isolates harbored mecALGA251 identified amongst 203 isolates. The recent results
documented the slightly percentage of mecC gene in MRSA isolates, similarly Peterson et al.[9]
described that mecC gene established in 1.5% of S. aureus isolates, while the frequency of goal
gene increased and reached to 1.9% in 2010 and 2.8% in 2011 in the Denmark.
In spite of the mecC gene was more detected in S. aureus isolated from animal samples and less
frequently detected in humans, but Doğan et al.[2] showed that all MRSA isolates harbored
mecA gene, whereas a mec C gene was not presence in completely isolates of S. aureus.
The molecular detection of mecA gene in staphylococci was usual mode, also Paterson et
al.[1] suggested the demonstration of this gene by PCR as gold standard method, but S. aureus
had mecC gene cannot detect via specific PCR through the discovery of mecA gene (Paterson et
al.[29] resulted from insufficiencies of phenotypic methods to the finding of mec C gene,
therefore the methods based on DNA used to limit the goal gene.
The mecC gene source was not tacit adequately[30], but Figueiredo and Ferreira,[31]
strongly suggested that the associations between humans and livestock had been maintained,
and an incidence of mecC gene cross-transmission between the last populations.
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CONCLUSION
The results of present study established the significance of mecC gene in MRSA recognition
than mecA gene and highlighted the increasing manner of its frequency in south of Iraq.
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Fig. (1): Agarose gel electrophoresis of mec C gene amplification, M: ladder, 1-6:
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1 2 3 4 5 6 7 M
M 1 2 3 4 5 6 7 8 9
100
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138 bp 100
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