www.als-journal.com/ ISSN 2310-5380/ November 2018
Full Length Research Article
Advancements in Life Sciences – International Quarterly Journal of Biological Sciences
ARTICLE INFO
Date Received:
21/06/2018;
Date Revised:
13/11/2018;
Date Published Online:
25/11/2018;
Authors’ Affiliation:
Department of Microbiology
and Molecular Genetics,
University of the Punjab Lahore,
Pakistan
*Corresponding Author:
Muhamad Sohail
Email:
drsohailmmg@gmail.com
How to Cite:
Sohail M, Latif Z (2018).
Molecular typing of
Methicillin Resistance
Staphylococcus aureus
(MRSA) isolated from
device related infections by
SCCmec and PCR-RFLP of
coagulase gene. Adv. Life
Sci. 6(1): 34-40.
Keywords:
Methicillin Resistant
Staphylococcus Aureus
(MRSA), Device related
infection,
SCCmec typing, agr typing,
RFLP, Panton–Valentine
leukocidin gene (PVL)
Open Access
Molecular typing of Methicillin Resistance Staphylococcus
aureus (MRSA) isolated from device related infections by
SCCmec and PCR-RFLP of coagulase gene
Muhamad Sohail*, Zakia Latif
Abstract
B
ackground: SCCmec and PCR-RFLP are productive and cost-effective methods for
epidemiological investigations and source tracking of MRSA. Aim of this study was to investigate
the epidemiology and molecular characterization of MRSA recovered from device related
infection.
Methods: A total of 626 MRSA were collected from prosthetic device related infections and subjected for
presence of mec gene and SCCmec typing. SCCmec characterized MRSA were subjected for agr typing
followed by RFLP genotyping and amplification of PVL gene. Following the Clinical & Laboratory
Standards Institute (CLSI), antibiotic resistance pattern was determined.
Results: Of 626 MRSA isolates, 488 (78%) were characterized by SCCmec typing. Most common type was
SCCmec IV (43%), followed by SCCmec II (25%) SCCmec III (22%) and only 10% was SCCmec V. SCCmec
characterized MRSA strains were 100% resistance to tobramycin and chloramphenicol, 96% resistance to
ciprofloxacin and 93% resistance to azithromycin. The gene PVL was only present in SCCmec IV. All agr
typed MRSA strains were resistance to gentamicin, tobramycin and chloramphenicol. PVL was present in
all strains of agr III, 80% of agrIV, 50% of agrII and absent in agrI. RFLP analysis resulted in 16 types of
non-duplicate unique bands pattern which were equally distributed among prosthetic device related
infection.
Conclusion: PVL harboring SCCmec or agr typed MRSA strains are less resistance to antibiotics. RFLP is
simple, productive and cost-effective method for molecular typing of MRSA. Correct use of antibiotics
and molecular surveillance is indispensable to detect the change in epidemiological and antibiotic
resistance trends of MRSA.
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Advancements in Life Sciences | www.als-journal.com | November 2018 | Volume 6 | Issue 1
34
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Molecular typing of Methicillin Resistance Staphylococcus aureus (MRSA) isolated from device related infections by
SCCmec and PCR-RFLP of coagulase gene
Introduction
Most of device related infections are associated with
Staphylococcus aureus, which harbors a lot of toxins
enzymes and virulent factors [1]. Device related
infections pose major problem toward the patient safety
especially when causative agent is methicillin resistant
Staphylococcus aureus (MRSA) which is notorious for
antibiotic resistance. Standard guidelines of
identification
and
antimicrobial
resistance
determination were used for better identification and
antibiogram of MRSA [2]. Minimum inhibitory
concentration (MIC) of vancomycin was assayed using
E-test following interpretation criteria of CLSI.
Expression of virulent factors of MRSA is under the
control of four major types of accessory gene regulator
(agr) system [3]. In addition to this agr system, density
dependent expression of proteins called quorum sensing
is also involved in the pathogenesis and different stages
of biofilm formation.
The timely and effectively
activation of agr system is essential to initiate and
establish the infection. This system is also known to
produce certain factors to escape the host immune
response [4]. Staphylococcal cassette chromosome mec
(SCCmec) is a mobile genetic element (21-67 kb) that
confers resistance to methicillin, is integrated into
chromosome of MRSA. Acquisition of mecA gene
causes the alteration of penicillin binding protein
(PBP2) gene and makes methicillin, synthetic penicillin,
cephalosporins and carbapenems ineffective against
MRSA. SCCmec typing is essential to determine
epidemiology and evolution. SCCmec genetic element is
divided into eight types and subtypes [5]. Efficient
typing of MRSA is essential to limit and monitor the
spread of pathogen among hospitals and communities.
There are many efficient, complex, expensive and timeconsuming methods for typing but RFLP getting
popularity due to cost effectiveness and simplicity [6].
Coagulation is produced by Staphylococcus aureus
essentially and it is a pathogenic and identification.
Coagulase gene (coa) was subjected for Restriction
Fragment Length Polymorphism (RFLP). MRSA
secretes some toxins like PVL which destroys the
immune cells and help to tolerate the immune response
[7]. Emergence of community acquired MRSA (CAMRSA) poses major problem to community, on other
hand hospital acquired MRSA (HA-MRSA) was threat
to hospital admitted, immunocompromised patients.
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Aim of this study was to investigate the molecular
typing of MRSA isolated from device related infections
on the basis of SCCmec typing, agr typing, PCR-RFLP
analysis and antibiotic resistance profiles. Selected
strains of MRSA were subjected for 16S rRNA
amplification followed by gene sequencing.
Methods
A total of 626 strains of MRSA were isolated from 6,424
prosthetic devices related infections following standard
microbiological guidelines as previously described [8].
These samples were collected from six types of
prosthetic devices and catheters including CVC (Central
Venous catheters), ETT (Endotracheal Tubes), VA/VP
shunts, urinary catheters, orthopedic implants and
peritoneal catheters.
Antibiogram: Antimicrobial sensitivity was done by
Kirby-Bauer disk diffusion method following the CLSI
guidelines [2]. MIC of vancomycin (BioMerieux-USA)
was determined by E-test following interpretive criteria
of CLSI. Staphylococcus aureus control strain
ATCC25923 was used as quality control for
antimicrobial sensitivity testing.
DNA extraction: Genomic DNA was extracted from all
bacterial cells following the protocol of CTAB/NaCl
extraction method with small modifications [9]. Briefly,
Overnight culture was centrifuged at 22,000 RCF for 10
minutes to get the pellet. The pellet was suspended in TE
buffer and incubated at 37C° for one hour after addition
of 10% SDS and proteinase K. After incubation NaCl
and CTAB/NaCl was added, mixed well and incubated
at 65C° for 10 minutes. Upper most layer was collected
and mixed with chilled phenol/chloroform/isoamyl
alcohol and centrifuged at 1400rpm for 10 minutes.
Supernatant was collected and mixed with isopropanol
to get DNA precipitated. After centrifugation DNA
pallet was washed with 70% alcohol and dissolved in
50ul of deionized water.
Molecular detection of mecA and PVL gene:
Amplification of mecA and PVL gene was done in all
MRSA strains. MRSA were confirmed by the presence
of mecA gene. Amplification of mecA was performed
with
primers
mecA-F
(5'GTGAAGATATACCAAGTGATT 3') and mecA-R
(5'ATGCGCTATAGATTGAAAGGAT3')
[10].
Presence of PVL gene is detected with primers Luk-PV-
Advancements in Life Sciences | www.als-journal.com | November 2018 | Volume 6 | Issue 1
35
You’re reading
Molecular typing of Methicillin Resistance Staphylococcus aureus (MRSA) isolated from device related infections by
SCCmec and PCR-RFLP of coagulase gene
F
(5'ATCATTAGGTAAAATGTCTGGACATGATCCA3
')
and
Luk-PV-R
(5'GCATCAAGTGTATTGGATAGCAAAAGC3') [11].
SCCmec typing: Multiplex PCR was used to amplify the
six types of SCCmec gene using the set of primers
already reported [12]. Multiplex PCR for subtypes of
SCCmec IV was performed separately and all others in
the same reaction. The reaction mixture of 50μl
contained master mixture, template DNA and primers.
agr grouping: agr grouping was performed by multiplex
PCR using primers reported in 2015 [12]. This PCR
grouped MRSA strains in four categories. There were
some strains which are not classified by these primers
and denoted as agr0.
Restriction Fragment Length Polymorphism (RFLP)
analysis: Bacterial genome DNA was extracted and coa
gene was amplified using the previously described set of
primer and conditions of amplification [13]. Amplified
product was digested using two restriction enzymes
(aluI and HaeII; NEB England). Restriction was done
following the manufacturer guidelines in the same
reaction tube. After restriction digestion gel
electrophoresis was done with 3% agarose to detect the
restriction fragments.
16S rRNA sequencing: Using the universal primers
785F (GGATTAGATACCCTGGTA) and 907R
(CCGTCAATTCMTTTRAGTTT) 16S rRNA was
amplified and sequenced from Macrogen, Korea. The
sequences obtained were BLAST in National Centre for
Biotechnology
Information
(NCBI)
database
(https://blast.ncbi.nlm.nih.gov/Blast) for identification
of bacterial strains and observed similarity to reported
sequences. The sequences obtained from Macrogen
were submitted to GeneBank for accession numbers.
After successful submission accession numbers were
obtained.
Phylogenetic tree: After getting accession number from
GeneBank, a phylogenetic tree was constructed using
Molecular Evolutionary Genetics Analysis version 7.0
(MEGA7) software on basis of similar sequences in
NCBI database keeping bootstrap value 1000 [14].
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Results
A total of 626 isolates were confirmed as MRSA by
amplification of mecA gene, isolated from prosthetic
device related infections. Of 626 isolates of MRSA, 98%
were resistance to ofloxacin, 97% were resistance to
ciprofloxacin, 92% were resistance to tobramycin, 88%
were resistance to erythromycin, 88% were resistance to
azithromycin and erythromycin and 87% resistance to
gentamycin. None of isolate was resistance to
vancomycin and linezolid [15].
SCCmec characterization: The distribution of SCCmec
determined by multiplex PCR. Out of 626 strains, 22%
(138/626) didn’t belong to any group. SCCmec IV was
most common group (43%) among MRSA followed by
type II 25%, type III 22%, type V 10%. A total of 488
strains were typed by SCCmec typing method (Figure 1).
Prevalence of antibiotic resistance among various
SCCmec characterized MRSA is given in the Table 1.
Results of antimicrobial resistance showed variable
resistance pattern among different groups of SCCmec
typing. Overall, all SCCmec groups were resistant to
tobramycin and chloramphenicol; sensitive to
vancomycin and linezolid, other antibiotics showed
variable resistance among different groups.
Figure 1: SCCmec and agr typing of MRSA
agr typing
Distribution of agr genes determined among MRSA
isolated form prosthetic devices. Some of MRSA, 14%
(68/488) didn’t belong to any agr group and denoted as
agr0 group. The most frequent type was agr type I which
was 61% followed by agr type IV 16%, agr type II 13%
and agr type III 10% (Figure 1). Out of 488 isolates of
MRSA 420 were types by agr typing method, remaining
68 isolates didn’t belong to any group. Distribution of
antimicrobial resistance among different agr types is
given in the Table 2. All of 55 isolates belonged to agr II,
were resistance to all antibiotics except vancomycin and
linezolid. Similarly, 67 isolates belonged to agr IV were
resistance to all tested antibiotics except vancomycin
Advancements in Life Sciences | www.als-journal.com | November 2018 | Volume 6 | Issue 1
36
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Antibiotics
Molecular typing of Methicillin Resistance Staphylococcus aureus (MRSA) isolated from device related infections by
SCCmec and PCR-RFLP of coagulase gene
SCCmec II
SCCmec III
SCCmec IV
25% (123/488)
22% (108/488)
43% (209/488)
SCCmec V
10% (48/488)
Amikacin
80%(98/123)
100%(108/108)
75%(157/209)
Gentamicin
100%(123/123)
100%(108/108)
75%(157/209)
Tobramycin
100%(123/123)
100%(108/108)
100%(209/209)
Azithromycin
80%(98/123)
90%(97/108)
100%(209/209)
Doxycycline
100%(123/123)
90%(97/108)
85%(178/209)
Ciprofloxacin
100%(123/123)
100%(108/108)
90%(188/209)
Ofloxacin
100%(123/123)
100%(108/108)
80%(167/209)
SXT
100%(123/123)
100%(108/108)
70%(146/209)
Clindamycin
80%(98/123)
90%(97/108)
10%(21/209)
Linezolid
0%(0/123)
0%(0/108)
0%(0/209)
Chloramphenicol
100%(123/123)
100%(108/108)
100%(209/209)
SXT= Trimethoprim/Sulfamethoxazole
Table 1: SCCmec typing and antibiogram of MRSA isolated prosthetic device related infection
100%(48/48)
100%(48/48)
100%(48/48)
100%(48/48)
42%(20/48)
100%(48/48)
100%(48/48)
48%(26/48)
42%(20/48)
0%(0/48)
100%(48/48)
Antibiotics
agr I
agr II
agr III
agr IV
Amikacin
61% (256/420)
90%(230/256)
13% (55/420)
100%(55/55)
10% (42/420)
80%(34/42)
16% (67/420)
100%(67/67)
Gentamicin
100(256/256)
100%(55/55)
100%(42/42)
100%(67/67)
Tobramycin
100(256/256)
100%(55/55)
100%(42/42)
100%(67/67)
Azithromycin
80%(205/256)
100%(55/55)
100%(42/42)
100%(67/67)
Doxycycline
60%(154/256)
100%(55/55)
80%(34/42)
100%(67/67)
Ciprofloxacin
100(256/256)
100%(55/55)
80%(34/42)
100%(67/67)
Ofloxacin
100(256/256)
100%(55/55)
80%(34/42)
100%(67/67)
SXT
100(256/256)
100%(55/55)
80%(34/42)
100%(67/67)
Clindamycin
80%(205/256)
100%(55/55)
0%(0/42)
100%(67/67)
Linezolid
0%(0/256)
0%(0/55)
0%(0/42)
0%(0/67)
Chloramphenicol
100(256/256)
100%(55/55)
100%(42/42)
100%(67/67)
SXT= Trimethoprim/Sulfamethoxazole
Table 2: Agr typing and antibiogram of MRSA isolated prosthetic device related infection
Figure 2: RFLP of MRSA isolated from prosthetic device related infections.
and linezolid. Other two groups (agr I and agr III)
showed the variable resistance towards tested
antibiotics.
Restriction Fragment Length Polymorphism: A total
of 420 strains of MRSA were typed by SCCmec and agr
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typing, were subjected for PCR-RFLP analysis. coa gene
was amplified by specific primers. Amplified products
were digested with HaeII and AluI restriction enzymes.
On the basis of pattern of bands from both restriction
enzymes 16 unique types of band pattern were identified
(Figure 2). One representative for each unique pattern
Advancements in Life Sciences | www.als-journal.com | November 2018 | Volume 6 | Issue 1
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Molecular typing of Methicillin Resistance Staphylococcus aureus (MRSA) isolated from device related infections by
SCCmec and PCR-RFLP of coagulase gene
was selected for further investigations. Antibiogram,
biofilm potential, SCCmec typing, agr typing was similar
for all pathogens showing same banding pattern.
Panton-Valentine
leukocidin
(PVL)
gene
characterization: The PVL gene was detected in SCCme
and agr typed MRSA. The PVL genes was found only in
SCCmec IV typed MRSA. All other SCCmec don’t have
PVL gene. On other hand PVL gene was present in 100%
agr type III, 80% of agr type IV and 50% of agr type II.
Phylogenetic analysis: On the basis of antimicrobial
sensitivity pattern, SCCmec typing, agr typing 420
strains of MRSA were selected. Which were subjected
for PFLP analysis and resulted in 16 unique nonduplicate DNA band patterns. Sixteen MRSA isolates
were selected form each unique DNA pattern for 16S
rRNA sequencing. Among selected strains, nine were
HA-MRSA and seven were CA-MRSA.
Figure 3: Phylogenetic analysis of CA-MRSA
Figure 4: Phylogenetic analysis of HA-MRSA
Phylogenetic tree was constructed separately for CAMRSA and HA-MRSA because these two groups had
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similarity among them. Phylogenetic tree showed
selected strain of CA-MRSA (ZS28C, ZS35C, ZS38C,
ZS41C, ZS43, ZS59C and ZS64C) were 99% to 100%
similar to the reported sequences. Similarly, HA-MRSA
(ZS39H, ZS40H, ZS42H, ZS44H, ZS45H, ZS46H,
ZS47H, ZS48H and ZS49H) were 99 to 100 % similar to
the reported sequences. Phylogenetic tree of CA-MRSA
and HA-MRSA with accession number is given in the
Figure 3 and 4 respectively.
Discussion
Most of catheter related infections are due to MRSA
which causes community acquired and hospital
acquired infections. Source identification is very
important while dealing with outbreak of infectious
disease. Most authentic and valuable tool for source
identification is molecular typing [16]. Pulse Field Gel
Electrophoresis (PFGE) and Multi Locus sequence
typing (MLST) are standard methods for tying of S.
aureus but these are expensive and laborious [17].
This study was designed to do the molecular typing of
MRSA isolated from prosthetic device related infections
and determine their antibiotic profile. None of MRSA
was characterized as SCCmec I, the most prevalent type
was SCCmec IV (43%) followed by SCCmec II (25%),
SCCmec III (22%) and SCCmec V (10%). These results
were consistent with previous studies describing
SCCmec IV most prevalent followed by type III, V and I
[18]. Some of MRSA (24%) isolated form prosthetic
devices were not typed by agr system. Among typed
MRSA 61% belonged to agr type I followed by agr IV,
agr II and agr III, these results were similar to the
findings reported in Iran in 2017[18]. The difference in
the prevalence of agr and SCCmec typing with some
studies was due to source of specimens [19-22]. PCRRFLP was done to genetically type the MRSA isolated
from prosthetic device related infections and sixteen
strains were selected for further testing. Different studies
showed different DNA banding patterns with same set
of enzymes. The variations were due to sample source
and geological location, but the similarity was that all
DNA bands were multiple of 80bp [23,24].
All SCCmec types isolates of MRSA were resistance to
tobramycin and chloramphenicol and sensitive to
linezolid and vancomycin which correlates the
antimicrobial sensitivity testing conducted in Iran
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Molecular typing of Methicillin Resistance Staphylococcus aureus (MRSA) isolated from device related infections by
SCCmec and PCR-RFLP of coagulase gene
[25,26]. For aminoglycosides (amikacin, gentamicin and
tobramycin), azithromycin some studies showed similar
results [27] and other showed different results [28].
SCCmec IV harboring PVL was less resistance to the
antibiotics compared to the SCCmec I, SCCmec II,
SCCmec III and SCCmec V that don’t have PVL gene.
MRSA belonged to agr III were less resistant to
antibiotics as compared to agr I that don’t have agr
typing gene. These results showed that PVL gene was not
associated with antibiotic resistance especially in the
MRSA isolated form prosthetic devices. MRSA isolates
harboring PVL were less resistant to the antibiotics [29].
All SCCmec IV have PVL gene and similar results of
association of SCCmec IV and PVL reported in China
[30]. MRSA types as SCCmec IV was the major MRSA
that spreads from community to the hospital setting and
same results reported in Libia [27]. The study described
that PVL gene is present in 40% of MRSA isolated from
prosthetic device related infection, these results were
similar to the study conducted in Oman (54%) and
different from the results reported in Kuwait hospital
(14.6%) [31,32]. MRSA containing PVL gene were most
commonly associated with skin and soft tissue infection
which justified the high prevalence of PVL containing
MRSA from prosthetic devices.
In Conclusion, MRSA isolated form device related
infections were more resistant to antibiotics than any
other type of infection. SCCmec IV along with PVL and
agr I was more prevalent genotype of MRSA involving
device related infections.
5.
6.
7.
8.
9.
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11.
12.
13.
Conflict of Interest Statement
The authors declare that there is no conflict of interest
regarding the publication of this paper.
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